904091_TRI_Symp_Umschlag.qxp 03.06.2009 11:23 Uhr Seite 1 Proceedings of the 4th International Baytril® Symposium 904091_TRI_Symp_Umschlag.qxp 03.06.2009 11:23 Uhr Seite 2 Bayer Animal Health GmbH 51368 Leverkusen Germany www.animalhealth.bayerhealthcare.com Impressum Publisher Bayer Animal Health GmbH Coordination Dr. Joy Olsen Global Veterinary Services E-mail: [email protected] Cover image Eye of Science Nicole Ottawa & Oliver Meckes GbR Reutlingen www.eyeofscience.com 904091_TRI_Symp_Vorspann.qxp 03.06.2009 10:35 Uhr Seite 3 Proceedings of the 4th International Baytril® Symposium Introduction Two decades ago, the introduction of Baytril® significantly changed the science and art of treating infectious diseases in small and exotic animals. The challenges associated with a wide range of infections, including the most severe ones, could be rapidly and more successfully met for the benefit of our four-legged patients.Today, Baytril continues to be a firmly established cornerstone and therapeutic standard in veterinary medicine. This success is rooted in the combination of two basic factors: the outstanding properties of Baytril as an antiinfective, and the clinical experience of veterinarians around the globe, acquired over time through innumerable treatments. When tackling infections, veterinary health professionals still trust that Baytril will work rapidly to restore the health of their patients, easing suffering and sometimes even saving lives. Veterinary medicine is continually evolving and offering ever more advanced and high quality care. Bayer Animal Health has strived to support these advancements not only by bringing pivotal products such as Baytril to the market, but also through actively supporting progressive clinical research and continuing education, helping to provide timely, relevant information and improve the management of infectious disease in companion animal practice. Symposia such as this 4th International Baytril Symposium underscore our commitment to the science behind our products and to the veterinary profession. It is fitting that here in Florence, Italy, a city rich in art, culture, and history, we reflect on the current state of the art in antimicrobial therapy for a variety of infections in our small animal patients.As the birthplace of Leonardi da Vinci and the Renaissance, Florence spawned masterpieces from artists such as Raphael, Botticelli, da Vinci, and Michelangelo, which remain singular benchmarks of art & culture today. We would like to express our sincere gratitude to our international panel of speakers for sharing their collective knowledge, insight and expertise in this forum. Special thanks and acknowledgement also go to Dr. Ralf Mueller, our symposium chairman, for guiding us through a program we trust is interesting for all. Ralf Ebert, DVM Joy Olsen, DVM Bayer Animal Health GmbH Leverkusen, Germany 3 904091_TRI_Symp_Vorspann.qxp 03.06.2009 10:35 Uhr Seite 4 Proceedings of the 4th International Baytril® Symposium Preface Medicine is the art and science of healing. It encompasses a range of health care practices which have evolved to both maintain and restore health through the prevention and treatment of illness. Medical and spiritual healers have been part of all cultures over many thousands of years. Shamans in various European and American Indian cultures, mudangs in Korea, ngakpas in Tibet, kahunas in Hawaii, and medicinal doctors in Western civilisations have tried to help injured and sick people. Contemporary medicine applies health science, biomedical research, and medical technology to diagnose and treat injury and disease, typically through medication, surgery, or some other form of therapy. One of the most prominent threats to health both in times past and nowadays is bacterial infection. Infection may occur through accidental or surgical trauma, a compromised immune system, or a variety of other causes. Only comparatively recently did it become possible to treat such bacterial infections with specifically targeted antimicrobial compounds that show a high enough efficacy and a concurrently low toxicity to be useful in human and veterinary medicine. Enrofloxacin (Baytril®) was the first fluoroquinolone to be introduced to veterinary medicine 20 years ago and few medications have had a similar impact on treatment in small animal practice. Bayer has conducted and supported high quality research on this antibiotic not only during the years of introducing Baytril to the veterinary profession but until this day. On this 4th International Baytril symposium, researchers from many European and North American countries present scientific information on enrofloxacin, underscoring the continuing importance of this antibiotic in veterinary medicine. As chairperson of this symposium I would like to thank the distinguished researchers for the time and effort they have put into the wide range of cutting edge information in both their manuscripts and presentations about enrofloxacin and its use in veterinary medicine. This symposium is now a well-established tradition and will provide competent, up-to-date and clinically relevant information, in line with the last three symposia. I would also like to acknowledge Bayer Animal Health for its support of veterinary medicine over the years and its relentless commitment to both research and evidence-based clinical medicine which ultimately benefits our patients. Such ongoing high quality research allows us to practice veterinary medicine at the highest possible level. For close to 100 years, Bayer has been a conductor or partner in scientific research and the veterinary community appreciates this continued commitment to high quality medicine. Dr. Ralf Mueller DVM, PhD, DACVD, FACVSc, DECVD Faculty of Veterinary Medicine Ludwig Maximilian University Munich, Germany Chairman 4 904091_TRI_Symp_Vorspann.qxp 03.06.2009 10:35 Uhr Seite 5 Proceedings of the 4th International Baytril® Symposium Contents 6 Baytril®: Historical Impact and Milestones of Veterinary Medicine’s Most Successful Antimicrobial Dr. David P. Aucoin 16 Diagnosis and Management of Bacterial Urinary Tract Infections in Dogs and Cats Dr. Jodi L. Westropp 24 Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Dr. Andreas Moritz 36 Update on Clinical Management of Pyoderma Dr. Antonella Vercelli 44 Bacterial Diseases and Antimicrobial Therapy in Exotics – Overview on the Use of Enrofloxacin Dr. Norin Chai 62 Use of Enrofloxacin in Cats with Resistant Mycoplasma spp. Infections Dr. Mike Lappin FRIDAY 19th JUNE 68 Unique Pharmacologic Characteristics of Baytril® Dr. Joy Olsen 74 Pharmacokinetics – What You Need to Know Dr. Gert Daube, Sandra Mensinger, Dr. Bernd Stephan 80 Baytril® Resistance Monitoring – Susceptibility Status after More Than 20 Years Dr. Hans-Robert Hehnen, Dr. Sonja M. Friederichs, Julia C. Heimbach, Dr. Anno de Jong, Dr. Bernd Stephan 86 Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Dr. Joseph M. Blondeau 5 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 6 Baytril®: Historical Impact and Milestones of Veterinary Medicine’s Most Successful Antimicrobial Historical perspective The developmental history of anti-infectives and Bayer is often not appreciated by today’s physicians and veterinarians. From the first sulfonamide (prontosil) to the latest 3rd generation fluoroquinolone, Bayer has led the way. Nothing is more demonstrative of the leadership role Bayer has played in anti-infectives than in the development of its most successful antimicrobials, ciprofloxacin in human medicine and enrofloxacin for veterinary use. These two antimicrobials introduced over 20 years ago have altered therapy like no others and continue today to be the gold standards other drugs are measured against. O Nalidixic Acid COOH N CH3 N CH2CH3 Norfloxacin The family of quinolone anti-infectives were discovered serendipitously at Bayer while working on the anti-malarial compound chloroquine. Nalidixic acid was restricted to urinary tract infections due to poor systemic absorption but was very active against most Gram-negative bacteria, including Pseudomonas species. Since then, Bayer is responsible for the synthesis of thousands of compounds from the basic quinolone structure and, using its chemistry expertise, demonstrated the role of structure-activity relationships in developing viable drug candidates. The introduction of a simple halogen molecule, fluorine, became the preferred structure in a newer generation of quinolones called fluoroquinolones (Fig. 1). In the late 1980s, norfloxacin became the first widely used 2nd generation fluoroquinolone in North America and was my first introduction to this new class of antimicrobials. However, it wasn’t until the release of enrofloxacin, the first veterinary fluoroquinolone released in North America, that there was a change in the approach and treatment of infectious disease. C2H5 HN N N COOH F O O Enrofloxacin O F OH N N Figure 1 Quinolone chemical structures. 6 N Until Baytril® was released in North America in the late 1980s, the selection of oral antimicrobials was restricted to amoxicillin, amoxicillin-clavulanic acid (Synulox, Clavamox), cefadroxil, and potentiated sulfonamides (Tribrissen). All other oral antimicrobials used were not approved in companion animals, but out of need used quite often. These included cephalexin, doxycycline, and chloramphenicol. Few if any clinical studies were available on these drugs and efficacy and toxicity were anecdotal at best. Moreover, none of these drugs had efficacy against the resistant Gram-negative bacteria that were becoming common in chronic urinary tract infections or against the Pseudomonas aeruginosa infections in chronic otitis externa. Clinicians like myself were relegated to using aminoglycosides, gentamicin 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 7 David P. Aucoin, DVM, Dip ACVCP VCA Antech Santa Monica, CA, USA and amikacin. Much of my early years as a clinical pharmacologist were spent determining safe dosing strategies with these drugs involving costly and expensive serum drug monitoring. Those days ended with the advent of Baytril. Baytril and its metabolites are eliminated into the urine where high concentrations and tissue penetration make it the drug of choice in urinary tract infections. Activity Baytril disposition and activity Baytril exhibits all of the general properties of fluoroquinolones and then some. Its oral bioavailability is both rapid and complete. So much so that the equivalent dose injected IM has the same peak serum concentrations as a dose given orally. This quick oral absorption is critical to its activity, which is based on its serum concentration. The quicker the absorption, the higher the serum concentration. Baytril is the most lipophilic of the veterinary quinolones, which contributes to its extensive disposition into all body tissues including privileged sites such as the prostate and brain. Studies specifically looking into tissue distribution show that tissue concentrations often exceed serum concentration. Baytril has been shown to accumulate into neutrophils and macrophages where it acts as a drug delivery vehicle into infected tissues. This single characteristic allows enrofloxacin to be used in any infection caused by susceptible bacteria. Baytril’s most unusual and distinctive feature is its transformation into its primary metabolite, ciprofloxacin. This fluoroquinolone continues to be the gold standard in human medicine after 20 years of use. Over 40 % of enrofloxacin is metabolized in the liver to ciprofloxacin which has equal or superior efficacy to enrofloxacin. Given ciprofloxacin’s variable and at times poor oral bioavailability in companion animals, it turns out that the best way to get ciprofloxacin into a patient is by using enrofloxacin. Baytril was unique due to its unprecedented activity against resistant Gram-negative bacteria. As a safe and effective replacement for costly and toxic aminoglycoside therapy, it quickly became a drug of choice for many chronic infections. As with many 2nd generation fluoroquinolones, Baytril has extensive activity against most aerobic Gram-negative bacteria. Most notable was the high degree of activity against Enterobacteriaceae such as E.coli and also Pseudomonas aeruginosa (Figs. 2, 3). Its broad activity includes many Mycoplasma and Ricketssia spp. Surprisingly it also has considerable activity against a few key aerobic Gram-positive bacteria such as staphylococcal species (Fig. 4). In vitro efficacy, however was not a simple yes-or-no interpretation. It was dose dependent. Baytril was the first veterinary antimicrobial that used the extensive clinical experience to modify its dosing options to achieve regulatory approval for what clinicians had been doing for years – modifying the dose depending on the organism and site of infection.The flexible dosing option approved in the United States was the first of its kind in veterinary medicine and represented a significant change in antimicrobial therapy. PK/PD relationships The relationship between in vitro activity and in vivo efficacy has been well documented through clinical trials. No other veterinary antimicrobial 7 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 8 Proceedings of the 4th International Baytril® Symposium Baytril®: Historical Impact and Milestones of Veterinary Medicines Most Successful Antimicrobial David P. Aucoin has undergone as many published studies in as diverse a list of species as Baytril. In a PubMed review, over 675 published articles with studies in over 20 animal species were found.The most important studies in the 1990s were establishing the dose-response relationship of fluoroquinolones, which revolutionized the approach to antimicrobial therapy. In determining dose and dose frequency, it became apparent that unlike almost all other antimicrobials, fluoroquinolones like enrofloxacin had a very rapid effect on susceptible bacteria. The killing curve for these drugs were extremely fast, on the order of a few minutes. This rapid killing was seen only with aminoglycosides and, like them, their efficacy seemed to correlate with some simple pharmacokinetic factors such as 100 86 87 88 80 60 40 20 12 0 ≤ 0.5 1 2 ≥4 Figure 2 Percent of E. coli (n = 53,121) susceptible at tested MIC concentrations of enrofloxacin. MIC (ANTECH Diagnostics 2008) 100 86 79 80 60 44 40 20 0 14 ≤ 0.5 1 2 ≥4 Figure 3 Percent of P. aeruginosa (n = 18,987) susceptible at tested MIC concentrations of enrofloxacin. 8 MIC (ANTECH Diagnostics 2008) 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 9 maximum serum concentration/MIC ratio or drug exposure (represented by the PK factor area under the curve or AUC). This simple correlation changed the way antimicrobials were characterized. Drugs were either a dose-dependent antimicrobial like Baytril or time-dependent like amoxicillin. Dose dependency meant changing the dose and not the frequency of dosing. Baytril was the first once-aday oral antibiotic and arguably the most effective. Indications for use Baytril, as most drugs approved through the arduous regulatory approval process, limits its label claims. Clinical use, however, has extensively broadened its clinical indications. Most notable has been the use of Baytril in pyoderma. In the 1990s, it was clear that almost all canine pyodermas were being treated by antimicrobials with excellent activity against Staphylococcus, the major pathogen involved in these chronic infections. Baytril has excellent activity against the major 94 100 Staphylococcus species including Staphylococcus pseudintermedius (formally known as Staphylococcus intermedius), Staphylococcus schleiferi ssp. coagulans and ssp. schleiferi and Staphylococcus hyicus). Chronic pyoderma also harbored Gram-negative bacteria, which were effectively treated by Baytril, but not by the 1st generation cephalosporins such as cephalexin or cefadroxil. Baytril became an accepted treatment amongst dermatologists for treatment of refractory canine pyodermas. Most surprising was the extended use of Baytril in the treatment of periodontal disease. Most 2nd generation fluoroquinolones have poor activity against the major anaerobic bacteria involved in this disease, especially Porphyromonas spp. However, it was noted that facultative aerobic Gramnegative bacteria were also involved in maintaining an anaerobic environment and their removal along with those of the anaerobic bacteria was effective in treatment. Baytril continues to have strong indication in all genito-urinary tract infections, especially those of chronic nature where tissue penetration or penetration into biofilmed colonies is important. 94 88 80 60 40 20 5 0 ≤ 0.5 1 2 ≥4 MIC Figure 4 Percent of Staphylococcus spp. (n = 14,158) susceptible at tested MIC concentrations of enrofloxacin. (ANTECH Diagnostics 2008) 9 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 10 Proceedings of the 4th International Baytril® Symposium Baytril®: Historical Impact and Milestones of Veterinary Medicines Most Successful Antimicrobial David P. Aucoin been common in this disease due the high prevalence of otitis media in these dogs. Unfortunately, improper dosing continues to be an issue in these cases and, unlike that in all other infected sites, the emergence of resistance is higher in these sites given the same bacteria. However, it would be wrong to say that Baytril has lost it efficacy through extensive use. The data supports the Its good to excellent activity against most Enterobacteriaceae and staphylococcal species makes it a drug of choice in these diseases. Fluoroquinolones remain the mainstay of treatment for otitis externa where multiresistant bacteria, especially Pseudomonas and Proteus species, have a high prevalence. Systemic treatment has 100 87 87 88 87 86 86 86 2002 2003 2004 2005 2006 2007 2008 80 60 40 20 0 Figure 5 Percent of E. coli cultured susceptible to enrofloxacin (MIC ≤ 2 µg/ml) from 2002 through 2008. A dose of 5 mg/kg once daily is recommended. Data from ANTECH Diagnostics. 100 84 86 2002 2003 84 82 2004 2005 84 82 80 79 60 40 20 0 2006 2007 2008 Figure 6 Percent of P. aeruginosa cultured susceptible to enrofloxacin (MIC ≤ 2 µg/ml) 2 µg/ml from 2002 through 2008. A dose of 10–15 mg/kg once daily is recommended. Data from ANTECH Diagnostics. 10 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 11 low potential to spread and is preventable with adequate dosing.We have been collecting data on antimicrobial susceptibility using the same CLSI methodology for the past 8 years and enrofloxacin has seen little to no change in its susceptibility to three key pathogens – Staphylococcus spp., E. coli, and Pseudomonas aeruginosa. concept that loss of efficacy absent P. aeruginosa has been through misuse, especially through inappropriate dosing. Resistance The vast majority of clinically relevant mechanisms for resistance to all fluoroquinolones occur through one of three ways, all mediated through chromosomal mutation rather than being plasmid-mediated: 1. lack of penetration into bacterium, 2. removal of drug from bacterium through an efflux pump, 3. change in its binding site at the type II topoisomerase (DNA gyrase) and topoisomerase IV. Of these, point mutations affecting binding to its site of action are most documented in veterinary isolates. A single base pair mutation at the binding site can reduce efficacy, but the type of resistance we see for enrofloxacin (> 4 µg/ml MIC) requires a change in binding in at least 4 sites. Specifically, looking at E. coli susceptibility, we see very little migration of MIC values within the testing range of ≤ 0.5 to 4 µg/ml. E.coli either is susceptible at ≤ 0.5 µg/ml or is resistant at ≥ 4.0 µg/ml (Fig. 2).This means that the standard dose of 5 mg/kg should be used in the treatment of all infections involving E. coli. At the national level we have not seen any changes to E. coli susceptibility to enrofloxacin over the past 7 years. The same is also seen for Staphylococcus spp. where, unlike with E. coli, we do see a significant increase in activity from ≤ 0.5 µg/ml to 1 µg/ml. In 2008, the jump was from 88 % to 94 %.This should not be looked at as a 6 % difference but rather a risk ratio. At 88 % susceptible 12 out of 100 organisms are resistant, while at 94 % the number is 6 out of 100. Therefore, using a higher dose decreases the The frequency of these point mutations is a subject for other presentations, however, it is important to point out that this type of mechanism has 100 91 92 92 92 92 92 94 2002 2003 2004 2005 2006 2007 2008 80 60 40 20 0 Figure 7 Percent of Staphylococcal spp. cultured susceptible to enrofloxacin (MIC ≤ 2 µg/ml) 2 µg/ml) from 2002 through 2008. A dose of 10 mg/kg once daily is recommended. Data from ANTECH Diagnostics. 11 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 12 Proceedings of the 4th International Baytril® Symposium Baytril®: Historical Impact and Milestones of Veterinary Medicines Most Successful Antimicrobial David P. Aucoin risk of failure by half! I currently recommend a dose of 10 mg/kg once daily. However, there has not been significant change in enrofloxacin’s activity against Staphylococcus during the past 7 years (Fig. 6). We have seen a slight decrease in activity to P. aeruginosa over the past 7 years (Fig. 7). Enrofloxacin has always shown less potency against this organism and this factor coupled with the lack of appropriate dose has contributed to the increase in resistance. A MIC of 2 µg/ml is considered susceptible but would require a dose of 20 mg/kg to insure clinical efficacy. However, since the accuracy of any given MIC is ± 1 dilution, I use a maximum of 1 µg/ml as a susceptible breakpoint. At this level, a dose of 10 mg/kg is sufficient. Resistance is a concern for all clinicians and appropriate use of any antimicrobial is needed. However, in the face of millions of doses of Baytril used during the past 20 years, it is remarkable how well this antimicrobial has retained its efficacy and still is considered the gold standard of all veterinary fluoroquinolones. Summary In the space of a few years Baytril changed antimicrobial therapy, offering a once-a-day oral dose for the vast majority of bacterial infections in a plethora of animal species. I could only touch on a few of the major achievements that this drug and Bayer has contributed to veterinary medicine. The antimicrobial with more firsts than any other drug in veterinary medicine. It will continue to be a mainstay of anti-infective therapy for many years to come. References Abd el-Aziz MI, Aziz MA, Soliman FA et al. (1997). Pharmacokinetic evaluation of enrofloxacin in chickens. Br Poult Sci; 38: 164–168. Boothe DM, Boeckh A, Boothe HW (2009). Evaluation of the distribution of enrofloxacin by circulating leukocytes to sites of inflammation in dogs. Am J Vet Res; 70: 16–22. Bailey TA, Sheen RS, Silvanose C et al. (1998). Pharmacokinetics of enrofloxacin after intravenous, intramuscular and oral administration in houbara bustard (Chlamydotis undulata macqueenii). 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Treatment of lesions of osteomyelitis in the hind flippers of six grey seals (Halichoerus grypus).Vet Rec; 145: 547–550. Jacobson E, Gronwall R, Maxwell L et al. (2005). Plasma concentrations of enrofloxacin after single-dose oral administration in loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med; 36: 628–634. Malbe M, Salonen M, Fang W et al. (1996). Disposition of enrofloxacin (Baytril®) into the udder after intravenous and intra-arterial injections into dairy cows. Zentralbl Veterinarmed A; 43: 377–386. Kaartinen L, Pyorala S, Moilanen M et al. (1997). Pharmacokinetics of enrofloxacin in newborn and one-week-old calves. J Vet Pharmacol Ther; 20: 479–482. Martin Barrasa JL, Lupiola Gomez P, Gonzalez Lama Z et al. (2000). Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa. J Vet Med B Infect Dis Vet Public Health; 47: 191–196. Kaartinen L, Salonen M, Alli L et al. (1995). Pharmacokinetics of enrofloxacin after single intravenous, intramuscular and subcutaneous injections in lactating cows. J Vet Pharmacol Ther; 18: 357–362. Kempf I, Gesbert F, Guittet M et al. (1995). Dose titration study of enrofloxacin (Baytril®) against respiratory colibacillosis in Muscovy ducks. Avian Dis; 39: 480–488. Klein H, Hasselschwert D, Handt L et al. (2008). A pharmacokinetic study of enrofloxacin and its active metabolite ciprofloxacin after oral and intramuscular dosing of enrofloxacin in rhesus monkeys (Macaca mulatta). J Med Primatol; 37: 177–183. Kordick DL, Papich MG, Breitschwerdt EB (1997). Efficacy of enrofloxacin or doxycycline for treatment of Bartonella henselae or Bartonella clarridgeiae infection in cats. Antimicrob Agents Chemother; 41: 2448–2455. Kumar N, Singh SD, Jayachandran C (2003). Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin and its interaction with diclofenac after intravenous administration in buffalo calves.Vet J; 165: 302–306. Kung K, Riond JL,Wanner M (1993). Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther; 16: 462–468. Kyriakis SC, Tsiloyiannis VK, Lekkas S et al. (1997). The efficacy of enrofloxacin in-feed medication, by applying different programmes for the control of post weaning diarrhoea syndrome of piglets. Zentralbl Veterinarmed B; 44: 513–521. Lautzenhiser SJ, Fialkowski JP, Bjorling D et al. (2001). In vitro antibacterial activity of enrofloxacin and ciprofloxacin in combination against Escherichia coli and staphylococcal clinical isolates from dogs. Res Vet Sci; 70: 239–241. 14 Martinez M, McDermott P, Walker R (2006). Pharmacology of the fluoroquinolones: a perspective for the use in domestic animals.Vet J; 172: 10–28. Meinen JB, McClure JT, Rosin E (1995). Pharmacokinetics of enrofloxacin in clinically normal dogs and mice and drug pharmacodynamics in neutropenic mice with Escherichia coli and staphylococcal infections. Am J Vet Res; 56: 1219–1224. Mengozzi G, Intorre L, Bertini S et al. (1996). Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and intramuscular administrations in sheep. Am J Vet Res; 57: 1040–1043. Novotny MJ, Shaw DH (1991). Effect of enrofloxacin on digoxin clearance and steady-state serum concentrations in dogs. Can J Vet Res; 55: 113–116. Oluoch AO, Kim CH, Weisiger RM et al. (2001). Nonenteric Escherichia coli isolates from dogs: 674 cases (1990– 1998). J Am Vet Med Assoc; 218: 381–384. Payot S, Cloeckaert A, Chaslus-Dancla E (2002). Selection and characterization of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin. Microb Drug Resist; 8: 335–343. Pellerin JL, Bourdeau P, Sebbag H et al. (1998). Epidemiosurveillance of antimicrobial compound resistance of Staphylococcus intermedium clinical isolates from canine pyodermas. Comp Immunol Microbiol Infect Dis; 21: 115–133. Piddock LJ, Jin YF, Ricci V et al. (1999). Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. J Antimicrob Chemother; 43: 61–70. Prescott JF, Yielding KM (1990). In vitro susceptibility of selected veterinary bacterial pathogens to ciprofloxacin, enrofloxacin and norfloxacin. Can J Vet Res; 54: 195–197. 904091_TRI_Symp_06_15_Aucin.qxp 03.06.2009 17:40 Uhr Seite 15 Randall LP, Cooles SW, Piddock LJ et al. (2004). Mutant prevention concentrations of ciprofloxacin and enrofloxacin for Salmonella enterica. J Antimicrob Chemother; 54: 688–691. Rubin J, Walker RD, Blickenstaff K et al. (2008). Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections.Vet Microbiol; 131: 164–172. Sanchez CR, Murray SZ, Isaza R et al. (2005). Pharmacokinetics of a single dose of enrofloxacin administered orally to captive Asian elephants (Elephas maximus). Am J Vet Res; 66: 1948–1953. Tyczkowska K, Hedeen KM, Aucoin DP et al. (1989). Highperformance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue. J Chromatogr; 493: 337–346. Walker RD, Stein GE, Hauptman JG et al. (1992). Pharmacokinetic evaluation of enrofloxacin administered orally to healthy dogs. Am J Vet Res; 53: 2315–2319. Wallmann J (2006). Monitoring of antimicrobial resistance in pathogenic bacteria from livestock animals. Int J Med Microbiol; 296(Suppl 41): 81–86. Schoevers EJ, van Leengoed LA,Verheijden JH et al. (1999). Effects of enrofloxacin on porcine phagocytic function. Antimicrob Agents Chemother; 43: 2138–2143. Wanke MM, Delpino MV, Baldi PC (2006). Use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial). Theriogenology; 66: 1573–1578. Schroder J (1989). Enrofloxacin: a new antimicrobial agent. J S Afr Vet Assoc; 60: 122–124. Wu G, Meng Y, Zhu X et al. (2006). Pharmacokinetics and tissue distribution of enrofloxacin and its metabolite ciprofloxacin in the Chinese mitten-handed crab, Eriocheir sinensis. Anal Biochem; 358: 25–30. Seguin MA, Papich MG, Sigle KJ et al. (2004). Pharmacokinetics of enrofloxacin in neonatal kittens. Am J Vet Res; 65: 350–356. Studdert VP, Hughes KL (1992). Treatment of opportunistic mycobacterial infections with enrofloxacin in cats. J Am Vet Med Assoc; 201: 1388–1390. Young LA, Schumacher J, Papich MG et al. (1997). Disposition of enrofloxacin and its metabolite ciprofloxacin after intramuscular injection in juvenile Burmese pythons (Python molurus bivittatus). J Zoo Wildl Med; 28: 71–79. 15 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 16 Diagnosis and Management of Bacterial Urinary Tract Infections in Dogs and Cats Bacterial urinary tract infections in dogs & cats The clinical signs of a bacterial cystitis can include stranguria, pollakiuria, inappropriate urination, dysuria, and hematuria. Bacterial urinary tract infections (UTIs) occur in approximately 14 % of dogs during their lifetime, with a variable age of onset.1 Spayed female dogs have been reported to be at increased risk for a UTI, which is likely due to anatomic differences as well as possible protective secretions from the prostate.2 While UTIs are uncommon in younger cats, the prevalence increases with increasing age. Most young cats that present with lower urinary tract signs (LUTS) do not have a positive urine culture. However, in older cats, a positive urine culture can occur as much as 15–20 % of the time; concurrent illnesses such as diabetes mellitus, chronic kidney disease, and hyperthyroidism are often present.3 A slight male predisposition has been reported in cats, which likely occurs because male cats can present with urethral obstruction and are more likely to be catheterized. In both species, other factors such as perineal or scrotal urethrostomies,4 a recessed vulva and perivulvar pyoderma,5 indwelling urinary catheter,6 or tube cystostomies7 can predispose to colonization of bacteria in the lower urinary tract. The entire urinary tract itself has several built-in “defense mechanisms” to prevent external pathogens from adhering to the urinary mucosa. Normal micturition itself and frequent and complete voiding can help remove bacteria. Furthermore, the proximal urethra is sterile and contains microplicae that expand as urine is voided and aid in the removal of the bacteria. Although the distal urethra does contain normal flora, some of these bacteria can help prevent access of the pathogenic bacteria to the urinary tract by producing bacteriocin, which can interfere with the metabolism of other pathogenic bacteria.8 16 In addition to anatomic structures and urine voiding, the mucosal surface of the urinary tract has intrinsic mucosal antimicrobial properties and the glycosaminoglycan layer can also act as a protective mechanism as well. High urine osmolality and high concentrations of urea can also inhibit bacterial growth.While some have stated that dilute (< 1.018) urine may predispose an animal to bacterial infection, it is likely an underlying disease that causes the animal to produce the dilute urine that allows the infections to occur. In cats, we did not find any correlation between decreasing urine specific gravity and positive urine culture.3 Also, it has been shown in preliminary data from our laboratory that there does not appear to be a correlation between decreasing specific gravity and positive urine cultures in the samples that we have obtained from dogs. Diagnosis of a UTI As mentioned previously, LUTS can be seen in many (but not all) dogs and cats that present with a bacterial cystitis. Many dogs and cats with concurrent diseases do not present with characteristic LUTS, however, a urine culture should be performed periodically in order to better manage the patient. A thorough physical examination is important and special attention should be given to the lower urinary tract. All dogs should have a rectal examination to evaluate the prostate for size, pain, and symmetry. The urethra should be palpated for irregularities, mass lesions, or calculi. In female dogs, the vulva should be examined to evaluate if it is recessed (“hooded”) and examined for the presence of urine leakage. In all patients suspected of having a UTI, a urinalysis and bacterial culture provide the most diagnostic information. If a UTI is present, pyruria, bacteruria, and hematuria can be identified on the sediment examination. Although not specific 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 17 Jodi L. Westropp, DVM, PhD, DACVIM University of California Davis, California, USA for a UTI, these factors were all associated with positive urine culture outcome in cats3 and presumably dogs.The sediment is more reliable than the dipstrips for evaluating these factors; the nitrate and leukocyte assays are unreliable in dogs and cats.9 The clinician should be aware that bacteria can often be difficult to see on sedimentation unless there are more than 10,000 bacteria/ ml for rods or 100,000 bacteria/ml for cocci.10 A quantitative urine culture obtained by cystocentesis is the gold standard for documenting a UTI. Ideally, the urine should be cultured within 30 minutes of collection; if this is not possible, it should be refrigerated and then plated for culture within 6–8 hours of collection.11 If one cannot obtain a urine sample by cystocentesis, urine collected by catheterization can be cultured. The quantitative urine culture allows the clinician to interpret if the numbers of bacteria present are considered significant or more likely represent an artifact. As a general rule, ≥ 1,000 cfu/ml of urine obtained by cystocentesis is considered significant in both cats and dogs. If urine is obtained by catheterization in male dogs and cats, ≥ 10,000 cfu/ml is considered significant.This number increases to ≥ 100,000 cfu/ml for catheterization of female dogs due to the possible contamination with bacteria when the catheter passes through the vestibule. Free catch samples are generally of no diagnostic value unless the culture is negative. In most uncomplicated UTIs, a complete blood count (CBC) and biochemical profile is not warranted, as the results of these tests are usually normal. However, if one suspects pyelonephritis or prostatitis, blood work should be evaluated because significant elevations in the white blood cell count and renal values can be seen in both of these conditions. Imaging studies such as radiographs and abdominal ultrasound would also be indicated if systemic organ involvement were suspected. Furthermore, if prostatitis is suspected, aspirates of the prostate can be obtained for cytology and culture. Advanced diagnostics are usually warranted in dogs and cats with recurrent infections (see below). Sensitivity testing There are two primary methods for obtaining this information, the minimal inhibitory concentration (MIC) determination and the Kirby-Bauer method (disk diffusion test).The MIC is the most preferred and widely used methodology and determines the least amount of an antimicrobial agent that causes the complete inhibition of growth of the infecting species or strain of bacteria. Discriminatory antimicrobial concentrations are used in the interpretation of results of susceptibility testing to define isolates as Sensitive (S), Intermediate (I), or Resistant (R). Clinical, pharmacological, and microbiological considerations are used to set these levels. MIC values are expressed in μg/ml. The average urine concentration of an antibiotic must exceed the growthinhibiting concentration (MIC value) for the infecting bacteria by at least fourfold. If the average urine concentration is greater than or equal to that of the MIC value x 4, the drug will be at least 90 % effective. Some commercial laboratories use Kirby-Bauer plates or run only trays set up with attainable serum levels of antibiotics. Attainable urine concentrations can be 100 times the attainable serum concentration. For example, enrofloxacin (and its active metabolite, ciprofloxacin) can achieve concentrations of 200 μg/ml in the urine, which is 100 times the attainable serum concentration after a standard oral dose of 5 mg/kg in healthy dogs. The clinician should consult the laboratory used to determine if serum concentrations or urine concentrations of the antibiotics were used in the susceptibility testing for urinary bacterial isolates.With regard to organisms isolated from urine cultures, if only serum level 17 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 18 Proceedings of the 4th International Baytril® Symposium Diagnosis and Management of Bacterial Urinary Tract Infections in Dogs and Cats Jodi L. Westropp and some Gram-positive uropathogens. Enrofloxacin (Baytril®) is absorbed readily from the gastrointestinal tract, is poorly bound to plasma proteins, and has good penetration in tissues to achieve the necessary high concentrations needed to eradicate urinary infections.The dose of enrofloxacin approved for dogs in the United States is 5–20 mg/kg once daily or divided12, but one should not exceed 5 mg/kg/day in the cat due to the possibility of ocular toxicity characterized by retinal degeneration and blindness. Once-daily dosing is preferred and the author recommends giving the antibiotic at night after the last void. This will allow the concentration of the antibiotic to remain in the bladder for an extended interval to achieve maximal effect against the pathogen. The fluoroquinolones currently available should not be used in young growing dogs, in which degeneration of articular cartilage could occur. trays for determining the MIC value or the KirbyBauer method is used, any antibiotic that is listed as sensitive will be effective. It is important to note, however, that the kidneys must be functioning properly for this previous statement to be true. If the infection is thought to be present in the kidneys or prostate, or kidney disease is diagnosed, serum MIC concentrations should be obtained to treat the animal appropriately. Treatment and management of uncomplicated and recurrent UTIs In order to select an appropriate antibiotic for a UTI, susceptibility testing should be performed, although empiric use of antibiotics for uncomplicated, first occurrence UTIs in dogs can be tried based on the bacteria isolated. Antibiotics for common pathogens are shown in Table 1. For any systemic or recurrent infections, for those animals that have received prior antibiotic therapy, or for infections that occur in cats, susceptibility testing should be performed. As shown in Table 1, the fluoroquinolones (e.g., enrofloxacin) offer good activity against many Gram-negative species Escherichia coli Appropriate antibiotic therapy and periodic urine cultures are ideal when treating dogs for bacterial UTIs. Several outcomes can arise which are depicted in Figure 1. Many animals will have a simple UTI, whereby the urine is sterilized during treatment, and it remains sterile after the ces- Proteus mirabilis Agent Coagulasepositive staphylococci Amoxicillin +a + + + + + + + + + + + + + Amoxicillin-clavulanate + Ampicillin Cephalexin + Chloramphenicol Klebsiella pneumoniae Pseudomonas aeruginosa Streptococcus Streptococcus viridans canis + + + + Enrofloxacin + + + + + Gentamicin + + + + +b Trimethoprimsulfamethoxazole + + + Table 1 Data obtained from the G. V. Ling Urinary Stone Analysis Laboratory. +a = > 90 % of strains susceptible based on MIC tests; +b Only 89 % of strains susceptible based on MIC tests 18 + + + + + + 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 19 Intact male or female dog/cat? Recent (past 3mo) UTI? Predisposing urogenital/Systemic disorder? (yes) (no) Uncomplicated UTI Complicated UTI Perform further diagnostics to look for underlying cause Tx UTI with appropriate antibiotic for 10–14 days Tx UTI with appropriate antibiotic for 4–6 weeks Culture 5–7 days after starting antibiotic (-) Culture 5–7 days post antibiotics (-) Cure (+) Relapsing/ Reinfection Culture 5–7 days after starting antibiotic (+) Evaluate owner compliance & antiobiotic choice (+) (-) Culture 5–7 days post antibiotics Good compliance Lack of compliance/wrong antibiotic Reinstitute therapy Persistent infection Figure 1 Algorithm for managing bacterial UTIs. sation of therapy. Although there are no studies in dogs and cats to determine duration of antibiotic therapy for simple, uncomplicated UTIs, by convention, these are usually treated for 10–14 days. Proper dosing and administration are essential to prevent the misuse of antibiotics which could promote antibiotic resistance. If the dog’s or cat’s urine is sterile during therapy, but the infection recurs weeks or months later, a reinfection or relapsing infection has occurred. Reinfections imply that a new organism or strain of bacteria has invaded the host, while a relapsing infection implies that the previous pathogen is still present. If the urine yields a positive culture while on antibiotics, the infection is said to be persistent. Antibiograms were thought to help determine different strains of bacteria, however, molecular probes using pulse gel electrophoresis appears to be a superior methodology to determine whether recurrent infections are due to the acquisition of new isolates or failure to eradicate previous isolates.13 In both reinfections and relapsing or persistent infections, a search for predisposing causes for the infection should be initiated. Before pursuing an extensive diagnostic workup, the clinician should question the client to be certain that the correct medication for the previous 19 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 20 Proceedings of the 4th International Baytril® Symposium Diagnosis and Management of Bacterial Urinary Tract Infections in Dogs and Cats Jodi L. Westropp a b Figure 2 Cytoscopic views of a bladder mucosal biopsy technique in a female dog with recurrent urinary tract infections. Tissue obtained should be submitted for aerobic culture; cultures for Mycoplasma spp. and anaerobes can also be considered for specific cases. UTI was given and that no doses were missed. Improper dosing can lead to bacterial resistance. For recurrent (> 3/year) or persistent infections, other diagnostics such as contrast radiography or ultrasound should be performed to evaluate for mass lesions or non-radiopaque stones. Cystoscopy with mucosal biopsy should be considered to evaluate the patient for deep-seated infections (Fig. 2). It has been reported for dogs that although urine cultures can be negative, the bladder mucosa or uroliths (if present) can yield positive growth.14 Furthermore, in mouse models, Escherichia coli have been noted to develop within the superficial epithelial cells of the mouse bladder, forming intracellular bacterial communities.15 Culture of the mucosal biopsies can help ascertain if this occurs in dogs and cats. If pyelonephritis or a deep-seated bacterial infection is suspected, antibiotics that achieve good tissue concentrations are warranted. Recurrent infections can also be due to other predisposing factors such as metabolic diseases (e.g., hyperadrenocorticism, diabetes mellitus), therefore a CBC and biochemical profile should be evaluated in all dogs and cats that have recurrent or persistent infections. Other differentials for recurrent infections include a multitude of ab20 normalities that can occur within the urinary system. In dogs, a recessed vulva can predispose the animals to UTIs; performing an episioplasty can prevent perivulvar pyoderma and improve anatomic defenses against uropathogens (Fig. 3).16 Antibiotics should be continued for at least 2–3 weeks after surgery before cessation. Micturition disorders such as urinary incontinence or urine retention should be addressed if present. If polypoid cystitis (Fig. 4) or a urachal diverticulum is noted with imaging studies, removal of these structures can help remove the nidus for infections. In older dogs that present with recurrent UTIs, a search for urinary tract neoplasms should be performed. Uroliths can also predispose an animal to UTIs by acting as a nidus for infection.The most common uroliths in cats and dogs are calcium oxalate and struvite. If an infection is found in an animal with a calcium-oxalate stone, the infection likely occurred secondary to the uroliths presence. However, struvite stones in dogs are usually formed secondary to a UTI with a urease-producing bacteria such as Staphylococcus intermedius or Proteus spp. Dissolution of these stones can be attempted with diet and antibiotic therapy. Penicillins and the fluoroquinolones can be good 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 21 choices for these pathogens, and enrofloxacin is usually quite effective against these bacteria. The antibiotics must be given throughout the dissolution protocol, which can take as little as a few weeks or last as long as 10–12 weeks. Many dogs will present with chronic UTIs, weight loss, or prepucial discharge. Prostatic abscesses can also occur after acute or chronic prostatitis, and can cause life-threatening peritonitis if the abscess were to rupture. The key to successfully managing both uncomplicated and complicated UTIs in dogs and cats is by evaluating urine cultures throughout therapy. Ideally, the urine should be collected by cystocentesis and cultured 5–7 days after therapy has started and 5–7 days after cessation of the antibiotic. This will allow the clinician to ascertain the difference between persistent infections and reinfections and guide further workup that may be necessary. Most dogs with bacterial prostatitis have a bacterial cystitis as well. The commonly isolated pathogens are very similar to isolates obtained from the bladder. Although in most dogs, a urine culture will suffice, cultures of the prostate can be necessary when there is a negative urine culture or the animal has clinical signs despite appropriate treatment. Diagnostic imaging such as an abdominal ultrasound (Fig. 5a) or retrograde contrast study (Fig. 5b) should be performed to evaluate the prostate for size, cysts, abscesses, as well to evaluate for findings compatible with neoplasia. Prostatic fluid can be obtained by ejaculation, prostatic massage, and most commonly by ultrasound-guided fine-needle aspirate of the prostate. The fluid should be analyzed for cytological abnormalities as well as cultured for pathogens. Bacterial prostatitis Bacterial prostatitis is a chronic or acute condition in sexually intact male dogs. Acute prostatitis can have serious systemic ramifications including depression, dehydration, and leukocytosis.Vomiting and diarrhea and septic shock may also occur. Chronic prostatitis can also occur and clinical signs can be vague. The prostate is usually symmetrical and non-painful upon palpation in chronic cases. a Treatment of prostatitis involves appropriate antibiotics and castration. If castration is not an option for a breeding animal, the 5-alpha-reductase b Figure 3 A severely recessed vulva with moderate perivulva pyoderma (a) in a 3.5-year-old FS Golden Retriever with recurrent UTIs. The same dog immediately post operatively after an episioplasty was performed (b). 21 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 22 Proceedings of the 4th International Baytril® Symposium Diagnosis and Management of Bacterial Urinary Tract Infections in Dogs and Cats Jodi L. Westropp inhibitor, finasteride, can be used to help decrease the size and secretions from the prostate.17 Surgical removal of the prostate is rarely performed due to the high morbidity associated with that procedure.18 However, prostatic abscesses often need to be surgically addressed and omentalization of the prostatic abscess is often performed to prevent fluid and purulent material from accumulating in the area. Figure 4 Cystoscopic view of a polypoid mass diagnosed in a dog with recurrent UTIs. a b Figure 5 An ultrasonographic image of an infected canine prostate and multiple intraparenchymal cysts (a) and a contrast urethrogram which illustrates the extravasation of contrast which can occur in prostatitis (b). 22 Antibiotic treatment for acute prostatitis should be continued for at least 4 weeks; longer treatment regimens are often warranted for chronic prostatitis. Due to the blood-prostate barrier, it can be difficult to achieve levels of antibiotics above the desired MIC for the bacterial pathogen. Although the blood-prostate barrier is often broken in acute prostatitis, antibiotics should still be chosen that will penetrate the blood-prostate barrier, which is important as the infection resolves. Due to this barrier, an antibiotic with high lipid solubility, low protein binding, and an appropriate pKa should be used. Non-ionized forms of antibiotics pass through lipid membranes, whereas the ionized forms do not. For Gram-negative infections in the prostate, trimethoprim/sulfa, chloramphenicol and the fluoroquinolones are the most appropriate choices. Enrofloxacin is considered the drug of choice for canine bacterial prostatitis due to its high lipid solubility, low protein binding, low MIC profile, and broad spectrum of activity against many uropathogens.19 Furthermore, unlike the other two antibiotics, side effects with enrofloxacin are rare. Oral enrofloxacin is readily absorbed from the GI tract and approximately 20–40 % is metabolized to its active metabolite, ciprofloxacin; the fractions of metabolized enrofloxacin were reported to be similar after intravenous and oral administrations of the drug.20 Oral ciprofloxacin should not be used as a substitute for enrofloxacin because the bioavailability of ciprofloxacin is only approximately 40 % in dogs and is widely variable. The routine dose of enrofloxacin for prostatitis is usually 10 mg/kg 904091_TRI_Symp_Westropp.qxp 03.06.2009 11:00 Uhr Seite 23 once daily. Higher doses may be needed for certain strains of Pseudomonas spp. Once-daily dosing is preferred, because higher maximum concentrations of the antibiotic are achieved compared to dividing the dose over the day. Summary owner compliance, the appropriate and judicious use of antibiotics, and periodic urine cultures to monitor for urine sterility. In complicated UTIs, a search for and eradication of an underlying cause will often prevent the occurrence of future infections. When infections are present in the kidney or prostate, a longer course of the appropriate antibiotic should be implemented and cultures are warranted to prove the efficacy of the treatment. Diagnosing and treating UTIs in dogs and cats can be very rewarding, but does require good References 1. Ling GV (1984).Therapeutic strategies involving antimicrobial treatment of the canine urinary tract. J Am Vet Med Assoc; 185: 1162–1164. 2. Seguin MA, Vaden SL, Altier C et al. (2003). Persistent urinary tract infections and reinfections in 100 dogs (1989–1999). J Vet Intern Med; 17: 622–631. 3. Bailiff NL, Westropp JL, Nelson RW et al. (2008). Evaluation of urine specific gravity and urine sediment as risk factors for urinary tract infections in cats. Vet Clin Pathol; 37: 317–322. 4. Griffin DW, Gregory CR (1992). Prevalence of bacterial urinary tract infection after perineal urethrostomy in cats. J Am Vet Med Assoc; 200: 681–684. 5. Crawford JT, Adams WM (2002). Influence of vestibulovaginal stenosis, pelvic bladder, and recessed vulva on response to treatment for clinical signs of lower urinary tract disease in dogs: 38 cases (1990–1999). J Am Vet Med Assoc; 221: 995–999. 6. Smarick SD, Haskins SC, Aldrich J et al. (2004). Incidence of catheter-associated urinary tract infection among dogs in a small animal intensive care unit. J Am Vet Med Assoc; 224: 1936–1940. 7. Stiffler KS, McCrackin Stevenson MA, Cornell KK et al. (2003). Clinical use of low-profile cystostomy tubes in four dogs and a cat. J Am Vet Med Assoc; 223: 325–329, 309–310. 8. Mooney JK, Hinman F (1974). Surface differences in cells of proximal and distal canine urethra. J Urol; 111: 495–501. 9. Klausner JS, Osborne CA, Stevens JB (1976). Clinical evaluation of commercial reagent strips for detection of significant bacteriuria in dogs and cats. Am J Vet Res; 37:719–722. 10. Ling GV, Biberstein EL, Hirsh DC (1980). Bacterial pathogens associated with urinary tract infections.Vet Clin North Am Small Anim Pract; 9: 617–630. 11. Lees GE (1996). Bacterial urinary tract infections. Vet Clin North Am Small Anim Pract; 26: 297–304. 12. Plumb DC (2005).Veterinary Drug Handbook. 5th edn., Pharm Vet Inc, Stockholm, WI; pp. 295–298. 13. Drazenovich N, Ling GV, Foley J (2004). Molecular investigation of Escherichia coli strains associated with apparently persistent urinary tract infection in dogs. J Vet Intern Med; 18: 301–306. 14. Gatoria IS, Saini NS, Rai TS et al. (2006). Comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis. J Small Anim Pract; 47: 727–732. 15. Anderson GG, Dodson KW, Hooton TM et al. (2004). Intracellular bacterial communities of uropathogenic Escherichia coli in urinary tract pathogenesis. Trends Microbiol; 12: 424–430. 16. Lightner BA, McLoughlin MA, Chew DJ et al. (2001). Episioplasty for the treatment of perivulvar dermatitis or recurrent urinary tract infections in dogs with excessive perivulvar skin folds: 31 cases (1983–2000). J Am Vet Med Assoc; 219: 1577–1581. 17. Sirinarumitr K, Johnston SD, Kustritz MV et al. (2001). Effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy. J Am Vet Med Assoc; 218: 1275–1280. 18. Wolfe DA (1978). Urethral prosthesis for treatment of prostatic abscess in a dog. J Am Vet Med Assoc; 172: 806–808. 19.Threlfall WR, Chew DJ (1999). Diagnosis and treatment of canine bacterial prostatitis. Comp Cont Educ Pract Vet; 21: 73–87. 20. Cester CC,Toutain PL (1997). A comprehensive model fro enrofloacin to ciprofloxacin transformation and disposition in dog. J Pharm Sci; 86: 1148–1155. 23 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 24 Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Introduction Infectious airway diseases in dogs and cats are associated with symptoms of cough and/or dyspnea, as well as general signs of disease such as fever, loss of appetite and apathy. The following article specifically addresses the interpretation of bacteriological findings in the respiratory tract of the dog, and their therapeutic relevance. A number of viral and bacterial pathogens have been detected in animals with kennel cough, but their etiological relevance remained unclear for many years, as they also occur frequently in healthy dogs.Viruses encountered in this connection include canine parainfluenza virus (CPIV), canine adenovirus 2 (CAV-2), canine herpesvirus 1 (CHV-1), reoviruses and influenza-A viruses, as well as canine distemper virus (according to some literature data). The viral pathogens of kennel cough usually cause only local infections of the respiratory tract mucosa. However, damage to the respiratory epithelium often paves the way for secondary bacterial infection. The bacterial pathogens especially involved include Bordetella bronchiseptica (even without prior viral infection it is itself an obligate pathogen in the lower respiratory tract), as well as Pasteurella, Streptococcus, Staphylococcus, Klebsiella, and Mycoplasma. It should be noted that canine infectious tracheobronchitis is a disease of underlying factors, i.e., environmental and host factors make a decisive contribution to the expression of the symptoms of disease. Singular infections of well-maintained dogs with the aforementioned pathogens usually lead to only mild or inapparent disease. Multiple infections and environmental factors such as temperature of the surroundings, relative humidity, stress (cramped quarters, crowding, etc.), and poor hygiene promote active disease. The number of cases increases in the summer (family dogs sent to kennels) and autumn. However, bacterial infections also arise as a result of other underlying 24 causes such as tracheal collapse, foreign body aspiration, tumors, congestion, etc. Bacteriological examination of the respiratory tract Bacteriological examination with susceptibility testing of tracheal swabs or bronchoalveolar lavage (BAL) is an important diagnostic measure, especially in dogs with chronic or recurrent respiratory tract diseases or in dogs with acute cough/ choking cough with suspected Bordetella bronchiseptica infection. However, when interpreting the findings, it must be taken into consideration that the respiratory tract is not sterile even in healthy animals. In a prospective study, we investigated the microbial flora of the upper and lower respiratory tract in healthy dogs, as knowledge of the microfloral composition is particularly relevant for the evaluation of bacteriological findings in patients with respiratory disease.The results of the bacteriological examination of tracheal swabs were hereby compared with those obtained by bronchoalveolar lavage in 43 healthy adult dogs (median age six years) (see Fig. 1). Bacteria isolated from the respiratory tract in 43 healthy adult dogs α-hemolytic Streptococcus Acinetobacter E. coli Neisseria Staphylococcus intermedius Erwinia β-hemolytic Streptococcus Klebsiella γ-hemolytic Streptococcus Pasteurella spp. aerobic bacillus Flavobacterium Staphylococcus epidermidis Enterobacter hemolytic E. coli Proteus Pseudomonas spp. Micrococcus S. aureus/intermedius Braunella Corynebacteria Enterobacteriaceae Figure 1 Bacterial species isolated from the respiratory tract of healthy dogs (Bauer et al. 2003). 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 25 Andreas Moritz, Dr med vet, PD, Dipl ECVIM-CA, Assoc. Member ECVCP Small Animal Clinic, Clinical Pathophysiology and Clinical Laboratory Diagnosis Justus-Liebig-Universität Giessen, Germany Bacteria isolated (%) from 151 dogs with respiratory diseases Bacteria Tracheobronchitis Pneumonia Foreign bodies Miscellaneous α-hemolytic Streptococcus 54.9 35.4 28.6 44.4 S. aureus/intermedius 43.6 31.2 28.6 33.4 γ-hemolytic Streptococcus 15.4 16.7 7.1 5.6 β-hemolytic Streptococcus 7.0 0 7.1 0 E. coli 25.3 35.4 50.0 22.2 Pseudomonas spp. 18.3 16.7 14.2 16.7 Pasteurella spp. 8.4 16.7 21.4 5.6 Klebsiella 9.9 12.5 14.3 11.1 Bordetella bronchiseptica 4.2 4.2 0 0 Proteus 1.4 6.3 14.3 0 hemolytic E. coli 2.8 14.5 0 5.6 Neisseria 15.4 4.2 7.1 16.7 0 6.3 14.2 5.5 No bacteria cultured Figure 2 Bacterial species isolated from the respiratory tract of sick dogs. While bacteria could be detected in 95.3 % of tracheal swabs (tracheal wash), only 44.2 % of BAL specimens showed bacterial growth. When bacteria were detected by BAL, only one bacterial species was detected in 61.1 % of the dogs, compared to 90.2 % of bacteriologically positive tracheal swabs with mixed flora. In all, the 17 different bacterial species obtained from the trachea and the eight obtained from BAL could be cultivated in only tiny amounts, with Streptococcus, bacteria of the Enterobacteriaceae family, and Staphylococcus dominating. The tracheal and BAL findings differed completely in 69.8 % of dogs, with the same results in only 4.7 % of cases and partially overlapping results in 25.6 % of cases. The results of our study confirmed that dogs often have different bacterial flora in the upper versus the lower respiratory tract. Therefore, the site from which specimens are obtained for bacteriological examination should absolutely be taken into account in the evaluation of the findings. In a retrospective study of 151 dogs (from years 1999–2000) with acute and chronic airway disease (Fig. 2), the patients were divided into the following groups on the basis of the clinical, laboratory diagnostic and radiographic findings: tracheobronchitis (47 %), pneumonia (12 %), foreign bodies (9 %), and miscellaneous (12 %, e.g., tracheal collapse, bleeding, tumors). It was shown that several bacterial species could be isolated in 76.5 % of cases and only one species in 18.5 % of cases (e.g., E. coli, Pseudomonas spp., Pasteurella spp., Staphylococcus aureus/intermedius, α-hemolytic Streptococcus, Klebsiella, as well as Bordetella bronchiseptica). 25 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 26 Proceedings of the 4th International Baytril® Symposium Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Andreas Moritz ratory tract. Bordetella bronchiseptica is a short, Gram-negative rod-shaped bacterium. It is peritrichously flagellated and forms numerous fimbriae as well as other adhesions for attachment of the ciliated respiratory epithelial cells (see Fig. 4). The bacterial load varied from slight to severe (see Fig. 3). 5.3 % of the specimens showed no bacterial growth. Patients with tracheobronchitis presented predominantly with Gram-positive bacterial species, whereas those with pneumonia had predominantly Gram-negative species. Fifty percent of dogs with tracheobronchial foreign bodies harbored E. coli. In simplified terms, it can be said that the deeper organisms are found in the respiratory tract, the higher the bacterial content (colony count) and the more relevant the finding is for the affected animal and for its treatment. This especially applies to animals in which infection with Bordetella bronchiseptica (with predominately high bacterial levels) could be detected. Resistance to external influences, especially drying, is limited. Nevertheless, the sources of infection include not only infected animals excreting the pathogen, but also the contaminated surroundings. Bordetella bronchiseptica exhibits a broad host spectrum: apart from dogs, species such as cats, rabbits, guinea pigs, and pigs as well as horses, seals, and humans (HIV-infected!) have been reported as frequently or occasionally infected. Pathogenesis Bordetella bronchiseptica The contagion usually passes from animal to animal via aerosols, and transmission from other animal species must also be taken into account.The spread of the pathogen is particularly favored by keeping animals in groups (e.g., kennels, dog- This very common infection in dogs manifests itself almost exclusively in the lower respiratory tract, but to some extent also in the upper respi- Bacteria isolated from 151 dogs with respiratory diseases E. coli Pseudomonas spp. Pasteurella spp. S. aureus/interm. α-hem. Streptococcus Klebsiella Bordetella bronchiseptica Proteus Acinetobacter hemolytic E. coli. Aeromonas Neisseria Enterobacteriacea γ-hem. Streptococcus 0 Colony count: 20 40 +, 5–50 60 ++, 50–200 Figure 3 Bacterial species isolated from the respiratory tract of sick dogs and respective colony counts. 26 80 % Isolates +++, > 200 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 27 Figure 4 BAL, three ciliated epithelium cells with cilia (left), two alveolar macrophages (round cells on the right in the picture). breeding units) or during dog shows.The pathogen adheres to the ciliated epithelium of the respiratory tract primarily via the hair-like fimbriae. Two other adhesion factors have also been described – a filamentous hemagglutinin and pertactin, acting as mediators via specific receptors. After successfully colonizing the mucosa, the pathogen then, by means of various exotoxins and endotoxins, damages the cilia of the epithelial cells (membrane proteins exhibit adenylate cyclase activity and thereby lead to lowering of cilial energy supply, with cilial arrest) so that elimination of the pathogen is no longer possible.The available phagocytes, responsiveness of the immune system, and mucociliary clearance are also affected. The bacteria also often penetrate into the host cells. Ultimately, it has not been definitively determined which factors are responsible for latent or clinically manifested Bordetella bron- chiseptica infection. Apart from pathogen-dependent and host-dependent factors, prior damage due to other microorganisms – viruses, mycoplasma (?), pyogenic bacteria – plays a significant role. This is especially clear in the case of kennel cough, when dogs are additionally infected with canine parainfluenza virus or canine adenovirus type 2. Case history and clinical symptoms The disease is highly contagious and often arises in larger dog populations. It is expressed as tracheitis, bronchitis, and tracheobronchitis, advancing as far as purulent-necrotic bronchopneumonia which can be fatal in young dogs. Sick animals are affected by a sudden onset of a highgrade cough (choking cough), which often sug27 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 28 Proceedings of the 4th International Baytril® Symposium Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Andreas Moritz Figure 5 Oropharyngeal contamination of a tracheal lavage specimen is detectable by the presence of pavement epithelial cells with Simonsiella bacteria. gests an inhaled foreign body as a differential diagnosis, especially when initial antibiotic therapy usually does not lead to the desired response. Tachypnea and mixed dyspnea, as well as fever may also arise as signs of bronchopneumonia. If only the upper respiratory tract is affected, apart from rhinitis, there may be latent colonization with the pathogen. pharyngeal, or nasal swabs are possible in principle but are less conclusive and difficult to evaluate due to the normal resident flora. If the cytological examination shows pavement epithelial cells with Simonsiella bacteria (Fig. 5), the specimen has certainly suffered oropharyngeal contamination. Neutrophilic granulocytes (with toxic changes) in the BAL, which have phagocytized bacteria, provide important diagnostic evidence for bacterially induced tracheobronchitis (Fig. 6). Diagnosis A definite diagnosis is possible only by culturing the pathogen, as other bacterial infectious agents (Streptococcus, Staphylococcus, Pasteurella spp., etc.) may also be involved in this type of localized disease process. The specimen should be obtained from the lower respiratory tract, for example, by bronchoalveolar lavage, endoscopically guided tracheal swab, or transtracheal probe. Laryngeal, 28 The BAL sample must be transported to the test laboratory without delay and with the use of a transport medium. Cultivation of Bordetella bronchiseptica can be difficult in mixed cultures and requires incubation for at least 48 hours and the use of selective media. In cases where Bordetella bronchiseptica is detected in nasal swabs and with low microbial counts, the possibility of latent infection should be considered. 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 29 Figure 6 BAL from a dog with cough, showing neutrophilic granulocytes with signs of toxicity and phagocytized bacteria. Therapy of bacterial tracheobronchitis While uncomplicated cases of tracheobronchitis/(choking) cough – without detection of Bordetella bronchiseptica – need not be treated with antibiotics, if there are dyspnoea and signs of systemic disease (apathy, fever), either fluoroquinolones, chloramphenicol, or cephalosporins (see Tab. 1 for dosage) should be used as the therapy of first choice. On antibiogram, Bordetella bronchiseptica is usually sensitive to tetracycline, doxycycline, chloramphenicol, enrofloxacin, and gentamicin. However, tetracycline-resistant isolates have been reported recently. Symptomatic treatments include theophylline to prevent bronchospasms, mucolytics, or inhalation (physiological NaCl solution, perhaps combined with antibiotic) and, in the case of severe dry cough, the controlled, and well-timed administration of antitussives, avoiding accumulation of secretions. The prognosis for tracheal and bronchial infection is good, but it must be borne in mind that Bordetella may be detectable in the respiratory tract for up to 140 days and the cough may persist for a long time (e.g., weeks) despite treatment. If there is pneumonia, the prognosis is guarded. Antimicrobial susceptibility testing Culturing of bacteria from BAL, with susceptibility testing, is very important for targeted antibiotic therapy. In the retrospective study mentioned above with 151 dogs presenting with various respiratory diseases, the bacteria isolated with high microbial counts from the lower respiratory tract were Bordetella bronchiseptica, Pseudomonas, E. coli, Pasteurella spp., Klebsiella, Staphylococcus aureus/intermedius, and hemolytic E. coli. Susceptibility testing was performed according to 29 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 30 Proceedings of the 4th International Baytril® Symposium Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Andreas Moritz DIN 58940. The results are presented in Fig. 7. From Figure 7 it can be seen that enrofloxacin is particularly effective against Bordetella bronchiseptica, Pasteurella spp., Klebsiella, and hemolytic E. coli, and is effective against Staphylocuccus aureus/intermedius infections; amoxicillin/clavulanic acid is effective against E. coli, hemolytic E. coli, Klebsiella, Staphylococcus aureus/intermedius, and Pasteurella spp. In addition, injectable tetracyclines are very effective against Bordetella bronchiseptica and effec- tive against Pasteurella spp. and hemolytic E. coli. To check whether the sensitivity of the bacteria to the various antimicrobials has changed in recent years, we again performed a retrospective analysis of the BALs from 77 dogs from the years 2004–2009 with various respiratory diseases. Antimicrobial susceptibility testing (Fig. 8) were performed according to DIN 58940 on bacteria isolated in larger numbers from the lower respiratory tract: Bordetella bronchiseptica (Fig. 9), Medicinal product Species Dose (mg/kg BW) Route of administration Interval (h) Amikacin D, C 10 IV, SC 8 Amoxicillin D, C 15–20 PO, SC, IV 8 Amoxicillin-clavulanic acid D, C 15 PO, IV 8 D, C 20 PO, IV 12 Ampicillin D, C 22–30 PO, SC, IV 8 Cefazolin (1st Gen.) D, C 20–25 IV 6–8 Cefotaxime (3rd Gen.) D, C 25–50 IV 6–8 Cefoxitin (2nd Gen.) D, C 15–30 IV 6–8 Cephalexin (1st Gen.) D, C 25 PO 12 D 30–50 PO, IV 8 C 30–50 PO, IV 12 D 10 PO, SC, IV 12 C 10–15 PO, SC, IV 12 Doxycycline D, C 5 PO 12 Enrofloxacin D, C 5–10 PO, SC 24 Gentamicin D 3–4 IV, SC 12 C 3 IV, SC 12 Ticarcillin-clavulanic acid D, C 30–50 IV 6–8 Trimethoprim-sulfonamide D, C 15 PO, SC, IV 12 Chloramphenicol Clindamycin D = dog, C = cat; taken from Reitemeier et. al. 2001, modified according to Green 1998 Table 1 Recommended doses, routes of administration and dosing intervals for antimicrobials used for respiratory diseases in dogs and cats. 30 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 31 Antimicrobial susceptibility (%) Bacteria Antimicrobial n Enro. Am/Clv. Chlora. Genta. Sulf/Tr. Tetra. Ampi. Pen. Bordetella bronchiseptica 8 100 63 13 88 25 100 63 0 Pseudomonas spp. 25 72 20 4 96 12 44 8 8 E. coli 12 42 100 33 58 50 0 8 0 Pasteurella spp. 10 100 80 90 80 30 90 70 80 Klebsiella 11 100 91 89 73 36 27 27 0 S. aureus/intermedius 23 87 91 59 70 70 30 22 30 hemolytic E. coli 6 100 100 100 100 67 83 100 0 Note: for treatment recommendation verify tissue concentrations! Figure 7 Antimicrobial susceptibility of bacteria isolated from 151 dogs with respiratory diseases. Shown are the number of bacteria tested and the % of bacteria which are susceptible to the particular antimicrobial agent. Figure 8 Antimicrobial susceptibility test of Bordetella bronchiseptica Figure 9 Bordetella bronchiseptica cultured (cultured on Mueller-Hinton agar plate) by agar diffusion method by using disks for 24 h (37 °C) on a blood agar plate, containing antimicrobials. small gray colonies without hemolysis. Pseudomonas spp., Pasteurella spp., Klebsiella, E. coli, and Staphylococcus aureus/intermedius. One therapeutically relevant bacterial species was isolated in 67/77 dogs, two in 9/77, and three in 1/77. The antibiogram (antimicrobial susceptibility test) results are presented in Figures 10–15. polymyxin B exhibit good efficacy against Bordetella bronchiseptica. Compared to the antibiogram of Bordetella bronchiseptica isolated 5–10 years previously, the bacteria were much more sensitive to amoxicillin/clavulanic acid and especially chloramphenicol. The fluoroquinolones enrofloxacin and marbofloxacin as well as tetracycline, doxycycline and With regard to Pseudomonas, it can be said that all isolates were 100 % sensitive to the fluoroquino31 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 32 Proceedings of the 4th International Baytril® Symposium Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Andreas Moritz lones enrofloxacin and marbofloxacin as well as polymyxin B, whereas sensitivity to sulfonamide/ trimethoprim, amoxicillin, chloramphenicol, and tetracycline had deteriorated. The susceptibility pattern for enrofloxacin had improved in comparison to the previous retrospective study. Whereas all previous Klebsiella isolated were 100 % sensitive to enrofloxacin, the current succeptibility was 62.5 %. Polymyxin B exhibited 100 % efficacy by itself. All Pasteurella spp. isolates were fully sensitive to enrofloxacin and marbofloxacin as well as amoxicillin, cephalexin, doxycycline, and polymyxin B. Sensitivity to sulfonamide/trimethoprim and amoxicillin/clavulanic acid had improved. The state of E. coli susceptibility pattern is problematic. None of the tested antibiotics/antimicrobials was 100 % effective. Sensitivity to enrofloxacin, sulfonamide/trimethoprim and especially amoxicillin/clavulanic acid had decreased, but that against tetracyclines has improved (previously 0 %, now 50 %). Bordetella bronchiseptica (n = 20, *n = 16) Pseudomonas spp. (n = 7, *n = 4) Enrofloxacin Enrofloxacin Marbofloxacin* Marbofloxacin* Polymyxin B* Polymyxin B* Sulf./Trim. Sulf./Trim. Clindamycin Clindamycin Lincomycin Lincomycin Gentamicin Gentamicin Cephalexin Cephalexin Amoxicillin* Amoxicillin* Amoxi./Clav. Amoxi./Clav. Penicillin* Penicillin* Oxacillin* Oxacillin* Chloramphenicol Chloramphenicol Tetracycline Tetracycline Doxycycline Doxycycline 0% susceptible, ++ 20% 40% 60% intermediate, + 80% resistant, – Figure 10 Antimicrobial susceptibility test of Bordetella bronchiseptica. 32 100% 0% susceptible, ++ 20% 40% 60% intermediate, + 80% 100% resistant, – Figure 11 Antimicrobial susceptibility test of Pseudomonas spp. 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 33 At present we can only speculate as to the reasons for the changes in sensitivities to the stated antimicrobials of the bacterial species studied here. It may be considered that the more frequent or rarer (e.g., chloramphenicol) use of some medicinal products has contributed. One should keep in mind, that the total number of isolates tested for each bacterial species was limited. However, it can be established that, as before, the fluoroquinolones such as enrofloxacin have very good efficacy against respiratory diseases of dogs. Additionally, enrofloxacin and its active metabolite ciprofloxacin have been demonstrated to have extensive distribution in the respiratory tract, achieving concentrations in the epithelial lining Pasteurella spp. (n = 27, *n = 23) Klebsiella (n = 8, *n = 4) The tested Staphylococcus aureus/intermedius isolates were fully sensitive to enrofloxacin, marbofloxacin, amoxicillin, cephalexin, doxycycline, and polymyxin B. Sensitivity to enrofloxacin, chloramphenicol and tetracyclines had improved. Conclusion Enrofloxacin Enrofloxacin Marbofloxacin* Marbofloxacin* Polymyxin B* Polymyxin B* Sulf./Trim. Sulf./Trim. Clindamycin Clindamycin Lincomycin Lincomycin Gentamicin Gentamicin Cephalexin Cephalexin Amoxicillin* Amoxicillin* Amoxi./Clav. Amoxi./Clav. Penicillin* Penicillin* Oxacillin* Oxacillin* Chloramphenicol Chloramphenicol Tetracycline Tetracycline Doxycycline Doxycycline 0% susceptible, ++ 20% 40% 60% intermediate, + 80% 100% resistant, – Figure 12 Antimicrobial susceptibility test of Pasteurella spp. 0% susceptible, ++ 20% 40% 60% intermediate, + 80% 100% resistant, – Figure 13 Antimicrobial susceptibility test of Klebsiella. 33 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 34 Proceedings of the 4th International Baytril® Symposium Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy Andreas Moritz fluid and alveolar macrophages that exceed plasma levels. As a supplement to systemically administered medications, gentamicin and, based on the results presented here, also polymyxin E (comparable sensitivity to polymyxin B can be assumed) can be recommended as inhalation products, especially for infections with Bordetella bronchiseptica. E. coli (n = 14, *n = 12) Staphylococcus aureus/intermedius (n = 18, *n = 15) Enrofloxacin Enrofloxacin Marbofloxacin* Marbofloxacin* Polymyxin B* Polymyxin B* Sulf./Trim. Sulf./Trim. Clindamycin Clindamycin Lincomycin Lincomycin Gentamicin Gentamicin Cephalexin Cephalexin Amoxicillin* Amoxicillin* Amoxi./Clav. Amoxi./Clav. Penicillin* Penicillin* Oxacillin* Oxacillin* Chloramphenicol Chloramphenicol Tetracycline Tetracycline Doxycycline Doxycycline 0% susceptible, ++ 20% 40% 60% 80% intermediate, + 100% resistant, – Figure 14 Antimicrobial susceptibility test of E. coli. 0% susceptible, ++ 20% 40% 60% intermediate, + 80% 100% resistant, – Figure 15 Antimicrobial susceptibility test of Staphylococcus aureus/intermedius. References Bauer N, Moritz A, Weiss R (2003). Vergleich der Keimflora im oberen und unteren Respirationstrakt gesunder Hunde (Comparison of bacterial growth in the upper and lower respiratory tract of healthy dogs). Tierarztl Praxis Kleintiere; 312: 92–99. 34 Boothe DM (2004). Drugs affecting the respiratory system. In: Textbook of respiratory diseases in dogs and cats (Ed: King LG), Saunders; pp. 229–252. ISBN 0-7216-8706-7. 904091_TRI_Symp_24_35_Moritz.qxp 04.06.2009 8:31 Uhr Seite 35 Boothe DM, Boeckh A, Boothe HW (2009). Evaluation of the distribution of enrofloxacin by circulating leukocytes to sites of inflammation in dogs. Am J Vet Res; 70(1): 16–22. Jones RL (2006). Laboratory diagnosis of bacterial infections. In: Infectious diseases of the dog and cat (Ed: Green CE), 3rd ed.; pp. 267–273. ISBN 1416036008. Brady CA (2003). Bacterial pneumonia in dogs and cats. In: Textbook of respiratory diseases in dogs and cats (Ed: King LG), Saunders; pp: 412–430. ISBN 0-7216-8706-7. King LG (1999). Management of bacterial bronchitis and pneumonia in small animals. Suppl Comp Cont Educ Pract Vet; 21(12): 60–64. Datz C (2003). Bordetella infections in dogs and cats: treatment and prevention. Comp Cont Educ Pract Vet; 25: 902–914. Weiss R, Moritz A (2007). Bakterielle Infektionskrankheiten und Systemmykosen. In: Klinik der Hundekrankheiten (Eds: Grünbaum EG, Schimke E), 3rd ed., Enke Verlag, Stuttgart; pp. 1082–1111. DIN 58940-7:1994-09: Medical microbiology – Susceptibility testing of microbial pathogens to antimicrobial agents – Determination of the minimum bactericidal concentration (MBC) with the method of microbouillon dilution, NA 063 Normenausschuss Medizin (NAMed) (New Document: Draft 2008-02). Wettstein K, Frey J (2004). Comparison of antimicrobial resistance pattern of selected respiratory tract pathogens isolated from different animal species. Schweiz Arch Tierheilkd; 146: 417–422. Hawkins EC, Boothe DM, Guinn A, Aaucoin DP, Ngyuen J (1998). Concentration of enrofloxacin and its active metabolite in alveolar macrophages and pulmonary epithelial lining fluid of dogs. J Vet Pharmacol Ther; 21: 18–23. 35 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 36 Update on Clinical Management of Pyoderma Introduction Dogs are considerably susceptible to bacterial skin infections and many studies have speculated about the causes related to host factors that enhance the probability of pyoderma. Current hypotheses for predisposing factors are: the pH of canine skin (relatively high), the small amount of intercellular lipids in the canine stratum corneum, the thin stratum corneum, and the scarce defenses in the canine follicular ostium. • Endocrine diseases (hypothyroidism, hyperglucocorticism – primary or iatrogenic) • Diseases of cornification (“primary seborrhoea”) • Genodermatoses (follicular dysplasia, color dilution alopecia, sebaceous adenitis) • Infectious skin diseases (dermatophytosis, Malassezia dermatitis) • Occult neoplasia (solar-induced squamous cell carcinoma, epitheliotropic lymphoma) • Autoimmune diseases (pemphigus complex) • Immunodeficiency (congenital, acquired). Underlying diseases, which predispose to disruption of the skin barrier, are considered fundamental to the development and recurrence of pyoderma. The role of staphylococci in pyoderma: carriage, colonization, infection Commonly recognized underlying diseases which play a consistent role in pyogenic infection of the skin are: • Allergic skin diseases (canine atopic dermatitis, food allergy, flea allergy dermatitis) • Parasitic skin diseases (sarcoptic mange, cheyletiellosis, demodicosis, trombiculosis) • Systemic parasitic diseases (leishmaniosis) To this point in time, Staphylococcus intermedius (recently renamed S. pseudintermedius), the most frequent isolate in cases of canine pyoderma, is not considered a potent pathogen but its virulence can be enhanced by many factors leading to infection. As a member of the residential microflora, S. intermedius may be regularly cultured from the oral and anal mucosae and perineal region of normal dogs, but this population is considerably higher in dogs with superficial bacterial folliculitis compared to healthy animals. For this reason, the paradigm of carriage of staphylococci, based on the human scheme, is still considered pertinent for both the pathogenesis and treatment of recurrent pyoderma. Recurrent staphylococcal skin infections are frequently seen in dogs. Some studies suggest that hypersensitivity to bacterial antigens may be involved in the pathogenesis of this clinical syndrome. Figure 1 Cytological sample of pustule from a case of recurrent superficial pyoderma. A large number of cocci are evident inside and outside the neutrophils, which are partially degenerated (Diff Quick stain 40 X). 36 Atopic dogs with pyoderma have detectable serum IgE against staphylococcal antigens, and it has also been demonstrated that staphylococcal antigens can penetrate the skin. 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 37 Antonella Vercelli, DVM, CES derm., CES opht. Ambulatorio Veterinario Associato Turin, Italy Even if uncommonly, other species of staphylococci have been isolated from dogs with pyoderma and there is growing concern about the role of more aggressive pathogens such as S. aureus, Staphylococcus schleiferi, and the appearance of multidrug-resistant cocci. In most text books, Gram-negative organisms such as Proteus sp., Pseudomonas sp., and Escherichia coli are discussed as secondary invaders in cases of deep or chronic pyoderma. However, in practice, Pseudomonas aeruginosa and other Gram-negative organisms can be the sole pathogen in some clinical cases. Unusual staphylococcal infections or Gram-negative organisms are more likely to be resistant to multiple antibiotics. Currently, most dermatologists agree on the fact that although the treatment of pyoderma was easily performed based on empirical choice of antibiotics in the past, now, especially in referral practice, managing the infection is a more complicated process, requiring bacterial culture and susceptibility testing and selection of appropriate treatment based on the individual patient requirements. methoprim/sulfamethoxazole is frequently observed. Methicillin-resistant Staphylococcus aureus (MRSA) is a well-recognized pathogen in human medicine and is mainly associated with human hospital-acquired infections worldwide; it can cause life-threatening infection after surgical procedures and the multidrug resistance increases health care costs due to prolonged hospitalization. In Staphylococcus aureus, methicillin resistance is conferred by a protein, a penicillin-binding protein, known as PBP2A. This protein is coded by the mecA gene which confers an intrinsic resistance to all beta-lactam antibiotics and their derivatives. Multidrug resistance to aminoglycosides, fluoroquinolones, fusidic acid, and mupirocin may be observed. The presence of individuals carrying these bacteria, without clinical disease on mucosae or the skin is now a growing public health concern. Resistance to cephalosporins and/or fluoroquinolones by Staphylococcus intermedius has remained low in Europe for many years, with effective drugs for systemic therapy in pets generally available. Facing MRSI, MRSA … zoonosis or anthropozoonosis At present, antimicrobial chemotherapy is the most practical way to treat staphylococcal infections and there have been many studies on the “in vitro effects” of antimicrobial agents against strains of S. intermedius isolated from the dog.The percentage of strains resistant to various classes of antibiotics varies in different continents, even countries, and changes with time. Irrespective of the place of isolation, most S. intermedius strains are susceptible to amoxicillin/clavulanic acid, gentamicin, oxacillin, cephalosporins, and enrofloxacin; however, resistance to penicillin, ampicillin, tetracycline, erythromycin, lincomycin, and tri- Figure 2 German Shepherd pyoderma: a multiresistant Staphylococcus intermedius was isolated from this 8-year-old male dog. 37 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 38 Proceedings of the 4th International Baytril® Symposium Update on Clinical Management of Pyoderma Antonella Vercelli improve identification of multiresistant isolates. In clinical practice, zoonosis must be a significant concern in any veterinary patient bearing large numbers of pathogenic bacteria that display worrisome antibiotic resistance, but, conversely, the possibility of anthropozoonosis should also be considered due to the presence of carriers among the human population. Figure 3 A macroscopic view of the ulcerative lesion of the same case. However, multiresistant, mecA-positive S. intermedius isolated from dogs and cats is now emerging in Europe. Prior antibiotic use is a known risk factor for selection of resistant strains of bacteria. A first report of multiresistant, mecA-positive Staphylococcus intermedius (MRSI) in Europe in a veterinary dermatology referral clinic in Germany indicated an incidence of 23 % among the isolates, and included resistance to cefalexin, methicillin, and enrofloxacin. More recently, the author has observed similar data in Italy, with an incidence of 18.5 % of MRSI/MRSA from selected cases of pyoderma in dogs previously treated with several antibiotics.Twenty-one of 113 cases were phenotypically identified as multidrug- and methicillinresistant staphylococci.They were represented by 9 cases involving S. aureus, 10 Staphylococcus intermedius, and 2 with Staphylococcus xylosus. Only one S. aureus was resistant to the entire panel of antibiotics tested, while the other 20 were resistant to more than four antimicrobial classes and sensitive at least to rifampicin. Based on these observations, treatment with antibiotics must be based on culture and sensitivity of swabs, and inclusion of oxacillin (methicillin) in antimicrobial susceptibility testing panels is advisable and may 38 In summary; • MRSA is primarily a human pathogen and infections in animals remain infrequent. • Some cases of MRSI/MRSA have been identified in referral dermatological practice, in dogs previously treated with antibiotics. • MRSA is a zoonotic pathogen that can have possible consequences for human health, but may also be transfered by humans to pets and vice versa. • MRSA transfer is considered a possible indicator of inadequate practice hygiene. • Human beings, dogs, and cats can carry MRSA or MRSI on skin and mucosae, without signs of clinical disease. Empirical treatment of staphylococcal infection has been the norm in veterinary dermatology. Historically, usually only refractory cases were cultured, leading to an underestimation of the occurrence of MRSA/MRSI in pets. A more rational approach would be the use of the right antibiotic based on bacterial culture and sensitivity testing. For the majority of cases, it is possible to identify the same pathogen in all the skin lesions, but in a small number of dogs there are bacteria with different susceptibility to antibiotics, which can lead to a clinical failure despite a presumably “good antibiotic choice”. 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 39 Common trends in treating skin infections The aim of the treatment is resolving the infection and the skin lesions, providing relief to the patient (controlling pruritus and pain) and preventing recurrence (with an extensive diagnostic workup). This goal is better achieved combining topical and systemic antimicrobial therapy. However, surface pyoderma, in mild or localized cases, can be managed with topical therapy, thus avoiding antimicrobial selective pressure in the intestinal tract. Weekly antibacterial shampoos, daily sprays or foams containing antibacterial ingredients are usually employed such as chlorhexidine, ethyl lactate, triclosan, and benzoyl peroxide, because they aid in decreasing the surface bacterial counts and limiting decolonization. Adverse reactions to topical agents can occur, but they are infrequent. Clipping enhances the probability of efficacy of the topical treatment even if it is not always well accepted by the owner. The use of antibiotic creams such as neomycin, fusidic acid, or mupirocin was suggested for lo- Figure 4 Chronic pododermatitis (furuncolosis) in a 3-year-old male Corso dog. calized lesions in the past, however, concerns about the use of mupirocin has emerged because this molecule is considered the “gold standard” in human medicine to treat nasal carriers of MRSA. Successful management of diffuse superficial pyoderma or extensive bacterial overgrowth and of all deep pyoderma requires systemic antibiotic therapy. The first step is the selection of an appropriate antibiotic and establishment of an appropriate dose for a sufficient duration to ensure cure, rather than transient remission.Time frames of one to three weeks of antimicrobial therapy beyond clinical cure are empirical concepts, although currently widely accepted. For this reason, it is very important to re-evaluate every case of pyoderma within two to three weeks after starting the treatment; this enhances the probability of discovering compliance problems due to owner or to the pet, and identifying factors that may contribute to the occurrence of clinical failure. Antimicrobial dosage recommendations for different infections may underestimate dosing needed in deep pyoderma. A concern about the “mutant selection window hypothesis” was first proposed in human medicine and is also beginning to be an important concept in the treatment of infections in pets.We know now that there may be a “dangerous drug concentration zone”, which enhances the survival probability of organisms having reduced susceptibility to the drug. The lower boundary of that selection window is represented by the MIC (minimum inhibitory concentration) and the upper boundary is the MPC (mutant prevention concentration). If we use a drug dosing that achieves concentrations above the MIC level but below the MPC, the majority of bacteria will be eliminated, but a small amount of them will constitute a subpopulation of resistant mutants. So using antibiotics within the mutant selection window will favor the increase of resistant mutant subpopulation. However, if drug 39 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 40 Proceedings of the 4th International Baytril® Symposium Update on Clinical Management of Pyoderma Antonella Vercelli concentrations above MPC are achieved, bacteria must acquire two different mutation genes at the same time, which is a very rare event. Therefore, newer drugs and pharmacological studies are oriented towards employing safe dosing above the MPC for the optimal management of infections. Further complication of the treatment of skin infections may be due to the “Darwinian evolution” of microorganisms – this concept states that mutations are continuously randomly occurring to improve their survival capability. Microbes are also able to elaborate biofilms that help in producing aggregates of organisms protected from the toxicity of antimicrobials and from host defenses, and develop signals that influence the settling behavior of other organisms, activating transcription of genes (the mechanism of “quorum sensing”). So, from the clinician’s point of view, we must also evolve our therapeutic model and start to use the antibiotic drug that has the highest probability of efficacy for a particular infection, with regard to antibacterial activity, as well as one with favorable ability to rapidly penetrate and diffuse into tissues, and that is easily administered to the patient (hit quickly, hit rapidly, hit for an adequate time). The choice of drug An antibiotic chosen empirically should have a known spectrum of activity against S. intermedius and should not be inactivated by beta-lactamases, and must have bactericidal activity. Presently, antibiotics considered useless for pyoderma include erythromycin, penicillin, amoxicillin, and tetracycline. Sulfonamides are usually not prescribed due to the potential for drug reactions, but could be used 40 Figure 5 Acral lick granuloma secondary to neuropathy in a mixed breed dog, 11 years old, female. in cases of MRSA/MRSI, if indicated by susceptibility tests. Drugs such as clindamycin and lincomycin can still be used, but a risk of 10–25 % of resistant strains of cocci should be expected in practice. Amoxicillin and clavulanic acid is currently employed, but some dermatologists advise using at a higher dose than on the label, three times a day. Cephalosporins have been the favorite antibiotic class in dermatology for a number of years, with different options for administration and posology. They are still very effective as an empirical choice, however, some resistant bacterial populations are identified both in superficial and deep pyoderma. Fluoroquinolones are still extremely active for the treatment of skin infections, including Gram-negative isolates; resistance when employed as firstline therapy is very rare, but some cases of resistance in recurrent superficial and deep chronic pyoderma have been observed in referral dermatological practice. 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 41 Suggested use of enrofloxacin for the treatment of pyoderma (more than twenty years later) Enrofloxacin is a fluorinated quinolone-carboxylic acid derivative developed exclusively for use in animals. It is licensed for use in many countries in Europe and the rest of the world at a dosage of 5 mg/kg once daily, and in the USA at a dose range of 5 to 20 mg/kg once daily for the treatment of skin and soft-tissue infections with a spectrum of activity including efficacy against S. intermedius, selected Pseudomonas spp. and other Gram-negative invaders. Enrofloxacin has been on the market for many years and is still a first-line choice antibiotic for the treatment of deep pyoderma and granulomatous lesions of the skin due to its ability to achieve high accumulation in tissues and inside macrophages and neutrophils in the dog, with relatively low incidence of clinical bacterial resistance. Fluoroquinolones are rapidly bactericidal, acting on the bacterial nuclear material (disruption of DNA replication) and, contrary to other antibiotic classes, they do not select for plasmid-mediated resistance. One of the metabolic products of enrofloxacin is ciprofloxacin, which has been shown to increase the production of interferon and that of interleukin-2 enhancing its killing effect on bacteria including Pseudomonas aeruginosa. A recent study determined that the concentration of enrofloxacin in the extracellular fluid in healthy dogs after oral and intravenous administration was equivalent to the plasma concentrations. to predict the efficacy of fluoroquinolones in tissues. Statistically significant higher skin levels of enrofloxacin were seen in dogs with deep pyoderma versus superficial pyoderma, and in contrast to levels in normal skin, thus indicating that the drug is transported into the tissue by inflammatory cells. Synergistic effects have been noted between fluoroquinolones and beta-lactam penicillins. The effects of penicillins on bacterial cell wall permeability may allow better penetration of fluoroquinolones. Fluoroquinolones should not be given in conjunction with drugs that inhibit protein or RNA synthesis (e.g., chloramphenicol and rifampin); these agents may diminish the activity of the fluoroquinolone. As a personal choice, I usually recommend enrofloxacin as a first-line antibiotic for the treatment of mucocutaneous pyoderma, for German Shepherd pyoderma, pododermatitis complex and in cases of acral lick granuloma, and confirmed by susceptibility testing. Precautions in use are related to possible damage to the articular cartilage in rapidly growing large breed dogs, so avoid use in giant breed dogs before 18 months of age and in other breeds prior to 12 months of age. In cats, it is advised not to use dosages exceeding 5 mg/kg due to the occurrence of retinal damage at elevated doses. Treatment with enrofloxacin would not be recommended for a bacterial organism intermediate or resistant in susceptibility to enrofloxacin as appropriate levels of enrofloxacin would not be expected to be attained. Enrofloxacin and ciprofloxacin binding to plasma proteins is low in dogs and does not impair diffusion to the interstitial space. Therefore, plasma concentrations may be used as a surrogate marker 41 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 42 Proceedings of the 4th International Baytril® Symposium Update on Clinical Management of Pyoderma Antonella Vercelli Conclusion The therapeutic choice of an antimicrobial in clinical practice is often empirical. In dermatology, the common opinion in the past was oriented towards the use of molecules with increasing potency in cases with therapeutic failure of the previous drugs. The use of the most effective and powerful antibiotic was reserved for the more serious clinical case, or the ones characterized by recurrences. In reality, over the past few decades, the empirical selection of the antibiotic, and the use of inappropriate dosing or duration of the treatment (sometimes secondary to lack of compliance), has potentially influenced the adaptability of bacteria in many cases, with appearance of resistant pathogens. Currently, this approach is being replaced by a more rational therapeutic attitude that immediately employs the use of the antibiotic with the greatest probability of potency and effectiveness against the involved bacteria, in order to avoid the selection of resistant strains and the appearance of chronic skin lesions. References DeBoer DJ (1995). Management of chronic and recurrent pyoderma in the dog. In: Kirk’s Current Veterinary Therapy XII (Ed: Bonagura JD), W. B. Saunders, Philadelphia; pp. 611–617. Miller WH Jr (1992).The use of enrofloxacin in canine and feline pyodermas and otitis in dogs. Proc 1st Int Baytril Symposium, Bonn; pp. 41–48. Ehrlich P (1913). Chemotherapeutics: scientific principles, methods and results. Lancet; ii: 445–451. Rantala M, Holso K, Lillas A et al. (2004). Survey of condition-based prescribing of antimicrobial drugs for dogs at a veterinary teaching hospital.Vet Rec; 155: 259–262. Ganiere JP, Medaille C et al. (2001). Antimicrobial activity of enrofloxacin against Staphylococcus intermedius strains isolated from bacterial pyodermas. Vet Dermatol; 12(3): 171–175. Riond JL, Wanner M (1993). Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther; 16: 462–468. Ihrke PJ (1996). Experiences with enrofloxacin in small animal dermatology. Suppl Comp Cont Educ Pract Vet; 18(2): 35–39. Tillotson GS (2003). Deja-vu: a ‘new’ approach to managing bacterial infections. Pulmon Perspec; 20: 7–9. Koch HJ, Peters S (1996). Antimicrobial therapy in German Shepherd pyoderma (GSP): an open clinical study.Vet Dermatol; 7: 177–181. Loeffler A, Linek M, Guardabassi L et al. (2007). First report of multiresistant, mecA-positive Staphylococcus intermedius in Europe: 12 cases from a veterinary dermatology referral clinic in Germany.Vet Dermatol; 18(6): 412–421. 42 Vercelli A, Carnevale M, Cornegliani L (2008). Multidrug and meticillin – resistance in Staphylococcus sp. Canine recurrent superficial and deep pyoderma in Italy. Scientific abstract of the 6th World Congress of Veterinary Dermatology, November 19–22, 2008.Vet Dermatol; 19 (Suppl 1): 37. 904091_TRI_Symp_Vercelli.qxp 03.06.2009 11:14 Uhr Seite 43 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 44 Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Introduction The importance of bacterial diseases in exotics and wild animals is well documented. Writing about “exotic animal medicine” means dealing with thousands of species. Even within the same animal class, the anatomy and physiology of the digestive, renal or respiratory systems differ widely among species. However, after taking into account these several specific considerations, medicating exotics is accomplished by the same methods of administration used in domestic mammals. Following their arrival in clinical medicine in the later 1980s, the fluoroquinolones have become a widely used group of synthetic antimicrobials in veterinary medicine. They have provided small animal clinicians with a truly exciting new class of antimicrobials. Never before had veterinarians a drug with such a broad spectrum of activity, combined with the pharmacokinetic properties that allow for oral administration on a once-aday basis. This has allowed clinicians to treat not only a larger number of patients, but also other species with more assurance. Enrofloxacin is a member of the family of 6-fluoro-7-piperazinyl-4-quinolones and was designed specifically as a veterinary drug, available for companion animals in a tablet form and an injectable preparation. Modification of the 4quinolone ring has enhanced the antimicrobial activity of this compound. It is widely used to treat bacterial infections in exotics and wildlife and has become the drug of choice for exotics and wild animals clinicians. Enrofloxacin is a highly effective bactericidal compound with relatively low minimum inhibitory concentrations (MIC). By altering the action of bacterial DNA gyrase, a type II topoisomerase (an enzyme involved in unwinding, cutting, and resealing DNA), it has excellent activity against Gram-negative and also Gram-positive pathogens. Enrofloxacin does not readily complex with plasma 44 proteins, which enables metabolites to readily cross cell membranes. This antibiotic has been used to control certain intracellular pathogens. Following some general considerations, this paper provides an overview on the main bacterial diseases encountered in exotic species. Special emphasis has been made to point out basic principles and to underscore some of the traps to be avoided. The main class of vertebrates will be approached and in each class, an overview on the use of enrofloxacin is discussed. General overview The skills, medications, and protocols used in medicine and surgery of domestic carnivores are applicable to exotics. Therefore, thorough physical examination should be followed by several clinical tests. Because most of the exotic species are highly dependent on their environmental conditions, husbandry records are extremely important. Reviewing information on both the sick individual and apparently healthy animals is part of the diagnostic process. Before any therapy is instituted, the clinician must carefully consider questions on the husbandry situation of the patient, especially its nutritional status. There is no sense in instituting antimicrobial therapy in exotic practice without correcting zoo-technical deficits. One should always have in mind that antimicrobial therapy is only a part of a more general therapeutic plan. The practitioner must also be careful to avoid the unnecessary prophylactic use of antimicrobials that can result in antimicrobial resistance and in the emergence of infections that were previously subclinical, or that might interfere with experimental studies if the patients are experimental models. 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 45 Norin Chai, DVM, MSc, MScVet, PhD Ménagerie du Jardin des Plantes, Museum of Natural History Paris, France Looking more specifically at antimicrobial therapy, the principles of antibiotic used in dogs and cats apply similarly to exotics. Ideally, before instituting antibiotics, the clinician should qualify the nature of the infection, predict the most likely pathogen(s), obtain a culture and a minimum database, and choose the antibiotic based on those results. Establishing these basic parameters allows the clinician to form a reasonable prognosis and a clear rationale for the therapeutic approach and length of treatment required. However, in the author’s experience, many animals are often presented late in the disease process. Immunosuppression in clinically ill subjects, the rapid progression to life-threatening diseases, and the suspected presence of mixed infections are indications for empirical use of combination antibiotics while waiting for culture and susceptibility results. The clinician should select not only the most efficient drug but also the safest and will use the most efficacious route of administration, knowing that restraint is often difficult and parenteral therapy can have its own limitations. Many dosage regimens have been designed largely on an empirical basis. In most of the cases, supportive care is as important as antimicrobial therapy, as well as correcting nutritional deficits. As a rule, optimal treatment of infectious diseases depends on accurate diagnosis, susceptibility to the selected drug, husbandry practices, and anatomical and physiological differences among the species. where bacterial infections are a common problem. They often result in walled-off abscesses with caseated pus in subcutaneous tissues and/or other sites, nervous symptoms for infection of the inner ear, and septicemia. Both Gram-positive and Gram-negative bacteria may be isolated. Aerobic bacteria are the most common. Ferrets General considerations Primary bacterial infections in pet ferrets are not common and should be lower on a list of differential diagnoses. If present, these infections are rather secondary to another primary disease process (traumatic, viral, or parasitic). A ferret with a bacterial disease will often present significant alterations of its hematologic profile, with white blood cell counts higher than 8,000, with primarily neutrophilia. Chronic affections tend to be characterized by monocytosis. Dental tartar, gingivitis, and periodontal disease are common in middle-aged and older ferrets. Tooth root abscesses are not common but can occur at any age. In ferrets, gastrointestinal disease is common. However, in practical terms, diarrheas are more Mammals Medication and diagnostic process used in domestic carnivores can be applied in ferret medicine. There are no specific intolerances to any drugs described. In ferrets, primary bacterial infections seem uncommon and are rather secondary to another primary disease process. In rabbits and rodents, there are numerous organ systems Figure 1 Primary bacterial infections in pet ferrets are not common and, if present, are rather secondary to another primary disease process (traumatic, viral, or parasitic). Here is a ferret presented with an advanced bronchopneumonia. 45 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 46 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai likely to result from non-infectious causes: foreign bodies (very common), trichobezoars, neoplasia. A fecal culture can be difficult to interpret. The prevalence of primary bacterial gastroenteritis is unknown. Helicobacter mustelae infection, the most commonly described, causes ulceration of the gastrointestinal mucosa, clinically characterized by lethargy, vomiting, anorexia, melena, and weight loss. Definitive diagnosis of Helicobacter disease is best made based on clinical signs, gastric Figure 2 Bacterial infection of the inner ear in a chinchilla. (Photo courtesy of Dr. Gersende Doumerc) biopsy, and response to treatment. Other gastrointestinal bacterial infections appear to be uncommon in ferrets. Urolithiasis may induce bacterial cystitis. Gramnegatives are commonly isolated in bacterial prostatitis, an infection closely linked with hypertrophy of the adrenal glands. The treatment is based on surgery (removing the affected adrenal glands, draining the cysts, and in some case, marsupialisation of the prostate to the body wall) and administration of appropriate antibiotics. The disease can endure even when there is no longer adrenal gland androgen influence on the prostate. Like the other organ systems, the respiratory tract is an uncommon area for bacterial infection in ferrets. Both canine distemper (CDV) and influenza virus can be complicated by secondary bacterial respiratory disease. In practical terms, bacterial respiratory disease secondary to influenza virus infection is rare in immune competent ferrets and bacterial pneumonia secondary to CDV infection is rarely treated, as CDV itself is fatal in ferrets. Primary bacterial dermatitis is uncommon compared to skin tumors, which are more frequently seen in ferrets. In all cases, ferret dermatology follows a classical diagnostic process: cytology or biopsy and culture of the affected area. Figure 3 Advanced pneumonia in a rat. Infectious respiratory diseases are the most common health problem in rats and have multifactorial causes, with Mycoplasma pulmonis often the major component. 46 (Photo courtesy of Dr. Gersende Doumerc) Antimicrobial therapy in ferrets Usually, the antibiotics that are safe to use in dogs and cats are safe to use in ferrets and the most appropriate antibiotic based on culture results can be used without regard to serious gastrointestinal upset. Antibiotic administration in ferrets includes the parenteral (intramuscular, subcutaneous, intraperitoneal, intravenous, or intraosseous), enteral, and topical routes. Because of their small body size, the intramuscular route is rarely used for an extended period of time in ferrets. The 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 47 intravenous or intraosseous routes are reserved primarily for the most severe cases of bacterial disease. The subcutaneous route of antibiotic administration is favored. In ferrets, the enteral route is used extensively, especially with antibiotics administered at home, given by syringe or in the food. The ideal antibiotic for use in ferrets is easily administered and bactericidal. Because it can be given orally with once-a-day administration and is effective against serious Gram-negative infections, the most commonly used drug is enrofloxacin. Enrofloxacin is used at 10–30 mg/kg q24h IM, SC, PO. Enrofloxacin may be used with metronidazole if anaerobic infections are suspected, at a dose of 20 mg/kg q12–24h PO. The incidence of anaerobic infections in ferrets is unknown. Rabbits and rodents Rabbits Unlike ferrets, bacterial infections are common in rabbits. However, the clinician should keep in mind before starting an antibacterial therapy that nearly all important diseases in rabbits are directly or indirectly related to diet and feeding practices or environmental conditions. The basic diagnostic approach first includes thorough investigation of the husbandry conditions. Bacterial infections induce an inflammatory reaction characterized by caseated pus and walled-off abscesses that are not necessarily associated with an increase in total white blood cell count. Many of them are attributed to Pasteurella multocida, even if numerous species of bacteria are cultured routinely from rabbits. Some additional selected bacterial species known to infect rabbits include pathogenic species of Staphylococcus and Streptococcus, Klebsiella, Proteus, Pseudomonas, Listeria, Actinomycoses, and Actinobacillus. A study demonstrated that anaerobic bac- teria, not Pasteurella, were common causes of bacterial disease in rabbit abscesses. As a rule, for abscess treatment, antibiotic administration alone will not cure the problem and surgical debridement of the area is always necessary. Respiratory tract infections are very common in rabbits. While the clinical diagnosis may be obvious, characterization of the disease is not (is this a recently developed upper respiratory bacterial disease or a chronic infection that becomes apparent only now?), and neither is the treatment. Furthermore, bacterial infections of the respiratory tract may act as the nidus for which bacteria spread to other areas of the body either by local invasion (for example, in the ear canal causing otitis) or septicemia. Bacterial otitis is common in rabbits.There is also a potential complication with infection of the central nervous system by invasion of the cranial nerves. Aural discharge, a vestibular syndrome, and hyperthermia are often observed. The treatment is based on antibiotic therapy, antihistaminic and antiinflammatory drugs administration, and careful nursing. Conjunctivitis, keratitis, naso-lacrimal duct and retrobulbar infections are also common in rabbits, as well as infection of the urinary tract. Bacterial cystitis might be an important pre-disposing cause for urolithiasis, and so in cases of urolithiasis in rabbits, the clinician should make a culture. Subcutaneous abscess are walled off with caseated pus. It appears rather spontaneously and the clinician should always search for a bacterial nidus of infection somewhere else, which might be challenging. Bacterial dermatitis is generally superficial and secondary to another infectious or non-infectious disease. Dermatology of rabbits follows a classical diagnostic process: cytology or biopsy and culture of the affected area. 47 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 48 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai Dental abscesses are common in rabbits and may involve the mandibular or maxillar teeth. In cases of odontogenic abscesses, multiple adjacent teeth may be affected. The most common bacterial species isolated are a mixture of anaerobic Gramnegative rods, such as Fusobacterium nucleatum, anaerobic Gram-positive non-spore-forming rods, predominantly Actinomyces species, and aerobic Gram-positive cocci of the Streptococcus milleri group. Other reported pathogens include Pasteu- rella and Staphylococcus species. Dental abscesses always have an origin in infection of tooth roots, with or without osteomyelitis of the mandible. Mandibular abscesses are non-resectable and therefore carry a poor prognosis. The clinician must remember that jaw abscesses are frequently due to organisms that travel from other sites; failing to recognize this may lead to antibiotic treatment failure. Enteritis complex due to proliferation of pathogenic bacteria is the most common gastrointestinal disease in rabbit medicine. Causes involve diet, antibiotics, stress, and genetic predisposition to gut dysfunction. Maintaining optimal husbandry, minimal stress, avoiding sudden changes in the diet, and offering good quality grass hay are the most important factors for prevention. Primary bacterial enteritis is less common and mostly involves pathologic strains of E. coli. Use of enrofloxacin (10 mg/kg, PO q12h) in early treatment, while awaiting culture and sensitivity test results, has provided positive clinical results. It also strongly advised to provide supportive care and to prevent the overgrowth of other pathogenic bacteria and the production of toxins. For this, administration of cholestyramine (2 g in 20 ml water q24 h PO), a strong anionic bile-acid exchange resin capable of binding bacterial toxins, has shown good results. Figure 4 Ocular bacterial infections are common in rabbits, presenting with blepharospasm, tearing, discharge, and swelling of the ocular tissues. Here is a rabbit with panophthalmia that required the enucleation of the eye. 48 Small rodents In rodents, there are numerous organ systems where bacterial infections are a common problem. Bacterial infections of the respiratory tract and gastrointestinal system are the most common in the majority of rodents. Guinea pigs are very susceptible to respiratory disease caused by Bordetella bronchiseptica (Gram-negative rod commonly carried by rabbits, dogs, and non-human primates) and Streptococcus pneumoniae (Gram-positive coccus). Salmonella typhimurium and S. enteritidis are the most common causes of bacterial enteritis in 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 49 guinea pigs. Stress and nutritional deficiencies increase susceptibility to diseases. Enteropathies are the most common problem seen in pet hamsters. Lawsonia intracellularis and Clostridum sp. are the common pathogens isolated in the young and adult, respectively. In rats, infectious respiratory diseases are the most common health problem, characterized by two major clinical syndromes: chronic respiratory disease and bacterial pneumonia. In most of the cases, these diseases have multifactorial causes with Mycoplasma pulmonis as the major component. In general, bacterial infections of the respiratory tract are secondary to a viral disease process in rodents. Ectoparasites represent the first primary predisposal cause of bacterial dermatitis in rodents. Following the example of the rabbit, dental infections are very common. do not have the same susceptibility. At low doses, lincomycin administration is fatal to hamsters and guinea pigs, but a much higher dose of this antibiotic given to rats for 30 days exhibited no toxic effects. Tetracyclines may induce dysbiosis in guinea pigs but apparently not in other species. Antimicrobial therapy in rabbits and rodents Antimicrobial therapy in rodents and rabbits entails greater risk than in most other species because inappropriate therapy can result in death of the patient due to enterotoxemia. Some antibiotics provoke a disruption of the normal enteric flora in rodents and rabbits, which can be potentially fatal.This dysbiosis is caused by a sudden loss of microbial diversity in the cecum of affected animals, which subsequently leads to the overgrowth of opportunists such as Clostridium sp. and E. coli. Ultimately, the production of toxins from these pathogens, specifically Clostridium spiroforme (iota toxins) leads to enterotoxemia. Antibiotics that may induce these gastrointestinal diseases include parenteral penicillin, oral or injectable cephalosporins, tetracycline, and erythromycin. Antibiotics that are highly likely to cause gastrointestinal dysbiosis include amoxicillin, ampicillin, clindamycin, and lincomycin. Lincomycin has been shown to consistently provoke cecal dysbiosis, leading to a fatal diarrhea. Ampicillin has been shown to kill up to 40 % of rabbits when administered orally. However, all rodents Medicating the drinking water is advantageous in that no stress of capture or restraint is involved and large numbers of animals can be treated easily. However, exact dosing is impossible since water intake, disease status, dehydration, age vary among individuals. Interspecies differences must also be considered. For example, guinea pigs consume relatively large amounts of water, whereas gerbils consume very little. Additionally, if the drinking water has an undesirable taste from the antibiotic, the patient may stop drinking and become dehydrated. Direct oral dosing is much preferred to medication of the drinking water, since the amount administered can be accurately determined based on the needs of the individual. The drug can be hidden in a small piece of fruit for instance or directly administered with a syringe after a proper restraint technique. The ideal antibiotic to use in rabbits and rodents is one easily administered, bactericidal, and which not cause gastrointestinal disease. The clinician should be aware of the high basal metabolism of small mammals, thus, it is important not to give too low of a dosage and to institute a twice-daily (or even three times daily) administration. In our rodent and lagomorph practice, it is common to complement the antibiotic therapy with probiotic administration such as Lactobacillus sp., which is appropriate in general. Enrofloxacin is one of the most commonly used antimicrobials to manage many different bacterial diseases in lagomorphs, from Pasteurella multocida to Mycoplasma sp. Oral dosing with enrofloxacin does not appear to lead to the development of 49 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 50 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai antibiotic dysbiosis. Enrofloxacin is well distributed through the tissues, including milk, and should be used cautiously in lactating rabbits. Dosing rabbits with 5 mg/kg q12h enrofloxacin PO or IM will achieve MIC levels appropriate for most bacterial pathogens. Some doses at 20 mg/kg SC, PO q12h have been given to rabbits as well. To date, there have not been any pharmacokinetic studies for enrofloxacin performed on rodents. Although it is empirical, doses for most rodents should be 5 to 20 mg/kg IM once, followed by oral administration every 12 or 24 hours. Small specimens would need a dose at 10 mg/kg PO, IM, q12h. Mice require higher dosing of 25–85 mg/kg q24h PO. Depending on the indication, it is common to give enrofloxacin with metronidazole at a dose of 20 mg/kg q12–24h PO. Birds General considerations Microbial diseases are common in companion and aviary birds. Bacterial infections may be primary, however, secondary infections due to poor husbandry conditions, stress, nutritional deficiencies, viral processes, or parasitic burden are the most common. Additionally, many secondary invaders are able to maintain a disease process independent of other infectious agents or predisposing conditions. Thus, with suspicion of bacterial infection, the clinician should always try to find the predisposing causes and should employ all practical diagnostic techniques to direct primary care before initiating any therapy. The status of certain patients highly suggests either an existing infection or the potential for one – birds presented with wounds, purulent si50 Figure 5 A very advanced pododermatitis in a pheasant; the main causes of the disease are environmental deficiencies and penetrations. Note that in birds, purulent material is rather solid. nus discharge, odiferous feces, increased warmth of the feet or beak. However, the differentiation between primary or secondary infection may be challenging as laboratory examinations (biochemical or serologic) are rarely of any help in this differentiation. Again, historical data and husbandry records are essential. Blood work may at least suggest the presence of an infectious process. Elevated white cell counts accompanied by a heterophilia or monocytosis support this hypothesis. Further diagnosis will be based on hematology, cytology, Gram’s strains, and specific stains (such as Ziehl-Neelsen staining for mycobacteriosis diagnosis), and culture and sensitivity testing. It is important to underscore here that there is not an obvious relation between the symptoms observed in the patient and the microbiological isolate obtained through standard culture techniques. Obtaining mixed cultures of different bacterial isolates suggests secondary infections are occurring and that the primary pathogen and/or the predisposing conditions still needed to be identified. Just as isolating a bacteria that is part of the autochthonous flora may suggest an opportunistic overgrowth. For this kind of interpretation, it is highly advantageous for the clinician to have databases or good literature on specific normal flora, from which bacterial results may be dis- 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 51 cussed with consistency. Finding viruses or other known pathogenic agents that play a significant role in the cause of illness in pet avian species (such as Chlamydophila or Mycoplasma) also assumes that the bacterium may be a secondary pathogen.Yet organisms such as Chlamydophila or Mycoplasma are not easily detected through routine in-house screening. Isolation of an almost pure culture may indicate that the bacterium is an important, or even the main, component in the disease process. Just as having a positive bacterial culture with (only) fungi and protozoa suggests its prominent pathogenic role.With necropsy or living biopsy, isolating small to moderate numbers of bacteria from the liver or kidney is not pathognomonic.These organs should be expected to contain autochthonous flora, due to the avian hepatic and renal portal circulations and the lack of lymph nodes that filter blood before it drains into the liver and kidney. The most common causes of primary and secondary bacterial infections in psittacine birds are Gram-negative bacteria (Escherichia coli, Klebsiella, Pseudomonas) and Chlamydophila psittaci. Gramnegative bacteria are frequently resistant to routine antibiotics, however, most isolates are susceptible to enrofloxacin. Enterobacteriaceae (Salmonella, Citrobacter, Proteus, Serratia), Enterococcus faecalis (canaries) are common as well. Less common infectious agents of psittacine birds are Staphylococcus aureus and Streptococcus spp., Mycoplasma, Bordetella, Mycobacterium, and Pasteurella multocida.The causative organisms of mycobacteriosis are Mycobacterium avium spp. avium, M. intracellulare, and M. genavense. The disease may be asymptomatic for long periods and the main clinical symptoms are chronic wasting, weakness, labored respiration, diarrhea, skin granulomas, lameness, and death. There is a zoonotic potential, particularly for immunocompromised individuals. M. genavense is of greatest zoonotic concern.Thus, therapeutic management must be considered with caution or per- haps not even recommended except for valuable and endangered species. M. avium is resistant to common antituberculosis drugs, however, combination therapy (isoniazid, ethambutol, and rifampicin) for extended periods (up to 18 months) has resulted in clinical remission in some exotic birds. Once the antimicrobial therapy is decided, it is important to ensure the antibiotic reaches therapeutic levels in all target sites. For example, direct flushing or nebulization is needed to bring effective concentrations of antibiotics in the upper respiratory tract. Avian abscesses are usually presented with solid pus and are completely unavailable to antibiotic penetration. Surgical excision or debridement followed by topical medication are often essential parts of the therapy. In all cases, antibiotic therapy is one part of the therapeutic process. Correcting husbandry and nutritional deficiencies, giving supportive care (placing the animal in its optimal thermal zone, fluid therapy and gavage if needed) are also essential. Antimicrobial therapy in birds – the use of enrofloxacin The ideal antibiotic to use in birds is bactericidal, readily absorbed and widely distributed with therapeutic concentrations in tissues, easily administered, and does not cause adverse effects. Because of the high basal metabolism of birds, it is important to ensure the availability of therapeutic concentrations. Thus, medicated water, a traditionally favored route for poultry, should not be adapted in companion and aviary birds and would rather be a last choice (e.g., multiple-bird flocks). Serious microbial infections and critically ill birds should be treated with parenteral medications (or PO by gavage) to establish effective drug concentrations quickly. It’s important not to give too low of a dosage and to institute a twice-daily, or even three times daily, administration. 51 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 52 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai Enrofloxacin is widely used to treat bacterial infections in companion birds. Enrofloxacin is readily absorbed and widely distributed, and partially metabolized to an active metabolite, ciprofloxacin, in many species, including psittacine birds. Enrofloxacin is bactericidal for many Gram-negative bacteria and species of Staphylococcus and Mycoplasma. Enrofloxacin is highly active against most Enterobacteriaceae recovered from psittacine birds. A recent study has compared the efficacy of enrofloxacin, oxytetracycline, and sulfadimethoxine for the control of morbidity and mortality caused by Escherichia coli in broiler chickens. Chickens that received enrofloxacin had significantly less mortality (p < 0.01), lower average gross pathology (colibacillosis) scores (p < 0.01), and better feed-conversion ratios (p < 0.05) than chickens that received either oxytetracycline or no medication. Chickens that received enrofloxacin had significantly less mortality and lower pathology scores than those that received sulfadimethoxine, and numerically lower feed conversion than the sulfadimethoxine group. Results from the study showed that enrofloxacin was superior to oxytetracycline and sulfadimethoxine for the control of morbidity and mortality caused by E. coli in broiler chickens. Enrofloxacin has variable activity against Pseudomonas aeruginosa and species of Streptococcus and Chlamydophila; it has little activity against anaerobes. It may also be used for the treatment of chlamydophilosis and reduces clinical signs in birds infected. The duration of treatment should be 21 days for all species. Practical experience indicates that total eradication with clearing the carrier state is often difficult and not routinely achieved. Route of Administration Selecting the route of drug administration in birds requires careful consideration. Available routes 52 Figure 6 Enrofloxacin is most effective if dosed per os or by intramuscular injection; intramuscular injection achieves greater peak concentrations than oral administration. IM dosing is performed in the pectoral muscle. include medicated water, medicated food, oral, intramuscular, intravenous, subcutaneous, intraosseous, intratracheal, inhalation, and topically. Water-based drug administration is easiest and less stressful route of administration. However, consumption is erratic and therapeutic serum concentrations are rarely achieved, especially during the night when less water is consumed. Several studies have measured the plasma concentrations of enrofloxacin achieved by offering drinking water medicated with an injectable enrofloxacin formulation. However, practical applications of these results must be taken with caution as the animal models were healthy animals. A debilitated patient 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 53 would have a different behavior and wouldn’t have the water consumption. One study concluded that 0.19 to 0.75 mg/ml enrofloxacin in drinking water should provide appropriate MIC levels for susceptible infections in African gray parrots (Psittacus erithacus).The study also showed that African gray parrots tend to consume less water as the doses get higher (0.09–3.0 mg/ml). Another study measured the plasma concentrations of enrofloxacin with 200 mg/l enrofloxacin in drinking water in 16 psittacine birds for 10 days. The birds included 6 cockatoos (Cacatua species), 4 conures (Aratinga species), 2 Senegal parrots (Poicephalus senegalus), 2 red-shouldered macaws (Ara nobilis), and 2 gray parrots (Psittacus erithacus). The authors concluded that water medicated with the injectable formulation of enrofloxacin at 200 mg/l maintains plasma concentrations in psittacine birds that are adequate only for treating systemic infections caused by highly susceptible bacteria. Administration ad libitum to 8 healthy sandhill cranes (Grus canadensis) of enrofloxacin in the drinking water at a concentration of 50 ppm would only be effective for treating infections of highly susceptible bacteria. In this study, enrofloxacin and ciprofloxacin concentrations were both below accepted therapeutic plasma concentrations for birds. Enrofloxacin is most effective if dosed per os or by injection. Given orally, the injectable formulation of enrofloxacin produces therapeutic plasma concentrations, even more, it induces higher peak plasma concentrations than the water-soluble formulation. The major disadvantage of parenteral administration is intramuscular pain and irritation at the site of injection. However, in the Amazon parrot and cockatoo, intramuscular injection achieves greater peak concentrations compared to oral administration. Enrofloxacin can be administered orally but is bitter, and many birds will refuse to accept it. It may be necessary to dilute the drug in a palatable vehicle such as fruit juice or lactulose syrup, or to deliver it via a gavage tube. Oral administration accomplished by feeding red-tailed hawks (Buteo jamaicensis) and great-horned owls (Bubo virginianus) a small freshly killed mouse that had received an intraperitoneal injection of enrofloxacin (15 mg/kg) had provided appropriate MIC levels for susceptible bacteria. Peak plasma levels were achieved between 4 to 8 hours for orally administered enrofloxacin (compared to 0.5 to 2 hours for IM dosing). Studies on the single-dose kinetics of enrofloxacin in healthy African gray parrots, blue-fronted and orange-winged Amazons, and Goffin’s cockatoos indicate that a dose of 7.5– 15 mg/kg administered IM or PO BID should maintain effective concentrations in these species. Again, intramuscular injection achieves greater peak concentrations (3–5 mg/ml versus 1–1.5 mg/ml with oral administration at 15 mg/kg). Enrofloxacin can be given with metronidazole to broaden the spectrum as well as to take advantage of the ability to control anaerobic bacteria. The dose of enrofloxacin in birds used by the author is 20 mg/kg PO, IM q24h for most of companion birds. For small specimens, a dose of 10–15 mg/kg PO q12h would be recommended. Larger birds such as ratites would need lower drug dosages: 1.5–2.5 mg/kg PO, IM q12h. Reptiles General considerations Infectious diseases are an important cause of illness and mortality in all reptilian species. Again, the therapeutic plan will begin first by correcting environmental and nutritional deficiencies, which are the most important predisposing causes of diseases in reptiles.Without this first step, there is no 53 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 54 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai pathogens, infections caused by Gram-negative bacteria are most common. Aeromonas hydrophila, Klebsiella oxytoca, Morganella morganii, Providencia rettgeri, Pseudomonas aeruginosa, and Salmonella arizonae are prominent among the microorganisms isolated from healthy and ill captive reptiles.These bacteria can remain dormant and become invasive when conditions decrease the immune resistance of the host and/or follow primary viral infection. Anaerobic infections are more common than once thought and may be involved in up to 40 % of all bacterial infections. Figure 7 Abscesses caused by traumatic injury, bite wounds, or poor environmental qualities are seen in all orders of reptiles. The pus is generally solid, as in birds. Mandibular abscess in a green iguana (top) and a frilled lizard. sense in starting other treatments. Environmental temperatures should be maintained near the upper limit preferred by the species to enhance immune function. Higher metabolic rates of anorectic reptiles may necessitate force feeding. Fluid therapy should be considered as well. Ideally, severely affected reptiles should be isolated and antibiotic therapy initiated. In general, good sanitation is paramount in prevention of all diseases. The enclosure should be set up to reduce stress, with addition of hide boxes. Arboreal animals should be furnished with a secluded branch on which to lay. Although a wide variety of bacteria have been incriminated as either primary or secondary 54 Abscesses caused by traumatic injury, bite wounds, or poor environmental conditions are seen in all orders of reptiles. Differential diagnoses include parasitic nodules, tumors, and hematomas. Isolates of the anaerobic organism Peptostreptococcus and of the aerobes Pseudomonas, Aeromonas, Serratia, Salmonella, Micrococcus, Erysipelothrix, Citrobacter freundii, Morganella morganii, Proteus, Staphylococcus, Streptococcus, Escherichia coli, Klebsiella, Arizona, and Dermatophilus have been recovered from reptilian abscesses, often in combinations. Antibiotic administration will always be combined with surgery that can be quite invasive in large abscesses, in order to remove as much material as possible. Aural abscesses are commonly seen in chelonians, most frequently in box turtles and aquatic turtles. Marked swelling is seen at the tympanic membrane, and caseous material is present. Proteus sp., Pseudomonas sp., Citrobacter sp., Morganella morganii, Enterobacter sp., and other bacteria have been isolated. The tympanic membrane must be incised, followed by an aggressive curettage and flushing of the area, with diluted povidone-iodine or chlorhexidine. All snakes and some lizards posess a transparent spectacle located over the cornea. Subspectacle infections are common in snakes. Drainage is achieved by surgically removing a small wedge from the spectacle and flushing with an antibi- 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 55 otic solution directly onto the globe and within the space. In the other orders, conjunctivitis is sometimes seen, ranging from mild inflammation to panophthalmitis, and may occur as a result of ascending infectious stomatitis.Topical antibiotic ointments are used in turtles, lizards without spectacles, and crocodilians. Trauma, local abscessation, parasitism, or environmental stress may induce septicemia, a common cause of death. Aeromonas and Pseudomonas sp. are frequently isolated in such cases. Petechiae may be found on the ventral abdomen, and chelonians develop erythema of the plastron. Infectious stomatitis is frequently seen in snakes, and less so in lizards and turtles.The disease course begins with petechiae in the oral cavity, followed by caseous material appearing along the dental arcade, and the infection can extend into the bony structures of the mouth. Aeromonas and Pseudomonas spp. are most frequently isolated. Debridement, irrigation with antiseptics, systemic antibiotics, and supportive therapy are indicated. Stomatitis is a secondary infection. The animal’s environment should be modified as necessary to aid in recovery. Respiratory infections are common in reptiles. The incidence can be influenced by respiratory or systemic parasitism, unfavorable environmental temperatures, unsanitary conditions, concurrent disease, malnutrition, and hypovitaminosis A. In snakes with neurologic symptoms, one should always consider involvement of a viral disease process.Turtles often have an underlying vitamin A deficiency. Increased temperatures are important not only to stimulate the immune system but also to help mobilize respiratory secretions. If the reptile does not respond to environmental correction and the antibiotic therapy, a culture and sensitivity along with histology should be performed. Figure 8 Advanced stomatitis in a boa constrictor (left) and a red-eared turtle. Stomatitis is most commonly seen in snakes and lizards, less frequently in chelonians. Pockets of caseous pus may occur in the soft tissues. Left untreated, the condition may progress to osteomyelitis of the mandibular and cranial structures, and teeth may be found loose within the necrotic tissue. Antimicrobial therapy Culture and sensitivity are essential in determining appropriate therapy. Because most infected reptiles have some level of immunosuppression, bactericidal drugs are preferable to bacteriostatic ones. Enrofloxacin has a wide spectrum of antimicrobial activity which includes the common reptile pathogens and a large volume of distribution and, thus, has become a commonly used antimicrobial in reptile medicine. Enrofloxacin is effective in the treatment of Mycoplasma infections such as conjunctivitis and soft tissue infections. A recent study showed that against Mycoplasma iguanae, a proposed species nova isolated from vertebral abscesses of two feral iguanas (Iguana iguana), clindamycin, doxycycline, oxytetracycline, and tylosin were bacteriostatic from 0.1 to 0.5 µg/ml, whereas enrofloxacin was bactericidal at 20 ng/ml.The MIC for common reptile pathogens is lower for enrofloxacin than other commonly used antimicrobials. Enrofloxacin, and its active metabolite ciprofloxacin, are capable of inhibiting growth of pathogenic bacteria at serum levels of approximately 0.1 µg/ml. Mixed or resistant infections may require combinations of antibiotics. Enrofloxacin can be given with metronidazole to broaden the spectrum as well as to take advantage of the ability to control anaerobic bacteria. 55 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 56 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai In most cases, antimicrobials will be given by injection, either SC or IM. Oral administration is reserved for primary infection of the gastrointestinal tract, for species that do not tolerate injections or extremely small specimens (some chameleons and geckos) that lack adequate muscle mass for a painless injection. Since most species of reptiles have a renal portal system, with blood from the caudal half of the body going to the kidneys before reaching systemic circulation, it has been recommended that SC and IM injections should be given in the cranial half of the body. However, there are few studies that have looked at this potential problem scientifically. For the dosing, if metabolic scaling may work in mammals and birds, it does not in reptiles. Differences in body temperature, season, reproductive status, nutritional, and overall specific physiology are just a few of the variables that may ultimately influence metabolic rates and thus make any equation of metabolic scaling invalid. Because IM injection is often painful and may induce adverse local effects, the pharmacokinetics of enrofloxacin disposition following oral administration have been investigated in green iguanas (Iguana iguana), savannah monitors (Varanus exanthematicus), red-eared sliders (Trachemys scripta elegans), American alligators (Alligator mississippiensis), and in loggerhead sea turtles (Caretta caretta). There is a markedly varied disposition among species, indicating that extrapolation between reptile species is likely to result in inaccurate dosing of enrofloxacin. Green iguanas given injectable enrofloxacin PO at 5 mg/kg had therapeutic plasma concentrations of enrofloxacin (> 0.2 µg/ml). However, enrofloxacin does not appear to be metabolized to ciprofloxacin in significant amounts in green iguanas. This result suggested that the parenteral route would be more suitable than oral administration for the treatment of critical infections in green iguanas. Savanna monitors fed mice 56 containing 10 mg/kg of injectable enrofloxacin showed similar but delayed therapeutic plasma concentrations as compared to IM injection. Thus, in Savanna monitors, an initial dose of enrofloxacin (10 mg/kg IM) followed by oral administration for continued therapy would be beneficial for acute infections. In American alligators, 5 mg/kg PO is not expected to achieve minimum inhibitory values for susceptible organisms based on a pharmacokinetic study in this species, however, IV administration every 36 hours would. In loggerhead sea turtles, a dosage rate of 20 mg/kg PO no more often than once per week may be recommended to treat susceptible pathogens. The use of enrofloxacin in reptiles depends then upon the species. The primary doses for most species are 5–10 mg/kg q24h PO, SC, IM. The following doses are adapted from Carpenter JW, Mashima TY, Rupiper D (2001), and Prescott JF, Baggot JD, Walker RD (2000): Green iguanas: 5 mg/kg PO, IM q24 h Monitors: 10 mg/kg IM q5d Pythons: 6.6 mg/kg IM q24h or 11 mg/kg IM q48h or 10 mg/kg IM, then 5 mg/kg q48h Herman’s tortoises: 10 mg/kg IM q24h Gopher tortoises: 5 mg/kg IM q24-48h Star tortoises: 5 mg/kg IM q12-24h Sea turtles: 5 mg/kg IM q48h or 20 mg/kg PO q1week (loggerhead sea turtles) Alligators: 5 mg/kg IV q36h Amphibians General considerations Amphibian medicine is an emerging field – an emerging field close to a “emergency” field. The amphibian patient is often presented late in the disease process and the most frequently apparent clinical signs are non-pathognomonic. Because amphibians are – more than any terrestrial verte- 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 57 Figure 9 Antibiotic therapy is always indicated for traumatic wounds. Here a young monitor (top and bottom left) that recovered uneventfully after a bite wound, and traumatic wounds on a Madagascarian boa (upper right) and a tortoise. brate – very dependent on their environmental conditions, husbandry records are critical for the clinician. Bacterial diseases have a high prevalence in amphibian facilities. Most of environmental bacterial agents become pathogens in stressed amphibians: transportation, bad husbandry, changes in the environment. Red-leg syndrome in amphibians is so named due to the hyperemia of the ventral skin of the thighs and abdomen of septicemia anurans, and is now synonymous with any generalized bacterial infection in amphibians. It is better to talk about bacterial dermosepticemia. If historically this syndrome is associated with Aeromonas hydrophila, many other infectious agents produce similar integumentary signs. For the diagnostic process, body fluids are taken when possible. Smears and fast staining are done in the first approach. Biopsy for histology, bacteriology, and sensitivity are conducted as rapidly as possible. Isolating ill animals, optimizing the husbandry conditions, combating dehydration, and putting the animal in the upper limit of its preferred thermal zone are the first steps of treatment. Antimicrobial therapy Safe, efficacious treatment for common anuran bacterial infections requires knowledge of specificity, pharmacokinetics, and toxicity of antibacterial agents in frogs. Three readily available antibiotic agents – tetracycline, enrofloxacin, amikacin – which have specificity for common anuran bacterial pathogens were selected for investigation. Tetracycline was the first and most commonly recommended antibiotic for treatment 57 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 58 Proceedings of the 4th International Baytril® Symposium Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin Norin Chai of bacterial disease in frogs. However, tetracyclineresistant organisms from clinically ill amphibians have been isolated and widespread bacterial resistance to tetracycline has also been reported in mammals and reptiles. On the other hand, bacterial resistance to enrofloxacin has only rarely been reported and amphibian pathogens have been uniformly susceptible. Enrofloxacin and its active metabolite ciprofloxacin are frequently effective in inhibiting growth of pathogenic bacteria at serum levels of approximately 0.1 µg/ml. In bullfrogs (Rana catesbeiana), a study has shown that dosages of 5 and 10 mg/kg once daily maintained the plasma concentration above this level throughout the dosing interval. A single 10 mg/kg intramuscular dose did not induce any significant hematological or biochemical abnormalities. Any route of administration is possible: PO, IM, percutaneous. All antibiotic therapy must last at least 7 days. In most amphibians, enrofloxacin is reported to be used at 5–10 mg/kg PO, SC, IM q24h.The weight is very variable depending on the state of hydration and one should not hesitate to reweigh the animal. and dose-related, and the drug has not been studied in all species. As a rule, with many animal species, this class of antimicrobial agents should not be administered to animals less than 8 months of age or to large-breed dogs less than 12 months of age. In reptiles, the injectable form can be extremely irritating, and this always needs to be considered when this route of administration is selected. Focal to diffuse areas of necrosis at intramuscular injection sites in snakes and chelonians, and excessive salivation in juvenile Galapagos tortoises (Geochelone elephantopus) that were administered a single IM injection have been reported. Conclusion Adverse effects Due to its bactericidal, wide distribution to tissues and the extracellular space, and because it can penetrate nearly every tissue in the body, enrofloxacin is among the most effective drugs for treating most bacterial infections in exotics. Enrofloxacin also offers the advantages of oral administration. Oral bioavailability of enrofloxacin is excellent in monogastric mammals and pre-ruminant calves, with up to 80 % of the ingested dose being absorbed into systemic circulation. However, the metabolism and elimination half-life of enrofloxacin varies greatly between species. More pharmacokinetic studies are required in veterinary medicine for non-empiric use in more species. The main adverse effects associated with fluoroquinolones are primarily associated with abnormal development of immature cartilage. Arthropathies have been reported in immature rats, beagles, guinea pigs, and foals. However, in birds, enrofloxacin has been widely used in psittacine nurseries without reports of side effects. Despite this fact, the drug should be used with caution in growing birds as toxic effects are species-specific Enrofloxacin is generally well tolerated. At therapeutic doses, it has proven to be relatively safe in all species, with few reported side effects. In addition, effective treatment with twice-daily, or once-daily in some species, administration is a clear advantage over some other antibiotics.The major disadvantage of parenteral administration is intramuscular pain and irritation at the site of injection. The author finds that the percutaneous route by bath is the less stressful. A dosage of 0.3 mg/ml water bath for 15 days has demonstrated routinely favorable results. 58 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 59 References Bercovier H,Vincent V (2001). Mycobacterial infections in domestic and wild animals due to Mycobacterium marinum, M. fortuitum, M. chelonae, M. porcinum, M. farcinogenes, M. smegmatis, M. scrofulaceum, M. xenopi, M. kansasii, M. simiae, and M. genavense. Rev Sci Tech Off Int Epiz; 20: 265–290. Harrenstein LA,Tell LA,Vulliet R et al. (2000). Disposition of enrofloxacin in red-tailed hawks (Buteo jamaicensis) and great-horned owls (Bubo virginianus) after a single oral, intramuscular, or intravenous dose. J Avian Med Surg; 14: 228–236. Berg J (1988). Clinical indications for enrofloxacin in domestic animals and poultry. In:Quinolones: A Symposium: A new veterinary medicine (Ed: Mobay Corporation), Shawnee, Kansas, USA; pp. 25–34. Helmick KE, Papich MG, Vliet KA et al. (2004). Pharmacokinetics of enrofloxacin after single-dose oral and intravenous administration in the American alligator (Alligator mississipiensis). J Zoo Wildl Med; 35: 333–340. Bowman MR, Waldoch JA, Pittman JM, Papich MG, Hartup BK (2004). Enrofloxacin and ciprofloxacin plasma concentrations in sandhill cranes (Grus canadensis) after enrofloxacin administration in drinking water. J Avian Med Surg; 18(3): 144–150. Hooper D, Wolfson J (1985).The fluoroquinolones: structures, mechanisms of action and resistance and spectra of activity in vitro. Antimicrob Agents Chemother; 28: 581–586. Boyer TH (1994). Emergency Care of Reptiles. Seminars Avian Exotic Pet Med; 3(4): 210–216. Broome RL, Brooks DL, Babish JG et al. (1991). Pharmacokinetic properties of enrofloxacin in rabbits. Am J Vet Res; 52: 1835–1841. Brown SA (1996). Fluoroquinolones in animal health. J Vet Pharmacol Ther; 19: 1–14. Burkhardt J, Hill MA, Carlton WW et al. (1990). Histologic and histochemical changes in articular cartilages of immature beagle dogs dosed with difloxacin, a fluoroquinolone. Vet Pathol; 27: 162–170. Chai N. Clinical techniques and pathology in amphibians. 15th Annual Association of Reptilian and Amphibian Veterinarians Conference. Los Angeles, USA, October 12–15, 2008; pp. 45–48. Converse KA (2007). Avian Tuberculosis. In: Infectious Diseases of Wild Birds (Eds: Thomas NJ, Hunter DB, Atkinson CT), Blackwell Publishing, Ames, IA, USA; pp. 289–302. Diver JM, Wise R (1986). Morphological and biochemical changes in Escherichia coli after exposure to ciprofloxacin. J Antimicrob Chemother; 18(Suppl D): 31–41. Flammer K, Aucoin DP,Whitt DA et al. (1990). Plasma concentrations of enrofloxacin in African grey parrots treated with medicated water. Avian Dis; 34: 1017–1022. Frye FL (1992). Reptile care: an atlas of diseases and treatment. TFH Publications, Inc., Neptune City NJ; pp. 111, 116–117. Hungerford C, Spelman L, Papich M (1997). Pharmcokinetics of enrofloxacin after oral and intramuscular administration in savannah monitors (Varanus exanthematicus). Proc Am Assoc Zoo Vet, 1997; pp. 89–92. Jacobson E, Gronwall R, Maxwell L, Merrit K, Harman G (2005). Plasma concentrations of enrofloxacin after singledose oral administration in loggerhead sea turtles (Caretta Caretta). J Zoo Wildl Med; 36(4): 628–634. James SB, Calle PP, Raphael BL, Papich M, Breheny J, Cook RA (2003). Comparison of injectable versus oral enrofloxacin pharmacokinetics in red-eared slider turtles, Trachemys scripta elegans. J Herp Med Surg; 13: 5–10. Jenkins JR (2001). Skin disorders of the rabbit. Vet Clin North Am Exotic Anim Pract; 4: 552–554. Maxwell LK, Jacobson ER (1997). Preliminary single-dose pharmacokinetics of enrofloxacin after oral and intramuscular administration in green iguanas (Iguana iguana). Proc Am Assoc Zoo Vet; p. 25. Meinen JB, McClure JT, Rosin E (1995). Pharmacokinetics of enrofloxacin in clinically normal dogs and mice and drug pharmacodynamics in neutropenic mice with Escherichia coli and staphylococcal infections. Am J Vet Res; 56: 1219–1224. Mitchell MA (2006). Enrofloxacin. Therapeutic review. J Exotic Pet Med; 15(1): 66–69. Prezant RM, Isaza I, Jacobson ER (1994). Plasma concentrations and dipsosition kinetics of enrofloxacin in gopher tortoises (Gopherus polyphemus). J Zoo Wildl Med; 25: 82–87. Fulton RM, Sanchez S (2008). Tuberculosis. 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Blood levels of some antiinfectives in the spur-tailed tortoise (Testudo hermanni). Proc 4th Int Colloq Pathol Ther Rept Amph; 1991; pp. 120–128. Stamper MA, Papich MG, Lewbart GA, May SB, Plummer DD, Stoskopf MK (1999). Pharmacokinetics of ceftazidime in loggerhead sea turtles (Caretta caretta) after single intravenous and intramuscular injections. J Zoo Wildl Med; 30: 32–35. Stamper MA, Papich MG, Lewbart GA, May SB, Plummer DD, Stoskopf MK (2003). Pharmacokinetics of florfenicol in loggerhead sea turtles (Caretta caretta) after single intravenous and intramuscular injections. J Zoo Wildl Med; 34: 3–8. 60 Tyrrell KL, Citron DM, Jenkins JR et al. (2002). Peridontal bacteria in rabbit mandibular and maxillary abscesses. J Clin Microbiol; 40: 1044–1047. Walker RD (2000). Fluoroquinolones. In: Antimicrobial Therapy in Veterinary Medicine (Eds: Prescott JF, Baggot JD, Walker RD), 3rd ed., Iowa State University Press, Ames, IA; pp. 315–318. Westfall ME, Demcovitz DL, Plourdé DR, Rotstein DS, Brown DR (2006). In vitro antibiotic susceptibility of Mycoplasma Iguanae proposed sp. nov. isolated from vertebral lesions of green iguanas (Iguana Iguana). J Zoo Wildl Med; 37(2): 206–208. Wright DH, Brown GH, Peterson ML, Rotschafer JC (2000). Application of fluoroquinolone pharmacodynamics. J Antimicrob Chemother; 46: 669–683. Young LA, Schumacher J, Papich MG, Jacobson ER (1997). Disposition of enrofloxacin and its metabolite ciprofloxacin after IM injection in juvenile Burmese pythons (Python molurus bivittatus). J Zoo Wildl Med; 28: 71–79. 904091_TRI_Symp_Chai.qxp 03.06.2009 11:43 Uhr Seite 61 904091_TRI_Symp_Lappin.qxp 03.06.2009 11:34 Uhr Seite 62 Use of Enrofloxacin in Cats with Resistant Mycoplasma spp. Infections Hemotropic Mycoplasma spp. The new names for Haemobartonella felis are Mycoplasma haemofelis (Mhf), ‘Candidatus M. haemominutum’ (Mhm), and ‘Candidatus M. turicensis’ (Mtc).1-3 The organisms are now classified as hemotropic mycoplasmas and clinical syndromes associated with the agents are collectively known as hemoplasmosis. In the studies of experimentally infected cats performed to date, Mhf is apparently more pathogenic than Mhm; however, some cats with Mhm alone develop clinical illness.4 Cats with chronic Mhm infection had more severe anemia and longer duration of anemia when experimentally infected with Mhf when compared to cats infected with Mhf alone.5 Cats infected with Mtc can be clinically ill or develop a chronic carrier state like Mhm and Mhf. In a recent study, we collected fleas from cats in the United States and attempted to amplify hemoplasma DNA from flea extracts as well as the blood of the cat using polymerase chain reaction assay (PCR).6 The prevalence rates for Mhf in cats and their fleas were 7.6 % and 2.2 %, respectively. The prevalence rates for Mhm in cats and their fleas were 20.7 % and 23.9 %, respectively. In another study of cats in the United States, the overall prevalences of Mhm, Mhf, and Mtc infection were 23.2 % (72/310), 4.8 % (15/310), and 6.5 % (20/310), respectively.7 In addition, fleas ingest Mhm and Mhf from infected cats when feeding. In one cat, we documented flea feeding to transfer Mhf.8 However, when we fed Mhf- or Mhm-infected fleas to cats, infection was not documented.9 In other studies, hemoplasmas have been transmitted experimentally by IV, IP, and oral inoculation of blood. Clinically ill queens can infect kittens; whether transmission occurs in utero, during parturition, or from nursing has not been determined. Transmission by biting has been hypothesized and the organisms are in the mouths of cats.2,10 Red blood cell de62 struction is due primarily to immune-mediated events; direct injury to red blood cells induced by the organism is minimal. Clinical signs of disease depend on the degree of anemia, the stage of infection, and the immune status of infected cats. Coinfection with FeLV can potentiate disease associated with Mhm.11 Clinical signs and physical examination abnormalities associated with anemia are most common and include pale mucous membranes, depression, inappetence, weakness, and, occasionally, icterus and splenomegaly. Fever occurs in some acutely infected cats and may be intermittent in chronically infected cats. Evidence of coexisting disease may be present.Weight loss is common in chronically infected cats. Cats in the chronic phase can be subclinically infected only to have recurrence of clinical disease following periods of stress. The anemia associated with hemoplasmosis is generally macrocytic, normochromic. Chronic non-regenerative anemia is unusual in cats with hemoplasmosis. Neutrophilia and monocytosis have been reported in some hemoplasma-infected cats. Diagnosis is based on demonstration Figure 1 Cytology of a blood smear of a cat with anemia after experimental infection with Mycoplasma haemofelis. 904091_TRI_Symp_Lappin.qxp 03.06.2009 11:34 Uhr Seite 63 Michael R. Lappin, DVM, PhD, Professor, Diplomate ACVIM (Internal Medicine) Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, USA Enrofloxacin 10 mg/kg/d 9 8 Log copy no of the organism on the surface of erythrocytes on examination of a thin blood film (Fig. 1) or PCR assay.12,13 Organism numbers fluctuate and so blood film examination can be falsely negative up to 50 % of the time. The organism may be difficult to find cytologically, particularly in the chronic phase. Thus, PCR assays are the tests of choice due to sensitivity. Primers are available that amplify a segment of the 16S rRNA gene common to both hemoplasmas. Real-time PCR to quantify hemoplasma DNA has now been titrated and can be used to monitor response to treatment.14 Many healthy cats are positive for hemoplasma DNA in blood and so not all PCRpositive cats are clinically affected. 7 6 5 4 3 2 1 0 7 14 21 28 35 42 Figure 2 Real-time PCR assay results showing the rapid decrease in Mycoplasma haemofelis copy numbers in the blood of a cat Doxycycline has fewer side effects than other tetracyclines in cats and so is the preferred drug in this class. Doxycycline is often administered at 10 mg/kg, PO, every 24 hours for 7 days. If there is a positive response to treatment at the day 3 or day 7 recheck evaluation and the cat is tolerating the drug, treatment is continued for 28 days. In the United States, doxycycline is administered as a flavored suspension or water is given after pilling to avoid esophageal strictures.15 Tetracyclines utilized to date appear to lessen parasitemia and clinical signs of disease but probably do not always clear the organism from the body and so recurrence is possible.16 Some cats with clinical hemoplasmosis appear to be resistant to administration of tetracyclines. In these cats, administration of antibiotics in the fluoroquinolone class appears to be frequently effective; enrofloxacin was the first drug studied.17-19 The drug was well tolerated in experimentally infected cats when administered 5 mg/kg or 10 mg/kg, PO, every 24 hours for 14 days.17 The drug was equally effective or more effective than doxycycline for the treatment of the clinical manifestations of hemoplasmosis in this study. administered enrofloxacin at 10 mg/kg, PO, daily. Administration of enrofloxacin rapidly decreased DNA copy numbers in blood, but some cats are still PCR-positive after a course of therapy (Fig. 2).14 Cats administered marbofloxacin also are PCR-positive after treatment. Pradofloxacin is a fluoroquinolone with potent effect against hemoplasmas that is currently under investigation. This drugs shows promise for the elimination of the hemoplasma carrier phase.20 Other antimicrobial agents that have been attempted for the treatment of hemoplasmosis in cats include azithromycin and imidocarb.5,21,22 Azithromycin was not effective for the treatment of hemoplasmosis in one study.5 Imidocarb has been administered safely to research cats harboring Mhf or Mhm and when administered at 5 mg/kg, IM, every 2 weeks for at least 2 injections was used successfully in the management of five naturally-infected cats that had failed treatment with other drugs.21,22 Hemoplasmosis and primary immune hemolytic anemia are difficult to differentiate based on clinical and laboratory findings.23 Thus, cats with se63 904091_TRI_Symp_Lappin.qxp 03.06.2009 11:34 Uhr Seite 64 Proceedings of the 4th International Baytril® Symposium Use of Enrofloxacin in Cats with Resistant Mycoplasma spp. Infections Michael R. Lappin vere, regenerative hemolytic anemia, particularly those with rapidly dropping PCV or autoagglutination, are often treated with glucocorticoids and antibiotics. In these cats, prednisolone is often prescribed at 1 mg/kg, PO, every 12 hours for a the first 7 days or until autoagglutination is no longer evident while waiting for hemoplasma PCR assay results to return. To attempt to prevent hemoplasma infections, it might be prudent to control fleas. Cats should be housed indoors to avoid other potential vectors and fighting. Blood donor cats should be screened by PCR assay prior to use.24 Mycoplasma spp.-associated bronchitis and rhinitis b Figure 3 Lateral (a) and VD (b) radiographs from a cat with bronchitis consistent with that seen with Mycoplasma spp. infection. 64 Almost all cats with mucopurulent or purulent nasal discharge have a bacterial component to their disease. In addition, bacterial bronchitis can occur in cats (Fig. 3). Primary bacterial disease is associated with Bordetella bronchiseptica, Mycoplasma spp., and Chlamydophila felis.25-29 Recently it was shown that Bartonella spp. are not causes of rhinitis in cats.30 Mycoplasma spp., are normal commensal organisms of the nose and mouth. However, some strains may be primary pathogens (i.e., M. felis), and other strains may be associated with clinical illness if another primary disease process has occurred. Primary infection with feline herpesvirus 1 or feline calicivirus with secondary bacterial infections are also very common causes of rhinitis. In these situations, Pasteurella spp., Streptococcus spp., and Staphylococcus spp. infections are likely isolates. Veterinarians in the United States frequently administer amoxicillin-clavulanate to cats with mucopurulent rhinitis or suspected bacterial bronchitis and this therapy is often effective. However, this class of antibiotic is ineffective for Mycoplasma spp. infections as these organisms do not have a cell wall. It is often difficult for commercial veterinary laboratories to culture Mycoplasma spp. successfully and antimicrobial susceptibility testing for Mycoplasma spp. is not 904091_TRI_Symp_Lappin.qxp 03.06.2009 11:34 Uhr Seite 65 routinely available.Thus, cats with suspected bacterial rhinitis or bronchitis that fail to respond to penicillins should be treated with an alternate drug class effective for Mycoplasma spp. The author has frequently prescribed enrofloxacin at 5 mg/kg, PO, daily to cats with mucopurulent rhinitis or bronchitis suspected to be from Mycoplasma spp. infection with clinical success. In addition, most B. bronchiseptica isolates in the United States are susceptible to enrofloxacin. Cats with acute mucopurulent rhinitis or bacterial bronchitis only need to be treated for 7 to 10 days. Cats with chronic rhinitis can have osteomyelitis and may need to be treated for several weeks. Doxycycline administered at 10 mg/kg, PO, once daily can also be effective for treatment of the primary bacterial pathogens. Most cases of bacterial rhinitis are secondary to other diseases including trauma, neoplasia, inflammation induced by viral infection, foreign bodies, inflammatory polyps, and tooth root abscessation. Cats with suspected bacteria bronchitis often have an underlying cause. Thus, if routine antibiotic therapy fails, a diagnostic workup should be performed. Mycoplasma spp.-associated polyarthritis Polyarthritis in cats from Mycoplasma spp. infections are rare.31-33 Most cats evaluated at Colorado State University have had fever and a stiff gait. Some cats have had a history of rhinitis. Multiple cats can be involved in the same household. Joint pain can be very severe. Cytology of synovial fluid reveals septic suppurative inflammation and both M. felis and other Mycoplasma spp. have been grown or amplified from affected joints by PCR assay. Doxycycline at 10 mg/kg, PO, daily has been clinically effective for the treatment of some cats. In others, administration of doxycycline failed to sterilize the joints. Enrofloxacin administered at 5 mg/kg, PO, daily has been used to successfully as a primary treatment as well as to eliminate Mycoplasma spp. from the joints of affected cats that failed treatment with doxycycline. References 1. Messick JB (2003). New perspectives about hemotrophic mycoplasma (formerly Haemobartonella and Eperythrozoon species) infections in dogs and cats. Vet Clin North Am Small Anim Pract; 33: 1453–1465. 5.Westfall DS, Jensen WA, Reagan W et al. (2001). Inoculation of two genotypes of Haemobartonella felis (California and Ohio variants) to induce infection in cats and response to treatment with azithromycin. Am J Vet Res; 62: 687–691. 2. Willi B et al. (2005). Identification, molecular characterization, and experimental transmission of a new hemoplasma isolate from a cat with hemolytic anemia in Switzerland. J Clin Microbiol; 43: 2581–2585. 6. Lappin MR, Griffin B, Brunt J et al. (2006). Prevalence of Bartonella spp., Mycoplasma spp., Ehrlichia spp., and Anaplasma phagocytophilum DNA in the blood of cats and their fleas in the United States. J Fel Med Surg; 8: 85–90. 3.Willi B, Boretti FS, Meli ML et al. (2007). Real-time PCR investigation of potential vectors, reservoirs, and shedding patterns of feline hemotropic mycoplasmas. Appl Environ Microbiol; 73: 3798–3802. 7. Sykes JE, Terry JC, Lindsay LL et al. (2008). Prevalences of various hemoplasma species among cats in the United States with possible hemoplasmosis. J Am Vet Med Assoc; 232: 372–379. 4. Reynolds C, Lappin MR (2007). “Candidatus Mycoplasma haemominutum” infections in client-owned cats. J Am Anim Hosp Assoc; 43: 249–257. 8. Woods J, Brewer MM, Hawley JR et al. (2005). Evaluation of experimental transmission of “Candidatus Mycoplasma haemominutum” and Mycoplasma haemofelis by Ctenocephalides felis to cats. Am J Vet Res; 66: 1008-1012. 65 904091_TRI_Symp_Lappin.qxp 03.06.2009 11:34 Uhr Seite 66 Proceedings of the 4th International Baytril® Symposium Use of Enrofloxacin in Cats with Resistant Mycoplasma spp. Infections Michael R. Lappin 9. Woods JE, Wisnewski N, Lappin MR (2006). Attempted transmission of “Candidatus Mycoplasma haemominutum” and Mycoplasma haemofelis by feeding cats infected Ctenocephalides felis. Am J Vet Res; 67: 494–497. 10. Dean RS, Helps CR, Gruffydd Jones TJ et al. (2008). Use of real-time PCR to detect Mycoplasma haemofelis and “Candidatus Mycoplasma haemominutum” in the saliva and salivary glands of haemoplasma-infected cats. J Feline Med Surg; 10: 413–417. 11. George JW, Rideout BA, Griffey SM et al. (2002). Effect of preexisting FeLV infection or FeLV and feline immunodeficiency virus coinfection on pathogenicity of the small variant of Haemobartonella felis in cats. Am J Vet Res; 63(8): 1172–1178. 12. Jensen WA, Lappin MR, Kamkar S et al. (2001). Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats. Am J Vet Res; 62: 604–608. 13.Tasker S, Binns SH, Day M J et al. (2003). Use of a PCR assay to assess prevalence and risk factors for Mycoplasma haemofelis and “Candidatus Mycoplasma haemominutum” in cats in the United Kingdom.Vet Rec; 152: 193–198. 14.Tasker S, Helps CR, Day MJ et al. (2004). Use of a Taqman PCR to determine the response of Mycoplasma haemofelis to antibiotic treatment. J Microbiol Methods; 56: 63–71. 15. Melendez L,Twedt D (2000). Suspected doxycycline-induced esophagitis with esophageal stricture formation in three cats. Feline Pract; 28: 10–12 16. Foley JE, Harrus S, Poland A et al. (1998). Molecular, clinical, and pathologic comparison of two distinct strains of Haemobartonella felis in domestic cats. Am J Vet Res; 59: 1581–1588. 17. Dowers KL, Olver C, Radecki SV et al. (2002). Enrofloxacin for treatment of cats experimentally infected with large form Haemobartonella felis. J Am Vet Med Assoc; 221: 250–253. 18. Tasker S, Caney SM, Day MJ et al. (2006). Effect of chronic FIV infection, and efficacy of marbofloxacin treatment on Mycoplasma haemofelis infection. Vet Microbiol; 31(117): 169–179. 19. Ishak AM, Dowers KL, Cavanaugh MT et al. (2008). Marbofloxacin for the treatment of experimentally induced Mycoplasma haemofelis infection in cats. J Vet Intern Med; 22: 288–292. 20. Dowers KL, Tasker S, Radecki SV et al. (2009). Use of pradofloxacin to treat experimentally induced Mycoplasma hemofelis infection in cats. Am J Vet Res; 70: 105–111. 66 21. Lappin MR, Radecki S (2002). Effects of imidocarb diproprionate in cats with chronic haemobartonellosis.Vet Ther; 3: 144–149. 22. Lappin MR, Foster A, Geitner K et al. (2002). Imidocarb diproprionate for the treatment of recurrent haemobartonellosis in cats. J Vet Int Med; 16: 364. 23. Ishak AM, Radecki S, Lappin MR (2007). Prevalence of Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum”, Bartonella spp., Ehrlichia spp., and Anaplasma phagocytophilum DNA in the blood of cats with anemia. J Feline Med Surg; 9: 1–7. 24. Wardrop KJ, Reine N, Birkenheuer A et al. (2005). Canine and feline blood donor screening for infectious disease. J Vet Intern Med; 19: 135–142. 25. Chandler JC, Lappin MR (2002). Mycoplasmal respiratory infections in small animals: 17 cases (1988–1999). J Am Anim Hosp Assoc; 38: 111–119. 26. Lappin MR, Veir J, Hawley JR (2007). Transmission of Mycoplasma spp. in specific pathogen-free kittens. Proceedings of the ACVIM Forum, Seattle, June 7. 27. Johnson LR, Foley JE, De Cock HE et al. (2005). Assessment of infectious organisms associated with chronic rhinosinusitis in cats. J Am Vet Med Assoc; 227: 579–585. 28. Foster SF, Martin P, Allan GS et al. (2004). Lower respiratory tract infections in cats: 21 cases (1995–2000). J Feline Med Surg; 6: 167–180. 29. Speakman AJ, Dawson S, Binns SH et al. (1999). Bordetella bronchiseptica infection in the cat. J Small Animal Pract; 40: 252–256. 30. Berryessa NA, Johnson LR, Kasten RW et al. (2008). Microbial culture of blood samples and serologic testing for bartonellosis in cats with chronic rhinosinusitis. J Am Vet Med Assoc; 233: 1084–1089. 31. Zeugswetter F, Hittmair KM, de Arespacochaga AG, Shibly S, Spergser J (2007). Erosive polyarthritis associated with Mycoplasma gateae in a cat. J Feline Med Surg; 9(3): 226–231. 32. Liehmann L, Degasperi B, Spergser J et al. (2006). Mycoplasma felis arthritis in two cats. J Small Anim Pract; 47: 476–479. 33. Ernst S, Goggin JM (1999).What is your diagnosis? Mycoplasma arthritis in a cat. J Am Vet Med Assoc; 215: 19–20. 904091_TRI_Symp_Lappin.qxp 03.06.2009 11:34 Uhr Seite 67 904091_TRI_Symp_68_73_Olsen.qxp 04.06.2009 8:36 Uhr Seite 68 Unique Pharmacologic Characteristics of Baytril® Introduction As the first fluoroquinolone developed exclusively for veterinary use, Baytril® (enrofloxacin) offered high potency, broad-spectrum activity with the advantages of both oral and parenteral administration, extensive tissue distribution, and a favorable safety profile, for the treatment of bacterial infections in companion animals.With numerous pharmacokinetic, microbiologic, and clinical investigations since its availability, and well over 1,100 publications in the scientific literature, it has become one of the most thoroughly investigated compounds in veterinary medicine. A few of the unique features of Baytril that continue to distinguish its utility will be highlighted here. Structure-activity relationships of enrofloxacin and fluoroquinolones The precursors to Baytril and other compounds in the fluoroquinolone class, the ‘early’ quinolones such as nalidixic and oxolinic acid, were used almost exclusively for urinary tract infections in humans and had limitations with respect to spectrum (soley Gram-negative bacteria, except Pseudomonas aeurginosa), potency, oral bioavailability, and tissue distribution.1 Advances in the understanding of chemical structure-function relationships for the quinolones led to synthesis of numerous derivatives and analogues of the original core quinolone structure, with markedly improved pharmacologic characteristics. The pivotal addition of a fluorine atom at position 6 of the basic 4-quinolone ring structure significantly broadened the antibacterial spectrum and gave rise to the modern ‘fluro’quinolones.2 This structural modification increased spectrum of activity to include Gram-positive bacteria and increased potency against Gram-negative organisms, as well as enhancing oral bioavailability and tissue penetration.The substitution of a piperazinyl ring at position 7 resulted in increased activity against bacteria such as Pseudomonas.3 Figure 1 highlights these structure-activity relationships for enrofloxacin as an example. Enrofloxacin Fluorine atom at C6 – enhances efficacy against Gram-negative bacteria – broadens spectrum against Gram-positive organisms Basic Quinolone Parent Structure co-planar carbonyl groups – hydrogen bond with DNA gyrase complex O O R6 R7 R4 COOH R3 piperazine ring at C7 – enhances antimicrobial activity, especially against Pseudomonas spp. R1 F OH N N O N N H5C2 N-ethyl group – enhances tissue penetration – decreases CNS toxicity amine (basic) and carboxylic acid (acidic) functional groups – endow amphoteric and zwitterionic properties – lipid solubility enhanced between pKa's of acidic and basic functional groups Figure 1 Chemical structure of enrofloxacin with key modifications to quinolone core molecule. 68 904091_TRI_Symp_68_73_Olsen.qxp 04.06.2009 8:36 Uhr Seite 69 Joy Olsen, DVM Global Veterinary Services Bayer Animal Health GmbH, Germany Enrofloxacin is structurally similar to ciprofloxacin, differing in the addition of an ethyl group on the piperazinyl ring, which enhances the absorption of enrofloxacin as compared to ciprofloxacin.4 Ciprofloxacin has been reported to have enhanced potency against organisms such as Pseudomonas aeruginosa. However, as will be later highlighted, enrofloxacin is partially metabolized to ciprofloxacin in many species, notably dogs.5 Low bioavailability following oral administration of ciprofloxacin has also been demonstrated in cats; a pharmacokinetic study in 2004 reported oral bioavailability of only 33 % in cats, with considerable differences between the drug exposure (AUC) observed with IV administration and that of oral administration (of pure drug substance).13 A highly active metabolite Bioavailability While the structural enhancements of the fluoroquinolones dramatically improved bioavailability in general, oral bioavailability may vary significantly both among different species and also between different fluoroquinolone drugs. Many compounds are characterized by favorable bioavailability with oral administration in monogastric animals, however, ciprofloxacin has been shown to have poor and variable oral bioavailability in dogs6–8 in contrast to that of enrofloxacin, which approaches 100 %.9 Underscoring the fact that dogs are not “small furry humans”, ciprofloxacin’s oral bioavailability is approximately 70 % in humans,10 whereas that reported in dogs is roughly 40 %.7,8 Mean peak serum concentrations (Cmax) of enrofloxacin (Baytril tablets) in dogs were four-fold higher than those of ciprofloxacin (Ciprobay tablets) following administration of each drug at a dosing rate of 5 mg/kg.11 Additionally, differences in rates of absorption may be seen as the dosage of ciprofloxacin is increased.7 Laboratory studies involving administration of radio-labeled ciprofloxacin [14C] to rats, dogs, and monkeys observed that following oral administration, ciprofloxacin was partially absorbed and achieved only 30– 40 % of the area under the serum concentration versus time curves (AUCs) of that obtained with intravenous dosing, indicating low overall exposure.12 A prominent unique pharmacologic feature of enrofloxacin is that it is partially metabolized in vivo by hepatic N-dealkylation to ciprofloxacin, a highly potent metabolite which contributes to overall antibacterial activity.9,14–16 This transformation has been documented in a variety of domestic animal species including dogs,9,16 cats,17,18 adult horses,19 sheep20, and cattle21. Pharmacokinetic studies in a diverse array of exotic animals have also identified additional species in which metabolism to ciprofloxacin occurs to a considerable extent, such as Burmese pythons,22 tortoises,22 rhesus monkeys,23 rabbits24, and psittacine birds,25 to name just a few examples. The resulting amount of ciprofloxacin conversion varies among species; therapeutic levels exceeding minimum inhibitory concentrations of relevant pathogens are produced in many animals following administration of enrofloxacin.8 Cester and Toutain conducted a pharmacokinetic study using a comprehensive model to quantitatively determine enrofloxacin to ciprofloxacin transformation in dogs following oral and IV administration.16 Their results indicated that approximately 40 % of enrofloxacin was metabolized to ciprofloxacin following both routes of administration. This conversion contributes to both the magnitude and duration of antimicrobial drug exposure.26 Additional pharmacokinetic investigations have demonstrated significant concentrations of cipro69 904091_TRI_Symp_68_73_Olsen.qxp 04.06.2009 8:36 Uhr Seite 70 Proceedings of the 4th International Baytril® Symposium Unique Pharmacologic Characteristics of Baytril® Joy Olsen fore, each square (representing one well) in the checkerboard configuration contains a unique combination of the two compounds.35 Drug concentrations encompassing a range of multiples (and fractions) of the minimum inhibitory concentration(s) (MIC) in serial two-fold dilutions are used. Each checkerboard plate also contains a row and a column in which a serial dilution of each respective agent alone is present. Bacteria are added to the test wells and, following incubation, observation for visible growth is done to determine the lowest combined inhibitory concentrations. floxacin in numerous tissues and body fluids following administration of enrofloxacin, including urine,27 alveolar macrophages and respiratory epithelial lining fluid,28 prostate gland,29 peripheral leukocytes,30 dermal tissues31, and bone32. Additive activity Enrofloxacin and its microbiologically active metabolite ciprofloxacin share the same mechanism of action, rapidly killing bacteria in a concentration-dependent manner. For this reason, in vitro studies have investigated what the interaction of enrofloxacin and ciprofloxacin is against various small animal pathogens.33,34 In these particular studies, a checkerboard assay method was used to ascertain the interaction and antibacterial activity of the combination of the two compounds. The “checkerboard” consists of a pattern of microtiter wells with multiple dilutions of the two drugs tested, such that each row (and column) contains a fixed amount of one drug and increasing amounts of the second drug. There- Pirro et al. investigated enrofloxacin and ciprofloxacin interactions on clinical isolates and reference strains of Staphylococcus intermedius, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Pasteurella multocida; a total of 56 Gram-negative and Gram-positive strains were included.33 Figure 2 illustrates an example of a checkerboard assay from this study for a strain of P. aeruginosa. For each combination experiment, the fractional inhibitory concentration (FIC) of ciprofloxacin enrofloxacin FIC 4 2 1 (MIC) 1/2 1/4 1/8 1/16 1/32 1/64 1/128 µg/ml 2.0 1.0 0.5 0.25 0.125 0.063 0.031 0.015 0.008 0.004 0 2 4.0 - - - - - - - - - - - ++ 1 (MIC) 2.0 - - - - - - - - - - - ++ 1/2 1.0 - - - - + + ++ ++ ++ ++ ++ ++ 1/4 0.5 - - - - ++ ++ ++ ++ ++ ++ ++ ++ 1/8 0.25 - - - - ++ ++ ++ ++ ++ ++ ++ - 1/16 0.125 - - - - ++ ++ ++ ++ ++ ++ ++ - 1/32 0.063 - - - ++ ++ ++ ++ ++ ++ ++ ++ - 0 - - - ++ ++ ++ ++ ++ ++ ++ ++ - growth control Figure 2 Checkerboard combination of enrofloxacin and ciprofloxacin with P. aeruginosa ATCC 27853. –: no visuable growth +: reduced growth ++: complete growth 70 (Adapted from Pirro et al. 1997, 14th Congress ESVD-ECVD) 904091_TRI_Symp_68_73_Olsen.qxp 04.06.2009 8:36 Uhr Seite 71 each drug was determined by dividing the inhibitory concentration when used in combination by the MIC of the agent alone, for example: FICenr = Cenr/MICenr. The FIC index (FICI) is the sum of FICs for the two compounds and defines the nature of the interaction. Criteria used for the assessment of the FIC indices in this particular study were as follows: synergistic interactions FICI of ≤ 0.5; additive interactions 0.5 < FICI ≤ 1; antagonism FICI > 2; and indifference 1 < FICI ≤ 2. Figure 3 is a plot of median FICs for some reference strains that graphically depicts the classification of the interactions. More recent investigations have used criteria of FICI > 4 to indicate antagonism,35 and FICI between 0.5 and 4 classification as additive.34 The FIC indices for all reference strains and field strains in this study were in the range of 0.5 to 1, clearly demonstrating additive inhibitory effects of the combination on all bacterial species tested. molytic, coagulase-positive Staphylococcus isolates cultured from dogs treated for dermal infectious, otitis externa, cystitis, pyometra, and respiratory tract infections; a total of 50 isolates of E. coli and 50 staphylococci were included.34 The results of MIC testing indicated that ciprofloxacin and enrofloxacin had equivalent potency against E. coli and staphylococci, and the results of the checkerboard assay again revealed that the compounds acted in an additive fashion against these bacteria. Both of the above studies demonstrated that the combination of enrofloxacin and ciprofloxacin in vitro has additive inhibitory effects on major Gram-negative and Gram-positive pathogens. Therefore, the in vivo transformation of enrofloxacin to ciprofloxacin may enhance the overall antibacterial activity. This activity is not reflected with traditional culture and susceptibility testing with enrofloxacin as it cannot account for presence of the highly active metabolite.26 A subsequent study reported in 2001 evaluated the in vitro interaction between enrofloxacin and ciprofloxacin against Escherichia coli and beta-he1 C enr C cip FICI = —–—— + —–—— MIC enr MIC cip FIC enrofloxacin 0.75 E. coli: P. multocida: P. aeruginosa: S. intermedius: E. coli ATCC 10536 FICI = 0.56 0.5 P. multocida DSM 5281 FICI = 0.625 0.25 P. aeruginosa ATCC 27853 FICI = 0.56 S. intermedius ATCC 29663 FICI = 0.53 0 0 0.25 0.5 0.75 0.5 0.5 0.06 0.03 + 0.06 + 0.125 + 0.5 + 0.5 = = = = 0.56 0.625 0.56 0.53 synergism addition indifference 1 FIC ciprofloxacin Figure 3 Median FICs of different reference strains. (Adapted from Pirro et al. 1997, 14th Congress ESVD-ECVD) 71 904091_TRI_Symp_68_73_Olsen.qxp 04.06.2009 8:36 Uhr Seite 72 Proceedings of the 4th International Baytril® Symposium Unique Pharmacologic Characteristics of Baytril® Joy Olsen Summary Over the past two decades, a substantial body of scientific and clinical research has been compiled on enrofloxacin, making it one of the most ex- tensively evaluated drugs in veterinary medicine. Despite the advent of additional compounds in the veterinary field, Baytril remains unique in both its pharmacology and breadth of scientific support and clinical applications. References 1. Emmerson AM, Jones AM (2003). The quinolones: decades of development and use. J Antimicrob Chemother; 51(Suppl S1): 13–20. 2. Tillotson G (1996). Quinolones: structure-activity relationships and future predictions. J Med Microbiol; 44: 320–324. 3. Martinez M, McDermott P, Walker R (2006). Pharmacology of the fluoroquinolones: A perspective for the use in domestic animals.Vet J; 172: 10–28. 4. Walker, RD (2000). Fluoroquinolones. In: Antimicrobial therapy in veterinary medicine (Eds: Prescott JF, Baggot JD, Walker RD), 3rd edn., Iowa State University Press, Ames, Iowa; pp. 315–338. 5. Tychowska K, Hedeen KM, Aucoin DP, Aronson AL (1989). High performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue. J Chromatogr; 493: 337–346. 6. McKellar QA (1996). Clinical relevance of the pharmacologic properties of fluoroquinolones. Suppl Comp Cont Educ Pract Vet; 18(2): 14–21. 7. Abadia AR, Aramayona JJ, Munoz MJ, Pla Delfina JM, Bregante MA (1995). Ciprofloxacin pharmacokinetics in dogs following oral administration. J Vet Med A; 42: 505–511. 8. Boothe DM, Boeckh A, Simpson RB, Dubose K (2006). Comparison of pharmacodynamic and pharmacokinetic indices of efficacy for 5 fluoroquinolones toward pathogens of dogs and cats. J Vet Intern Med; 20: 1297–1306. 9. Küng K, Riond JL, Wanner M (1993). Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther; 16: 462–468. 72 10. Drusano GL, Standiford HC, Plaisance K, Forrest A, Leslie J, Caldwell J (1986). Absolute oral bioavailability of ciprofloxacin. Antimicrob Agents Chemother; 30(3): 444– 446. 11. Heinen E (1999). Comparative pharmacokinetics of enrofloxacin and difloxacin as well as their main metabolites in dogs. Suppl Comp Cont Educ Pract Vet; 21(12): 12–18. 12. Dalhoff A, Bergan T (1998). Pharmacokinetics of fluoroquinolones in experimental animalsanimals. In: Quinolone antibacterials (Eds: Kuhlmann J, Dalhoff A, Zeiler H-J), Springer, Berlin; pp. 179–206. 13. Albarellos GA, Kreil VE, Landoni MF (2004). Pharmacokinetics of ciprofloxacin after single intravenous and repeat oral administration to cats. J Vet Pharmacol Ther; 27: 155–162. 14. Tychowska K, Hedeen KM, Aucoin DP, Aronson AL (1989). High performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue. J Chromatogr; 493: 337–346. 15. Küng K, Riond JL,Wolffram S,Wanner M (1993). Comparison of an HPLC and bioassay method to determine antimicrobial concentrations after intravenous and oral administration of enrofloxacin in four dogs. Res Vet Sci; 54: 247–248. 16. Cester CC,Toutain PL (1997). A comprehensive model for enrofloxacin to ciprofloxacin transformation and disposition in dogs. J Pharm Sci; 86(10): 1148–1155. 17. Richez P, Monlouis JD, Dellac B, Daube G (1997).Validation of a therapeutic regimen for enrofloxacin in cats of the basis of pharmacokinetic data. J Vet Pharmacol Ther; 20(Suppl 1): 152–153. 904091_TRI_Symp_68_73_Olsen.qxp 04.06.2009 8:36 Uhr Seite 73 18. Seguin MA, Papich MG, Sigle KJ, Gibson NM, Levy JK (2004). Pharmacokinetics of enrofloxacin in neonatal kittens. Am J Vet Res; 65(3): 350–355. 19. Papich MG, Van Camp SD, Cole JA, Whitacre MD (2002). Pharmacokinetics and endometrial tissue concentrations of enrofloxacin and the metabolite ciprofloxacin after i.v. administration of enrofloxacin to mares. J Vet Pharmacol Ther; 25: 343–350. 20. Mengozzi G; Intorre L; Bertini S; Soldani G (1997). Pharmacokinetics of Enrofloxacin and Its Metabolite Ciprofloxacin after Intravenous and Intramuscular Administrations in Sheep. Am J Vet Res; 57(7): 1040–1043. 21. De Lucas JJ, San Andrés MI, González F, Froyman R, Rodríguez C (2008). Pharmacokinetic behaviour of enrofloxacin and its metabolite ciprofloxacin after subcutaneous administration in cattle.Vet Res Commun; 32(4): 275–279. 22. Papich MG (1999). Pharmacokinetics of enrofloxacin in reptiles. Suppl Comp Cont Educ Pract Vet; 21(12): 110–114. 23. Klein H, Hasselschwert D, Handt L, Kastello M (2008). A pharmacokinetic study of enrofloxacin and its active metabolite ciprofloxacin after oral and intramuscular dosing of enrofloxacin in rhesus monkeys (Macaca mulatta). J Med Primatol; 1–7. 24. Elmas M, Yazar E, Bas AL, Tras B, Bayezit M, Yapar K (2002). Comparative pharmacokinetics of enrofloxacin and tissue concentrations of parent drug and ciprofloxacin after intramuscular administrations of free and liposome-encapsulated enrofloxacin in rabbits. J Vet Med; 49: 507–512. 25. Flammer K, Aucoin DP, Whitt DA (1991). Intramuscular and oral disposition of enrofloxacin in African grey parrots following single and multiple doses. J Vet Pharmacol Ther; 14: 359–356. 26. Boothe DM, Boeckh A, Boothe HW, Wilkie S, Jones S (2002). Plasma concentrations of enrofloxacin and its activie metabolite ciprofloxacin in dogs following single oral administration of enrofloxacin at 7.5, 10 or 20 mg/kg.Vet Ther; 34(4): 419. 27. Monlous JD, De Jong A, Limet A, Richez P (1997). Plasma pharmacokinetics and urine concentrations of enrofloxacin after oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther; 20(Suppl 1): 61–63. 28. Hawkins EC, Boothe DM, Guinn A, Aucoin DP, Ngyuen J (1998). Concentration of enrofloxacin and its active metabolite in alveolar macrophages and pulmonary epithelial lining fluid of dogs. J Vet Pharmacol Ther; 21: 18–23. 29. Dorfman M, Barsanti J, Budsberg SC (1995). Enrofloxacin concentrations in dogs with normal prostate and dogs with chronic bacterial prostatitis. Am J Vet Res; 56(3): 386–390. 30. Boeckh A, Booth D, Wilkie S, Jones S (2001).Time course of enrofloxacin and its active metabolite in peripheral leukocytes of dogs.Vet Ther; 2(4): 334–344. 31. Cole LK, Papich MG, Kwochka KW, Hillier A, Smeak DD, Lehman AM (2009). Plasma and ear tissue concentrations of enrofloxacin and its metabolite ciprofloxacin in dogs with chronic end-stage otitis externa after intravenous administration. Vet Dermatol; 20(1):51–59. 32. Ehinger AM, Meyer-Lindenberg A, Vick M, Nolte I, Kietzmann M (2002). Concentration of enrofloxacin and its metabolite ciprofloxacin in canine matrices of the locomotor system. J Vet Med; 49: 99–104. 33. Pirro F, Scheer M, De Jong A (1997). Additive in vitro activity of enrofloxacin and its main metabolite ciprofloxacin. Proceedings 14th Annual Congress of the ESVDECVD; p. 199. 34. Lautzenhiser SJ, Fialkowski JP, Bjorling D, Rosin E (2001). In vitro antibacterial activity of enrofloxacin and ciprofloxacin in combination against Escherichia coli and staphylococcal clinical isolates in dogs. Res Vet Sci; 70: 239–241. 35. Eliopoulos GM, Moellering RC (1996). Antimicrobial combinations. In: Antibiotics in laboratory medicine (Ed: Lorian V), Williams & Wilkens, Baltimore, MD; pp. 330– 396. 73 904091_TRI_Symp_Daube.qxp 03.06.2009 11:53 Uhr Seite 74 Pharmacokinetics – What You Need to Know Pharmacokinetic variables describe absorption, distribution, metabolism, and elimination (ADME) of a drug. This short overview will focus on the most important topics veterinary practitioners need to know about the pharmacokinetic behavior of antimicrobial drugs with special focus on fluoroquinolones, such as enrofloxacin. Antimicrobials used in companion animal medicine generally are administered via the oral route, although parenteral formulations are also available. Absorption After oral administration, most drugs are absorbed in the proximal gastrointestinal tract. Systemic availability, i.e., the rate and extent to which a drug appears in the circulation, is usually determined by measurement of the concentration of the active ingredient in plasma or serum as a function of time. Ideally, a comparison of plasma concentrations after oral application and injection of an equivalent intravenous dose should be made. This will allow the determination of the absolute systemic exposure (bioavailability). Uptake into the circulation is dependent on the disintegration of the oral formulation, liberation of the active substance, dissolution in the gastrointestinal fluid, intestinal blood flow, molecular size, lipophilic properties, ionization of the drug, and, finally, the pH value at the site of absorption.1 Enrofloxacin possesses a carboxylic acid in position 3 and a basic amine functional group in position 1 of the molecule, making the compound amphoteric and zwitterionic. At a pH range of the medium between 6 and 8, the drug is sufficiently lipophilic to be well absorbed and to penetrate cell membranes, resulting in high intracellular concentrations.2 Following oral administration, the intestinal absorption of the fluoroquinolones 74 is rapid and nearly complete. The oral bioavailability of enrofloxacin is high with almost 100 % in dogs and 95 % in cats,2 and plasma concentrations after oral and subcutaneous administration are nearly identical.3 Absorption of the drug is best after administration on an empty stomach.4 Distribution Drug concentration-time curves describe the behavior of a drug in serum or plasma and represent the basis of subsequent pharmacokinetic analysis. The highest concentration achieved in the reference compartment is denominated Cmax, whereas Tmax is the time interval between application and peak concentration.These two parameters can be directly read from the curve. AUC, the area under the concentration-time curve, is calculated by means of an integral function. It is directly proportional to the amount of drug in the organism and thus is a measure of the total exposure to the drug. AUClast is based on the time interval between application and time of the last measurable concentration, whereas AUCinf represents the extrapolated area from time zero to infinity5 (Fig. 1). Cmax AUC Concentration PK Parameters 0 24 time (h) Tmax Figure 1 Concentration-time curve with Cmax, Tmax, AUC. 904091_TRI_Symp_Daube.qxp 03.06.2009 11:53 Uhr Seite 75 Gert Daube, Dr med vet · Sandra Mensinger · Bernd Stephan, Dr med vet Clinical Research and Development Antiinfectives Bayer Animal Health GmbH, Germany Parameter Doga Cat Maximum serum concentration Cmax (µg/l)b 1,711 2,454 1 0.83 6,291 29,376 Volume of distribution Vdssb 3.7 4.0 Half-life T1/2 (h)b 3.24 8.86 Protein binding (%) < 30 – 95 100 Time of maximum serum concentration Tmax (h)b Area under the curve AUClast (µg h/l)b Bioavailability, oral (%) Table 1 Pharmacokinetic parameters of Baytril after oral administration of tablets at 5 mg/kg BW. a Sum of concomitant enrofloxacin and ciprofloxacin concentrations; For optimum clinical efficacy, aminoglycosides and fluoroquinolones should achieve high Cmax values. For these drugs, the rate and extent of bacterial killing and the clinical efficacy increases, as the concentration of the drug increases. Members of this group are therefore denominated concentration- or dose-dependent antibiotics.6 After administration of Baytril tablets, the mean serum peak concentration in cats and dogs was 2,454 µg/l and 1,711 µg/l, respectively (Tab. 1). For other classes of antimicrobials, e.g., beta-lactams, sufficiently high drug concentrations have to be maintained above the minimum inhibitory concentration of the pathogen for almost the entire dose-to-dose interval.This group is therefore denominated as time-dependent antibiotics.6 b Daube et al. 2007, unpublished data thetical volume of body fluid that would be necessary to accommodate the whole amount of drug based on the concentration actually measured in serum or plasma7 (Fig. 2). A Vd of above 0.6 l/kg is generally regarded as indicator of intracellular penetration.1 In pharmacokinetic studies with enrofloxacin in cats and dogs, a Vd of 4.0 and 3.7 l/kg was calculated, suggesting an excellent penetration of the drug into the target tissues.8 This has been confirmed by numerous publications on high tissue concentrations after administration of Baytril, not only in readily accessible target organs, but also in skin, bones, inflamed tissues, and phagocytic cells. Vd = Distribution of a drug into the tissues is dependent on its lipophilic properties, molecular size, ionization, the permeability of membranes, blood perfusion, pH values at the infection site, and plasma protein binding of the drug.1 An indicator of the distribution within the organism is the volume of distribution (Vd).Vd does not represent any physical or anatomical structure, but a hypo- Total amount of drug administered Concentration in plasma (l/kg) Vd = 0.05 l/kg – Distribution in plasma Vd = 0.2 l/kg – Distribution in extracellular space Vd = 0.6 l/kg – Distribution in extra- and intracellular space Vd > 0.6 l/kg – Accumulation in tissues Figure 2 Volume of Distribution Vd . 75 904091_TRI_Symp_Daube.qxp 03.06.2009 11:53 Uhr Seite 76 Proceedings of the 4th International Baytril® Symposium Pharmacokinetics – What You Need to Know Gert Daube · Sandra Mensinger · Bernd Stephan Distribution of a substance also is influenced by its protein binding. Drugs bound to plasma or tissue proteins do not have antimicrobial activity, cannot cross barriers, nor can they be metabolized or eliminated. Protein binding is strongly influenced by animal species, age, disease, and nutritional status (e.g., cachectic animals).1 However, binding in general is reversible following a concentration gradient. In dogs, less than 30 % of enrofloxacin is bound to plasma proteins.Therefore, it is considered to have low protein binding.8 Metabolism In general, substances undergoing metabolism are inactivated, detoxified, and prepared for excretion. Enrofloxacin is dealkylated to ciprofloxacin, a compound with similar antimicrobial activity.8 In dogs, around 40 % of enrofloxacin is transformed to ciprofloxacin,9 whereas in cats the percentage of the metabolite in serum is much lower. Elimination and excretion Further pharmacokinetic parameters routinely provided include the elimination half-life (T1/2), clearance (Cl), and Mean Residence Time (MRT). Half-life is the time period during which the concentration of a drug is reduced to half of its initial value. T1/2 has some influence on possible drug accumulation and is therefore important for estimation of the dosing interval.10 The term clearance describes the rate of excretion of a drug. In the majority of cases, the clearance is determined in plasma, as this matrix can easily be obtained. It represents the volume of body fluid which is completely cleared of a substance per time interval by an elimination process. Clearance can also be defined for each elimination organ, for example renal or hepatic clearance. Total body clearance is the sum of all organ clear76 ances. Half-life strongly depends upon the Cl and Vd parameters.11 Mean Residence Time represents the average time a drug molecule remains in the circulation, between (intravenous) administration and elimination from the central compartment.1 Fluoroquinolones are excreted via the bile after conjugation with glucoronic acid, and via the urine mainly by glomerular filtration. In dogs, 70 % of enrofloxacin is excreted in the bile and around 30 % in urine.2 Due to the renal concentration process, however, a sufficiently high amount of active drug is present in the urine. In the intestines, segregation of glucuronic acid from the fluoroquinolone molecule by bacterial enzymes subsequently leads to re-absorption thereby enabling enterohepatic re-circulation of the drug.1 PK/PD relations – what do they mean? The aim of each antimicrobial therapy should not only be the clinical cure of the patient, but also elimination of the target pathogens in order to minimize the selection of resistant bacteria. In the case of incomplete eradication, less susceptible bacterial subpopulations may develop and lead to therapeutic failure. Therefore PK/PD calculations are conducted, whereby pharmacokinetic parameters are linked to pharmacodynamic processes.12 They enable a rough estimation of the in vivo antimicrobial effect to be expected and offer the possibility of a prospective dose setting for patients in clinical studies (Fig. 3). For drugs with concentration-dependent antimicrobial activity, e.g., aminoglycosides and fluoroquinolones, the parameters used for PK/PD calculations (Cmax and AUC0-24) are derived from dose linearity studies in healthy animals.The data are subsequently linked to the in vitro minimum inhibitory concentration (MIC90) of each target 904091_TRI_Symp_Daube.qxp 03.06.2009 11:53 Uhr Seite 77 Pharmacokinetics Pharmacodynamics Dose Concentration Concentration Effect PK / PD Dose Effect Figure 3 PK/PD relationships. pathogen by simple division. In 1993, Forrest et al.13 proposed that the ratios of Cmax/MIC ≥ 10 or AUC0-24/MIC ≥ 125 should be considered as predictors of clinical efficacy, based on observations in critically ill human patients in intensive care units. Most of these patients suffered from Gram-negative infections, predominantly caused by Pseudomonas aeruginosa. Meanwhile it has become evident that these values first proposed more than 15 years ago are highly conservative and generally relate to Gram-negative organisms only. Critical review of several experimental studies resulted in the finding that the magnitude of the PK/PD ratios necessary for successful therapy is reduced in animal models due to the presence of neutrophils, as antimicrobial drugs and immune mechanisms act in concert.14–17 Today, most data from controlled animal studies suggest that an AUC0-24/MIC ratio of ≥ 40 is sufficient to predict clinical efficacy when Gram-positive infection is present.14 In case of Gram-negative infections, an AUC0-24/MIC ratio above 125 is now regarded as conservative, as ex vivo experiments in bovines and studies in cats have also shown good results with ratios lower than initially proposed.18,19 Pharmacokinetics and bioequivalence Registration of generic products in general is based on the proof of bioequivalence between the pioneer approved product and the generic copy. Bioequivalence studies are statistically based comparisons of two drug formulations to demonstrate that both are interchangeable.The basic assumption underlying the bioequivalence concept is that essentially similar drug concentration-time courses in the circulation lead to essentially similar pharmacological and therapeutic effects. Most bioequivalence studies are conducted in vivo under good laboratory practice (GLP) requirements on a homogenous group (age, breed, gender) of healthy animals of the target species. The reference product usually is the pioneer approved product with the most extensive dossier. In bioequivalence studies in general, each product is administered as a single dose within a complete cross-over design, so that each animal serves as its own control.The blood sampling schedule has to be adapted to the pharmacokinetic characteristics of the products, so that absorption, distribution, and elimination phases are properly represented. The serum- or plasma-analytic procedure needs to 77 904091_TRI_Symp_Daube.qxp 03.06.2009 11:53 Uhr Seite 78 Proceedings of the 4th International Baytril® Symposium Pharmacokinetics – What You Need to Know Gert Daube · Sandra Mensinger · Bernd Stephan follow recognized standards. Bioequivalence calculation today is done with computer programs, e.g., WinNonlin®, Pharsight Corp., Mountain View, CA, USA. In general, the maximum serum concentration Cmax and the area under the concentration-time curve (AUClast) of the originally approved product and the generic are compared to each other after log-transformation. The 90 % confidence interval of the ratios of Cmax reference product/Cmax test product and AUClast reference product/AUClast test product must then be entirely contained within the reference limits of 80–125 %. Due to the high variability of Cmax values, a wider range of limits may be acceptable, if sufficiently justified in advance20 (Fig. 4). Thus, by means of a bioequivalence study only, generic copies of an original product can officially be licensed without generating much more than basic pharmacokinetic data. Additionally, this bioequivalence is granted across a range of 45 % variability compared to the original substance. In contrast, the entire body of information on pharmacokinetics, pharmacodynamics, safety, toxicology, and clinical efficacy laid down in dossiers or reported in the literature was generated using the original product. + 25 % Deviation* generic 100 % Original product Plasma concentration Cmax – 20 % Deviation* generic Original product Generic copy Figure 4 Bioequivalence between original and generic copy. 78 * Deviation calculated from AUC generic/ AUC original Time 904091_TRI_Symp_Daube.qxp 03.06.2009 11:53 Uhr Seite 79 References 1. Kietzmann M (2008). Richtlinien für die Durchführung pharmakokinetischer Studien in der Entwicklung von Tierarzneimitteln. Proceedings „Seminar Pharmakokinetische Studien bei Tierarzneimitteln“, Hannover; pp. 1–29. 12. Bäumer W (2008). Vergleich von Formulierungen, Bioverfügbarkeit, Bioäquivalenz. Proceedings „Seminar Pharmakokinetische Studien bei Tierarzneimitteln“, Hannover; pp. 70–84. 2. Brown SA (1996). Fluoroquinolones in animal health. J Vet Pharmacol Ther; 19:1–14. 13. Forrest A, Nix DE, Ballow CH, Goss TF, Birmingham MC, Schentag JJ (1993). Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrob Agents Chemother; 37: 1073–1081. 3. Kroker R (2006). Pharmaka zur Behandlung und Verhütung bakterieller Infektionen. In: Pharmakotherapie bei Haus- und Nutztieren (Eds: Löscher W, Ungemach FR), Parey Verlag, Stuttgart; pp. 234–258. 4. Küng K,Wanner M (1993). Einfluss zweier verschiedener Futter auf die Pharmakokinetik von oral appliziertem Baytril® (Enrofloxacin) beim Hund. Kleintierpraxis 38: 95–102. 5. Pabst G (2003). The area under the concentration-time curve. In: Parameters for compartement-free pharmacokinetics (Ed: Cawello), Shaker Verlag, Aachen; pp. 65–80. 6. Meinen JB, McClure JT, Rosin E (1995). Pharmacokinetics of enrofloxacin in clinically normal dogs and mice and drug pharmacodynamics in neutropenic mice with Escherichia coli and staphylococcal infections. Am J Vet Res 56: 1219–1224. 7. Cawello W (2003).The physiological basis of pharmacokinetics. In: Parameters for compartement-free pharmacokinetics (Ed: Cawello W), Shaker Verlag, Aachen; pp. 7–22. 8. Kietzmann M (1999). Overview of the pharmacokinetic properties of fluoroquinolones in companion animals. Proceedings of the Third International Veterinary Symposium on Fluoroquinolones. Suppl Comp Cont Educ Pract Vet; 21(12): 7–11. 9. Cester CC, Toutain PL (1997). A comprehensive model for enrofloxacin to ciprofloxacin transformation and disposition in dog. J Pharm Sci; 86(10): 1148–1155. 14. Wright DH, Brown GH, Peterson ML, Rotschafer JC (2000). Application of fluoroquinolone pharmacodynamics. J Antimicrob Chemother; 46: 669–683. 15. Nicolau DP (1999). Using pharmacodynamic and pharmacokinetic surrogate markers in clinical practice: Optimizing antimicrobial therapy in respiratory-tract infections. Am J Health Syst Pharm; 56(3): S16–S20. 16. Dudley MN, Ambrose PG (2000). Pharmacodynamics in the study of drug resistance and establishing in vitro susceptibility breakpoints: ready for prime time. Curr Opin Microbiol; 3: 515–521. 17. Ambrose PG, Grasela DM, Grasela T, Passarell J, Mayer HB, Pierce PF (2001). Pharmacodynamics of fluoroquinolones against Streptococcus pneumoniae in patients with community-acquired respiratory tract infections. Antimicrob Agents Chemother; 45: 2793–2797. 18. Lees P, Aliabadi FS (2002). Rational dosing of antimicrobial drugs: animals versus humans. Int J Antimicrob Agents; 19: 269–284. 19. Coulet M, Cox P, Lohuis J (2005). Pharmacodynamics of ibafloxacin in microorganisms isolated from cats. J Vet Pharmacol Ther; 28: 29–36. 20. Guideline for the conduct of Bioequivalence Studies for Veterinary Medicinal Products, EMEA/CVMP/016/00corr-Final, 11. Juli 2001. 10. Gieschke R (2003). Half-life. In: Parameters for compartement-free pharmacokinetics (Ed: Cawello W), Shaker Verlag, Aachen; pp. 39–64. 11. Toutain PL, Bousquet-Melou A (2004). Plasma clearance. J Vet Pharmacol Ther; 27: 415–425. 79 904091_TRI_Symp_Hehnen.qxp 03.06.2009 12:03 Uhr Seite 80 Baytril® Resistance Monitoring – Susceptibility Status after More Than 20 Years Introduction Baytril® has been successfully marketed for more than 20 years in most European countries. Several oral and parenteral formulations are available for the treatment of infectious diseases in both companion animals and livestock. Excellent pharmacokinetic properties and high bactericidal activity against a broad spectrum of aerobic Gram-negative and Gram-positive bacteria as well as mycoplasmas make it highly suitable for the treatment of serious bacterial infections.To ensure the long-term efficacy of enrofloxacin, the active ingredient of Baytril, knowledge on the ongoing susceptibility status of bacterial pathogens is important. Bayer has established several susceptibility monitoring programs, starting in 1992, involving veterinary pathogens as well as zoonotic and commensal bacteria. In all, more than 40,000 individual bacterial strains have been collected up to this time, comprising target pathogens (> 20,000) and food-borne bacteria (22,000) such as Salmonella spp. Regarding pathogens of dogs and cats, more than 12,000 isolates have been collected, the majority from dogs. Additionally, several external monitoring surveys have been organized by Bayer Animal Health. In the last decade, various national resistance monitoring programs have been initiated (e.g., CIPARS, DANMAP, MARAN, NARMS, SVARM) which include a wide variety of bacterial pathogens from food-producing and companion animals. However, the focus of these programs is on resistance monitoring of zoonotic/commensal organisms, and relatively few pathogens from companion animals have been collected. One exception is the German GERM-Vet program which was initiated in 2001 by the German Federal Office of Consumer Protection and Food Safety (BVL), supplemented by an industry association (BfT) sponsored project (BfT-GermVet), which focused on pathogens 80 from both livestock and companion animals (Schwarz et al. 2007a). The current susceptibility status for enrofloxacin with respect to major pathogens relevant for dogs and cats is presented here. Results of the Bayer monitoring programs are compared to recent literature data, particularly that of the BfT-GermVet monitoring program. Materials and methods Two different European programs have been conducted by Bayer Animal Health. In one program starting in 1992, samples were taken from four major clinical indications in three large regions of Germany. These regions (South, Central, North) are considered representative of Germany. In this survey it was not possible to collect historic data and it is assumed that in many cases the patients had received antimicrobial treatment prior to sampling. In the second program, samples of the same four indications have been collected from 2003 onwards in various veterinary practices from animals not recently exposed to antimicrobial treatment. Four countries (Hungary, Poland, Sweden, United Kingdom) were involved. Bacterial isolates were recovered by local diagnostic laboratories and transferred to Bayer Animal Health laboratories in Monheim am Rhein, Germany. The isolates were identified by colony morphology, Gram stain, and biochemical tests (e.g., API). The minimum inhibitory concentration (MIC) to enrofloxacin was determined by agar dilution according to the principles of the Clinical and Laboratory Standards Institute (CLSI; M31-A3, 2008). MIC50, MIC90, and MIC90s values as well as the percentage of resistance were calculated for each bacterial species, divided into different indications if data from a sufficient number of strains was collected. 904091_TRI_Symp_Hehnen.qxp 03.06.2009 12:03 Uhr Seite 81 Hans-Robert Hehnen, Dr med vet · Sonja M. Friederichs, Dr rer nat Julia C. Heimbach · Anno de Jong, Dr · Bernd Stephan, Dr med vet Clinical Research and Development Antiinfectives Bayer Animal Health GmbH, Germany According to Guideline EMEA/CVMP/627/ 01-FINAL, the MIC90 should be determined against the susceptible population reporting the percentage of resistant strains separately, where field isolates show a bimodal or multimodal distribution distinguishing susceptible and resistant isolates.The MIC90s is thus defined as the MIC90 of the susceptible bacterial population. To differentiate between enrofloxacin resistant and susceptible strains, the CLSI breakpoint of 4 µg/ml was used throughout all calculations although CLSI has not confirmed this value for each bacterial species and indication yet. Escherichia coli strain ATCC 25922 was used as quality control strain. n = 12,032 E. coli Staphylococci Pseudomonas Proteus Pasteurella Salmonella Bordetella Klebsiella Others Streptococci Enterococci Figure 1 Distribution of canine and feline bacterial pathogens sampled from 1992 onwards in the German Bayer monitoring program. Clinical indication Bacterial species Number of isolates MIC50 (µg/ml) MIC90 (µg/ml) MIC90s (µg/ml) Resistance (%) Respiratory tract Bordetella bronchiseptica 357 0.5 1 1 2.5 E. coli 342 0.03 32 0.125 15 P. multocida 289 0.015 0.03 0.03 0 Staphylococcus spp. 251 0.125 0.25 0.125 4 E. coli 1,607 0.03 16 0.06 15 Klebsiella spp. 127 0.125 64 0.25 39 Proteus spp. 230 0.25 8 0.25 21 P. aeruginosa 238 1 16 2 21 P. multocida 126 0.015 0.03 0.03 0 Proteus spp. 226 0.25 8 0.5 17 P. aeruginosa 611 1 16 2 28 Staphylococcus spp. 1,638 0.125 0.5 0.125 4 E. coli 821 0.03 16 0.06 16 Urinary/genital tract Skin/ear/mouth Gastrointestinal tract Table 1 Antimicrobial susceptibility to enrofloxacin of canine and feline pathogens from the German Bayer monitoring program (2000–2008 subset; n = 6,863). Results and Discussion In total, 12,000 isolates were investigated encompasing the most important bacterial diseases of dogs and cats in the German Bayer program. The frequency of recovery of the pathogens is depicted in Figure 1. The antimicrobial suscepti- bility to enrofloxacin for a subset of the collection (2000–2008) is summarized in Table 1 and includes MIC50 and MIC90 values as well as the prevalence of resistance. The MIC50 and MIC90s values underline the high susceptibility of the major key pathogens to enrofloxacin. Prevalence of resistance varied from 0 to 39 %; the resistance 81 904091_TRI_Symp_Hehnen.qxp 03.06.2009 12:03 Uhr Seite 82 Proceedings of the 4th International Baytril® Symposium Baytril® Resistance Monitoring – Susceptibility Status after More Than 20 Years Hans-Robert Hehnen · Sonja M. Friederichs · Julia C. Heimbach · Anno de Jong · Bernd Stephan level was notable for strains from infections of the urinary and genital tract. It should be noted, however, that collecting samples in diagnostic laboratories without knowledge of the treatment history can be considered as a worst-case scenario. We analyzed the data set for possible trends, but low annual numbers per bacterial species precluded conclusions on any trends. However, a combination of several years showed that the rate of resistance is constant for the major pathogens. It is of interest to compare our findings with the results of BfT-GermVet survey, 2004–2006 (Tab. 2), the isolates of which also reflect the sus- ceptibility status in Germany. It can be seen that MIC50 and MIC90s values are very similar to our data and underline the high susceptibility of pathogens from all German regions. Prevalence of resistance varied from 0 to 29 % and is predominantly seen for isolates from urinary/genital tract and skin/ ear/mouth infections. For all pathogens but one (Proteus spp.), resistance rates of our collection exceeded those of the BfT-GermVet study. The fact that pathogens recovered from untreated animals – as usually applies to the BfTGermVet study – have a higher susceptibility than frequently treated animals is reassuring. Clinical indication Bacterial species Number of isolates MIC50 (µg/ml) MIC90 (µg/ml) MIC90s (µg/ml) Resistance (%) Respiratory tract Bordetella bronchiseptica 42 0.25 0.5 0.5 0 Schwarz et al. 2007b E. coli 28 0.03 0.06 0.03 7 Grobbel et al. 2007a P. multocida 72 0.015 0.03 0.03 0 Schwarz et al. 2007b Staphylococcus spp. coagulase-positive 57 0.125 0.5 0.25 4 Schwarz et al. 2007c E. coli 100 0.03 0.5 0.06 7 Grobbel et al. 2007a Klebsiella spp. 17 0.03 ≥ 32 0.25 29 Grobbel et al. 2007b Proteus spp. 37 0.125 8 0.25 22 Grobbel et al. 2007b P. aeruginosa 28 1 2 1 11 Werckenthin et al. 2007 P. multocida 20 0.015 0.06 0.06 0 Schwarz et al. 2007b Proteus spp. 30 0.125 4 0.125 27 Grobbel et al. 2007b P. aeruginosa 71 1 ≥ 32 2 24 Werckenthin et al. 2007 Staphylococcus spp. coagulase-positive 101 0.125 0.25 0.25 2 Schwarz et al. 2007c E. coli 100 0.03 0.06 0.06 2 Grobbel et al. 2007b Urinary/genital tract Skin/ear/mouth Gastrointestinal tract Table 2 Summary of findings of BfT-GermVet: Antimicrobial susceptibility to enrofloxacin of canine and feline pathogens. 82 Reference 904091_TRI_Symp_Hehnen.qxp 03.06.2009 12:03 Uhr Seite 83 n = 2,378 E. coli Staphylococci Pseudomonas Proteus Pasteurella Klebsiella Enterococci Figure 2 Distribution of canine and feline bacterial pathogens during 2003–2004 of the European Bayer monitoring program. The results obtained from the European study are summarized in Table 3 for isolates recovered in 2003–2004; the distribution frequency is shown in Figure 2. Isolates of different indications are combined.The spectrum of species differs from the German program due to the inclusion of a relatively high number of samples from skin infections. It is shown that the overall susceptibility is very high. MIC50 values of seven species ranged from 0.016 to 0.125 µg/ml; only for P. aeruginosa and Enterococcus spp. 0.5 and 1 µg/ml were determined, respectively. Resistance levels of major pathogens barely exceeded 5 % and usually did not exceed 2 %. The resistance rate for E. coli amounted to 5.1 %. Bacterial species Number of isolates MIC50 (µg/ml) MIC90 (µg/ml) MIC90s (µg/ml) Resistance (%) E. coli 472 0.03 0.25 0.06 5.1 P. multocida 177 0.016 0.016 0.016 0 Staphylococcus spp. 290 0.125 0.5 0.5 1.7 Staphylococcus aureus 49 0.125 0.25 0.25 0 Staphylococcus intermedius 1150 0.125 0.125 0.125 1.0 Klebsiella spp. 27 0.03 0.5 0.5 0 Proteus mirabilis 74 0.125 0.25 0.25 1.4 P. aeruginosa 88 0.5 2 2 5.7 Enterococcus spp. 51 1 1 1 3.9 Table 3 Antimicrobial susceptibility to enrofloxacin of canine and feline pathogens of the European Bayer monitoring program. Conclusions The data demonstrate that the susceptibility of major canine and feline pathogens to enrofloxacin is usually excellent even after two decades of therapeutic use of fluoroquinolones in veterinary medicine. This is consistent with the findings of other national monitoring surveys. In spite of this favorable status, prudent and rational use of key antibiotics such as fluoroquinolones, and all antibiotics in general, is crucial for maintaining high susceptibility in the future. Ongoing monitoring will allow detection of any emerging resistance or shifts in susceptibility. Acknowledgement The excellent technical assistance of Nora Schröter, Ilona Fietz, and Stefan Buschmann is gratefully acknowledged. 83 904091_TRI_Symp_Hehnen.qxp 03.06.2009 12:03 Uhr Seite 84 Proceedings of the 4th International Baytril® Symposium Baytril® Resistance Monitoring – Susceptibility Status after More Than 20 Years Hans-Robert Hehnen · Sonja M. Friederichs · Julia C. Heimbach · Anno de Jong · Bernd Stephan References Grobbel M, Lübke-Becker A, Alesik E, Schwarz S, Wallmann J, Werckenthin C, Wieler LH (2007a). Antimicrobial susceptibility of Escherichia coli from swine, horses, dogs and cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120: 391–401. Grobbel M, Lübke-Becker A, Alesik E, Schwarz S, Wallmann J, Werckenthin C, Wieler LH (2007b). Antimicrobial susceptibility of Klebsiella spp. and Proteus spp. from various organ systems of horses, dogs and cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120: 402–411. Schwarz S, Alesik E, Grobbel M, Lübke-Becker A, Wallmann J, Werckenthin C, Wieler LH (2007a). The BfTGerm-Vet monitoring program – aims and basics. Berl Munch Tierarztl Wochenschr; 120: 357–362. Schwarz S, Alesik E, Grobbel M, Lübke-Becker A,Werckenthin C, Wieler LH, Wallmann J (2007b). Antimicrobial susceptibility of Pasteurella multocida and Bordetella bronchiseptica from dogs and cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120: 423–430. 84 Schwarz S, Alesik E, Werckenthin C, Grobbel M, LübkeBecker A, Wieler LH, Wallmann J (2007c). Antimicrobial susceptibility of coagulase-positive and coagulase-variable staphylococci from various indications of swine, dogs and cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120: 372–379. Werckenthin C, Alesik E, Grobbel M, Lübke-Becker A, Schwarz S, Wieler LH, Wallmann J (2007). Antimicrobial susceptibility of Pseudomonas aeruginosa from dogs and cats as well as Arcanobacterium pyogenes from cattle and swine as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120: 412–422. 904091_TRI_Symp_Hehnen.qxp 03.06.2009 12:03 Uhr Seite 85 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 86 Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Introduction Enrofloxacin (Baytril®) is a fluoroquinolone antimicrobial agent with broad-spectrum activity against a wide variety of Gram-positive and Gram-negative bacteria, and is rapidly bactericidal against key companion animal pathogens. Its pharmacokinetic/pharacodynamic profile is favorable for optimal dosing against susceptible bacterial strains. Clinical trial data indicate enrofloxacin is clinically efficacious in dogs and cats for a range of bacterial infections, including treatment of infections of the respiratory and urinary tracts and for skin and soft tissue infections. More recent data suggest enrofloxacin has a low potential to select for resistance. This manuscript is organized to provide an overview of antimicrobial resistance issues, pharmacology, resistance selection/prevention, and kill studies measuring bactericidal activity. A summary of this data in reference to achievable and sustainable enrofloxacin drug concentrations will be presented. Antimicrobial resistance is a worldwide problem requiring worldwide solutions. Increasing awareness of the issues surrounding resistance has demanded a rethinking about antimicrobial use in infectious diseases. Many variables complicate our understanding of the interaction of pathogenic microorganisms and antimicrobial agents, especially when one considers that susceptibility testing is conducted in vitro, yet the infection is treated in vivo. In vitro measurements are unable to replicate the role that immunity plays in combating infection, however, we do believe that these measurements are useful in guiding appropriate therapy. It is becoming increasingly clearer that traditional in vitro measurements of susceptibility or resistance may not fully predict the heterogenous nature of bacterial populations associated with infection, as the number of bacterial cells tested by minimum inhibitory concentration testing is less than the number of bacterial cells 86 typically associated with infection. We have argued that susceptibility testing utilizing higher bacterial burdens may more accurately measure the amount of drug required to inhibit bacterial pathogens. This is especially true when one considers the amount of drug required to inhibit high density bacterial growth with the pharmacokinetic and pharmacodynamic characteristics of the drug or drugs considered for therapy. Optimal versus suboptimal therapy has been debated, as we continue to evolve our understanding of infection, antimicrobial therapy and antimicrobial resistance selection. For optimal therapy, the combined goals of infectious diseases therapy need to be a successful clinical outcome and reducing the likelihood of resistance selection to the therapeutic drug(s) used. Are such combined goals possible in today’s environment and with our current antimicrobial compounds? The answer appears to be both yes and no. Clearly, one difficulty is the syndromic empiric approach to antimicrobial therapy. For respiratory tract, urinary tract and skin and skin structure infections, the etiology can be one of several different pathogens that may be either Gram-negative or Gram-positive. It is well known that not all antimicrobial agents exert the same degree of in vitro microbiological potency (defined by in vitro minimum inhibitory concentration (MIC) measurements), nor the same degree of pharmacological potency as defined by pharmacokinetic (PK) or pharmacodynamic (PD) measurements/principals. Ideally, the pathogen(s) identification and drug susceptibility/resistance would be known prior to antimicrobial administration and in all instances, the correct drug(s) would be used at the correct dosage, dosing intervals, and duration of therapy to ensure pathogen eradication and minimization of resistance. 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 87 Joseph M. Blondeau, MSc, PhD, RSM(CCM), SM(AAM), SM(ASCP), FCCP Departments of Microbiology, Microbiology and Immunology and Pathology, Royal University Hospital and University of Saskatchewan, Canada Pharmacokinetics and pharmacodynamics of antimicrobial therapy Pharmacokinetics (PK) determines the fate of the drug in the body (i.e., absorption, transformation, distribution, elimination). Pharmacodynamics is the effect of the drug on the body and includes its mechanism of action and efficacy. Much emphasis has been placed on PK/PD parameters and antimicrobial agents in an attempt to establish optimal antimicrobial therapy. Figure 1 summarizes some of the key points. For compounds classified as concentration-dependent agents (e.g., fluoroquinolones, aminoglycosides), two relationships have defined their antibacterial activity: 1) the maximum serum drug concentration (Cmax) to MIC ratio and 2) the area under the drug concentration curve (AUC) to MIC ratio (AUC/ MIC), which is sometimes referred to as the AUIC or area under the inhibitory curve. From investigations by Forrest et al. (1993), it was suggested that a Cmax-to-MIC ratio of 8/12 and an AUC/MIC ratio of > 125 correlated with favorable clinical outcomes and minimization of resistance. Considerable controversy has emerged regarding an AUC/MIC ratio of ≥ 125.1-4 Some argue that for Gram-positive organisms (particularly Streptococcus pneumoniae), the AUC/MIC ratio needs only be in the range of 30–50. File et al. (2009) investigated human patients with chronic obstructive pulmonary disease.5 Such patients may frequently have acute infectious exacerbations (AECB) of their chronic lung disease. The study investigated various factors that may influence the progression from an acute exacerbation to community-acquired pneumonia (CAP). Factors that were associated with progression to CAP included infection with S. pneu- Concentration (mg/l) Cmax = Peak serum concentration AUC = Area under the curve MPC = Mutant prevention concentration MIC = Minimal inhibitory concentration T > MIC Concentration-dependent – Peak/MIC > 8–12 – AUC/MIC = AUIC • > 125 Gram – • ~ 30–50 Gram + (??)* • > 100 Gram +** – AUC/MPC = 22*** • AUIC-PMA Time-dependent – T > MIC; 40–50 % * Schentag et al. (2001). CID; 32 (Suppl. 1): S39–46. Drusano et al. (2001). CID; 32: 2091–2092. Schentag et al. (2001). CID (Response); 33: 2092–2096. ** File et al. (2009). For human pathogen S. pneumoniae, reported that patients with an acute exacerbation of chronic bronchitis were statistically less likely to progress to pneumonia If the AUIC was >100. Int J Antimicrob Agents; 33: 58–64. *** Oloffson et al. (2006). J Antimicrob Chemother; 57(6): 1116–1121. Time (h) Blondeau, updated 2009 Figure 1 PK/PD relationships: surrogate markers. 87 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 88 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau moniae versus other pathogens (p < 0.001), an AUIC > 100 was more likely in patients with AECB, whereas patients with an AUIC of < 100 were associated with CAP (p < 0.001).The authors concluded that for S. pneumoniae-infected patients, achieving an AUIC > 100 can attenuate progression to CAP. To date, the absolute value for this AUC/MIC ratio remains unresolved, however, this most recent study suggests that for S. pneumoniae-infected patients a higher ratio is preferential to a lower ratio.The antibacterial activity of time-dependent antimicrobial agents (i.e., betalactams) is linked to the length of time-drug concentrations remain in excess of the MIC over the dosing interval. It has been suggested that this value needs to be in the range of 40–60 % of the dose where the drug concentration exceeds the MIC. Figure 2 summarizes the PK/PD classification of antimicrobial agents used in veterinary medicine. The pathogenesis of infection begins when organisms invade otherwise sterile body sites and are not initially eliminated by non-specific immune processes. The host is often damaged as a direct result of invasion by the pathogen, by liberation of exotoxins, by endotoxin, and by stimulating the immune response. While recovery from infection requires a functioning immune system, symptoms associated with infection may be partly or mostly related to the inflammatory response. As such, antimicrobial agents are adjunct therapies to the bodies own natural defences for fighting infection.This in no way lessons the importance of adjunctive antimicrobial therapies but does stress the importance of correctly using these agents for a successful clinical outcome and not inadvertently contributing to antimicrobial resistance. Unfortunately, most decisions regarding antimicrobial drug selection are based solely on clinical outcome (as determined in clinical tri- Concentration (mg/l) Cmax = Peak serum concentration AUC = Area under the curve T > MIC MIC = Minimal inhibitory concentration Time (h) Figure 2 PK/PD relationships: surrogate markers. 88 Cmax/MIC: streptomycin, gentamicin, tobramycin, omikacin, danofloxacin, enrofloxacin, marbofloxacin, difloxacin, sarafloxacin, metronidazole AUC/MIC: streptomycin, gentamicin, amikacin, tobramycin, danofloxacin, enrofloxacin, marbofloxacin, difloxacin, sarafloxacin, metronidazole, colistin, oxytetracycline, chlortetracycline, doxycycline, azithromycin, clarithromycin, vancomycin T > MIC: benzylpenicillin, amoxicillin, cloxacillin, carbenicillin, cephalixin, ceftiofur, cephapirin, florphenicol, chloramphenicol, erythromycin, tilmicosin, tulathromycin, aivlosin, clindamycin, sulfadiazinesulfadoxime, trimethoprim 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr als) with little or no consideration for microbiological or pharmacological potency. In today’s environment where few new antimicrobial agents are under clinical development, a renewed interest in existing agents seems necessary and nonclinical outcome parameters require investigation for optimal drug use. In human medicine, numerous guidelines have been assembled and published by expert working groups, whereby recommended therapies for various infectious diseases are based on the best evidence available.6 In the most recent CAP guidelines, Mandel et al. wrote: “Because overall efficacy remains good for many classes of agents, the more potent drugs are given preference because of their benefit in decreasing the risk of selection for antibiotic resistance. Other factors for consideration of specific antimicrobials include pharmacokinetics/pharmacodynamics, compliance, safety, and cost”. Such statements suggest that the potential for the prevention of resistance selection is important and that PK/PD parameters are also important considerations: clinical outcome alone should not be the sole criteria for drug selection for therapy. Resistance prevention In vitro susceptibility testing has been the cornerstone of measuring the activity of antimicrobial agents against various pathogens, and in an ideal world, the results of such testing impact of appropriate therapy. Current routine susceptibility testing is based on utilizing a bacterial inoculum of 105 colony forming units per mililiter (cfu/ml) and is standardized for a number of variables. Such testing may not fully appreciate the true dynamics of higher bacterial populations present during infection, where bacterial numbers likely fluctuates. During acute infections, bacterial loads may exceed 109 cfu7-11 – a value relevant in the context of antimicrobial resistance, as the frequency with which a spontaneous mutant that Seite 89 confers drug resistance occurs ranges from 1 x 10-7 to 1 x 10-9 or 1 spontaneous mutant for every 107 to 109 bacterial cells.12 As such, an acute infection with ≥ 109 cfu is likely to harbor a resistant cell (or resistant cells) and in the presence of an antimicrobial agent, may allow for the selective amplification of the resistant subpopulation as the susceptible population is being eliminated by the drug. Resistance prevention is being recognized as a goal of antimicrobial therapy. Dong et al. published the mutant prevention concentration (MPC) approach in 1999.13 As stated above, high bacterial burdens may be present during infection and from these higher bacterial burdens, mutant cells may be present spontaneously within the population and under antimicrobial selective pressure may be selectively amplified during therapy. In the landmark experiments by Dong et al., increasing bacterial populations of Staphylococus species and Mycobacterium species were exposed to increasing fluoroquinolone drug concentrations in an in vitro assay. For these experiments, approximately 1010 bacterial cells were inoculated to the surface of agar plates containing increasing drug concentrations of the fluoroquinolones being investigated. Following incubation under ideal conditions, the lowest drug concentration blocking all growth was termed the mutant prevent concentration (MPC). It was readily demonstrated in the experiments by Dong et al. that mutant bacterial cells could be readily selected off drug-containing agar plates over drug concentrations ranging between the measured MIC and MPC drug concentrations. As such, the drug concentration that blocked all growth (including those of the mutants) was termed the MPC. Since the initial description of MPC by Dong et al., a number of other reports have appeared in the peer reviewed literature expanding on this concept and reporting on the results for the testing of numerous different antimicrobial and microorganism combinations.14, 15 89 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 90 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau Figure 3 is a schematic representation demonstrating how mutant cells may be selectively amplified in the presence of the MIC drug concentration. This schematic representation assumes that the MIC drug concentration is insufficient to block the growth of mutant cells that may be present in the population. As shown in the figure, spontaneously resistant cells naturally present in the population may be amplified in the presence of the MIC drug concentration as the susceptible cells are being eliminated.This would occur if the achievable drug concentration is insufficient to block the mutant cells. In a patient with a healthy and functioning immune system, all of these organisms would be cleared. In patients that may be immunocompromised, had prior infection, had prior antibiotic exposure or patients that appeared to be failing therapy for acute infection, continued proliferation of the bacterial population to a point where it breaches the immune threshold may result in a patient being colonized or infected with the mutant population. As shown in the bottom right hand section of the figure, mutant cells that were selectively ampli- fied in the presence of the drug may ultimately be eliminated over time with factors such as competitive inhibition with normal flora and this in some way may relate to the relative fitness of the mutant cells. Figure 4 shows a schematic representation of the blocking of mutant cells in the presence of the MPC drug concentration. This schematic assumes that the MPC drug concentration is high enough to block the growth of the mutant cells present in the population. As shown in the figure, MPC drug concentrations would essentially eliminate the mutant cells along with the susceptible bacteria and control resistance to the spontaneous rate seen in high density bacterial populations or eliminate these mutant cells from the population. As depicted, some microorganisms tested by MPC may require a centrifugation step in order to concentrate the number of bacterial cells. As well, the number of starter plates required to be inoculated per organism varies. One limitation with MPC testing relates to the fact that it is technically more demanding than performing a MIC test (Fig. 5). In comparison, TIME (hours to days) 20 in 1 billion MIC 2 in 1 billion 200 in 1 billion Immunocompromised state Prior infection Prior antibiotic exposure Patient colonized or infected with mutant population Acute infections/ failed therapy HEALTHY IMMUNE SYSTEM Potential Clearance Mutant cells may be cleared over time, i.e., competitive inhibition from normal flora (fitness) IMMUNE THRESHOLD BREACHED Blondeau et al. (2004). J Chem, updated 2009 Figure 3 Selective amplification of resistant mutants at MIC. 90 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 91 the MPC assay is based on testing of ≥ 109 cfu on drug containing agar plates, whereas MIC testing is conducted in a microbroth assay utilizing 105 cfu/ml. More recently, Hesje and Blondeau reported on MPC testing using a modified microbroth dilution assay.16,17 Of the data generated to date, it appears that the modified microbroth dilution assay yields consistent results to those demonstrated by the agar dilution method that initially defined the MPC approach. Further testing of the modified microbroth dilution method is necessary in order to confirm this method as suitable assay for generating MPC results. The mutant selection window (MSW) defines the antimicrobial drug concentration that falls between the MIC and MPC drug concentrations and it has been previously argued that the time the drug concentration remains within the MSW or above the MPC has an impact on the selective amplification of resistant subpopulations that may be present in high density heterogeneous bacterial populations.14,18–20 Figure 6 is a schematic representation of the MSW. When drug concen- trations are below the MIC drug concentration, neither susceptible nor mutant cells would be inhibited and as such, the mutant fraction is not selectively amplified. Similarly, for drug concentrations in excess of the MPC, susceptible and mutant populations are inhibited or killed. However, when drug concentrations remain within the MSW – i.e., above the MIC but below the MPC – the susceptible cells are inhibited and the mutant fraction selectively amplified in the presence of the drug. While clinical evidence for the selection window hypothesis is lacking, two in vitro experiments using fluctuating fluoroquinolone concentrations support the idea. In one, Firsov et al.21 simulated dosing of fluoroquinolones with S. aureus. They found that organisms with elevated values of MIC were obtained only when the drug concentration remained inside the selection window, not when it was above the MPC or below the MIC. Similar results were found with quinolones and S. pneumoniae.20 Croisier et al. showed in a rabbit pneumonia model that when drug concentrations remained within the MSW for 45 % or greater of the dos- TIME MPC 2 in 1 billion Colorization or new infection may occur Mutant and susceptible cell inhibited Immunocompromised state Prior infection Prior antibiotic exposure Acute infections/ failed therapy HEALTHY IMMUNE SYSTEM Potential Clearance IMMUNE THRESHOLD BREACHED Hansen & Blondeau (2002); Blondeau et al. (2004). J Chem, updated 2009. Figure 4 Blocking of resistant mutants at MPC. 91 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 92 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau Basic method: (varies by organism) • Inoculate plates and incubate • Transfer to fresh media (~ 100 ml) • Centrifuge and resuspend in fresh media • Inoculate drug containing plates with 1010 cfu 1) 1) Inoculate 3* plates per organism; incubate 18–24 h at 35–37 °C in O2 2) 2) Transfer contents of plates to flask with 100 ml fresh media. Incubate 18–24 h at 35–37 °C in O2 3) Centrifuge** culture media at 5,000 xg for 30 min at 4 °C Centrifuge** 3) 4) Resuspend in 3 ml of media 5) Inoculate drug containing plates with 1010 organisms; incubate for 18–24 h in O2, examine for growth, reincubate for 18–24 h in O2 and reexamine. The lowest drug concentration preventing = MPC 4) 5) 0.06 0.12 16 8 0.25 0.5 1 MPC 2 4 Organism # Starter plates inoculated* E. coli 2–3 S. intermedius 2–3 P. multocida 3–4 P. aeruginosa 2–3 M. haemolytica 4–5 A. pleuropneumonia 7–8 Centrifugation required** No No No No Yes Yes Figure 5 Schematic method of MPC testing. Serum or tissue drug concentration • Above MPC – both susceptible and 1st-step resistant cells inhibited – no selective amplification of resistance subpopulation. • Double mutants may not be inhibited. MPC MSW MIC Sub-MIC – neither susceptible bacteria nor 1st-step resistant mutants killed/ inhibited – no selective amplification of resistant subpopulation. Time post administration Figure 6 Mutant selection window (MSW). 92 • Susceptible cells killed/inhibited. • 1st-step or 2nd-step resistant cells not inhibited – selective amplification may occur. • Longer times in MSW = greater risk for mutant selection/amplification. 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 93 sponse clearly contributes to organism killing and eradication is currently unknown! ing interval, it correlated 100 % with the selection of a mutant subpopulation conferring quinolone resistance.22 Smith et al. previously argued that while the MPC approach applied to the testing of fluoroquinolone compounds, it does not apply to betalactam, macrolide, or aminoglycoside compounds as the principal mechanisms or resistance to these compounds were not as a result of de novo resistance but rather acquisition of various resistance determinants.28 Zhao argued that when a mutant was present within a bacterial population, the issue is really how do you prevent that mutant from being selectively amplified during drug therapy, not how the mutant came to be.29 Prevention of mutant amplification and not mutation prevention is the goal of this approach and, as such, might readily be applied to any scenario where restricting mutant growth could be achieved. It may be that for some pathogens and drugs, prevention of mutant amplification could only be achieved with combinations of drugs.30 To date, MPC measurements have been made Based on the MSW approach, drug dosages should be administered to be in excess of the MPC (i.e., above the MSW) for as long as necessary to affect a substantial reduction in viable organisms. One unknown is how long do drug concentrations need to remain in excess of the MPC and what percentage of viable organisms needs to be killed – 99 % versus 100 %. Is 1 % or less of 1–10 billion cells surviving following drug exposure too many – especially if they are all mutants? From kill experiments carried out with human and veterinary pathogens and fluoroquinolone and non-fluoroquinolone compounds, it was shown that at least 6–12 hours above the MPC was necessary to yield a > 99 % reduction in viable organisms when 106 to 109 bacteria were exposed to MPC drug concentrations.23–27 How such observations translate to infection in animals, where the impact of the immune reAntimicrobial agent MIC50 MIC90 MIC Range MPC50 MPC90 MPC Range Amikacin 2 4 1–8 32 32 16–32 b Ampicillin 2 2 1–2 16 32 16–>128 Cefazolinb 2 2 1–8 64 64 16–128 Cefotaxime 0.063 0.125 0.031–0.25 8 16 0.5–32 Ceftriaxone 0.063 0.125 0.031–0.25 2 16 0.5–16 Doxycycline 0.5 1 0.25–8 64 > 64 > 8–>64 Enrofloxacin 0.008 0.016 < 0.008–0.063 0.125 0.25 0.063–0.25 Gentamicin 0.5 1 0.25–1 0.5 16 0.25–>8 Marbofloxacin 0.016 0.016 0.004–0.016 0.125 0.5 0.063–0.5 Nitrofurantoin 8 16 4–32 64 ≥ 64 64–≥64 Tobramycin 1 1 0.5–16 8 16 8–≥8 Table 1 Comparative in vitro activity of several antimicrobial agents tested against E. coli a isolates from companion animals (studies combined). a Some isolates complements of Dr. Heinz Wetzstein of Germany. b For organisms with MICs ≤ 2 µg/ml; MPCs for organisms with MICs ≥ 4 µg/ml ranged from 64–≥ 256 µg/ml 93 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 94 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau with macrolides 31, aminoglycosides30, tetracyclines, beta-lactams, glycopeptides32, and glycylcycline.33,34 In some instances, the mechanisms associated with the observations have not been fully elucidated and further investigations are necessary (reviewed in Hesje et al.).15,35,36 MPC values were higher against organisms with MICs ≥ 4 µg/ml. Comparative MIC and MPC data for enrofloxacin and comparator antimicrobial agents tested against companion animal isolates of S. intermedius are shown in Table 2. For enrofloxacin, the MIC90 value was 0.063 and the MPC90 was 0.5 µg/ml; by comparison, values for marbofloxacin were 0.5 and 1 µg/ml, respectively. For amikacin, gentamicin, and tobramycin, MIC90 values ranged from 0.25 to 2 µg/ml being lowest for gentamicin and highest for amikacin: MPC90 values respectively were 32, 4 and 8 µg/ml. For cefazolin, cefotaxime and ceftriaxone, MIC90 values were 0.063, 2, and 1 µg/ml and MPC90 values were 16, 4, and 8 µg/ml, respectively. Veterinary data Table 1 is a summary of MIC and MPC data for enrofloxacin and comparator antimicrobial agents tested against companion animal isolates of E. coli. For enrofloxacin, the MIC90 value was 0.016 µg/ ml and the MPC90 was 0.25 µg/ml. By comparison, MIC90 values for amikacin, gentamicin, and tobramycin were 4, 1, 1 µg/ml, respectively, and MPC90 values were 32, 16, 16 µg/ml. For cefotaxime and ceftriaxone, MIC90 values were 0.125 µg/ml and MPC90 values were 16 µg/ml. The MPC90 value for cefazolin was 64 µg/ml. Enrofloxacin MIC and MPC testing has also been completed against a more than 100 strains of Pasteurella multocida. The MIC90 value was 0.008 µg/ml and the MPC90 was 0.125 µg/ml. Antimicrobial agent MIC50 MIC90 MIC Range MPC50 MPC90 MPC Range Amikacin 1 2 1–2 32 32 16–>32 Ampicillin 0.12 0.25 0.031–0.25 > 128 > 128 0.125–>128 Cefazolin 0.063 0.063 0.031–0.063 0.25 16 0.125–16 Cefotaxime 0.5 2 0.25–2 1 4 0.5–8 Ceftriaxone 0.5 1 0.5–2 4 8 4–8 < 0.063–0.125 8 16 4–6 Doxycyclineb ≤ 0.063 ≤ 0.063 Enrofloxacin 0.063 0.063 0.031–0.063 0.5 0.5 0.5–1 Erythromycin 0.25 0.5 0.125–0.5 >8 >8 0.5–>8 Gentamicin 0.125 0.25 0.063–0.5 2 4 2–8 marbofloxacin 0.25 0.5 0.125–0.5 1 1 0.5–2 Nitrofurantoin 8 8 8 32 32 32–64 Tobramycin 0.25 0.5 < 0.125–0.5 8 8 2–≥8 Table 2 Comparative in vitro activity of several antimicrobial agents tested against S. intermediusa isolates from companion animals (studies combined). a Some isolates complements of Dr. Heinz Wetzstein of Germany. organisms with MICs ≥ 2 µg/ml had MPCs ≥ 64 µg/ml. 94 b For organisms with MICs ≤ 2 µg/ml; 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 95 Figure 7 shows enrofloxacin serum drug concentrations in dogs for 3 dosages.The mutant selection window is bordered by a MIC90 of 0.016 and MPC90 of 0.25 µg/ml for E. coli. For S. intermedius, the mutant selection window is bordered by a MIC90 of 0.063 µg/ml and a MPC90 of 0.5 µg/ml. As such, serum drug concentrations for all dosages remain in excess of the mutant selection window for 7–>12 hours. From kill studies conducted by our laboratory with enrofloxacin and E. coli, 98–99 % of a 106–107 cfu/ml bacterial burden were killed in the presence of enrofloxacin following 1–2 hours of mutant prevention drug concentrations in kill assays. Serum drug concentrations would remain in excess of the MPC90 of P. multocida for ~ 12 hours. Figure 8 shows serum drug concentrations for enrofloxacin in cats to be 5 mg/kg dosage. For E. coli, the mutant selection window was bordered by a MIC90 of 0.016 µg/ml and the MPC90 of 0.25 µg/ml. For S. intermedius, the mutant selection window is bordered by a MIC90 of 0.063 µg/ml and a MPC90 of 0.5 µg/ml. As such, serum drug concentrations would be in excess of the mutant selection window for ~ 9–>12 hours of the dosing interval. Serum drug concentrations would remain in excess of the MPC90 for P. multocida for >12 hours. Previously, it was mentioned that PK/PD parameters have been utilized to characterize various antimicrobial agents and their antibacterial activity. To date, most measurements related to pharmacology have included MIC measurements of the respective organisms being investigated. Given the recent swell of data related to MPC measurements, what impact does such a measurement have on our understanding of resistance prevention with the ratio derived from Cmax/ MIC or AUC/MIC? To date, limited data is available defining Cmax/MPC or AUC/MPC ratios. Olofsson et al. working with ciprofloxacin and E. coli reported that an AUC/MPC ratio of ≥ 22 Concentration µg/ml Dose-dependent 8 E. coli MIC90 0.016 µg/ml MPC90 0.25 µg/ml AUC/MIC = 656 AUC/MPC = 42 7 6 S. intermedius MIC90 0.063 µg/ml MPC90 0.5 µg/ml AUC/MIC = 166 AUC/MPC = 21 5 4 Elimination half-life = 4 h 20 mg/kg PO OD 10 mg/kg PO OD 5 mg/kg PO OD Mutant Selection Window 3 MAX SUSCEPTIBILITY 2 MPC90 0.25 µg/ml 1 2 µg/ml MIC 90 0.016 µg/ml 0 0.5 1 2 3 4 5 6 Hours 7 8 9 10 11 12 Figure 7 Enrofloxacin – E. coli/S. intermedius. 95 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 96 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau correlated with the prevention of resistance development (Fig. 1).37 Clearly, additional studies are required considering MPC rather than MIC measurements for defining PK/PD relationships and to help predict or prevent the selection of resistance subpopulations. Recently, the idea of “best-in-class” has emerged as a potential way to optimizing therapy: maximize successful patient outcome while minimizing resistant mutant selections. Once the class of agent to be used for therapy has been decided, the most potent (microbiologically, pharmacologically) agent in the class would be used based on the organism(s) most likely to be the cause of infection. One major problem with this approach is that patients are most often treated empirically; consequently, microbiological investigation may not be performed to identify the pathogen(s) and its/their susceptibility profile. A more fundamental problem is that two compounds can have very similar activity with susceptible cells, but very different activities against resistant mutants.12,38,39 The more mutant-active agent is more likely to restrict the enrichment of mutant subpopulations. Ideally, “best-in-class” should include a strong consideration of anti-mutant activity and not be based solely on MIC measurement. In vitro kill studies Fluoroquinolones such as enrofloxacin may be characterized as having excellent bactericidal activity and tissue penetration and, in general, have broad in vitro and in vivo activity against Grampositive and -negative pathogens. In previous sections, in vitro susceptibility testing was discussed and the MPC concept reviewed. Measurements such as MIC testing and MPC testing are measurements of inhibition of bacterial growth and not a measurement of killing. Traditional measurements of in vitro killing are performed by ex- Concentration µg/ml Dose-dependent 8 E. coli MIC90 0.016 µg/ml MPC90 0.25 µg/ml AUC/MIC = 656 AUC/MPC = 42 7 6 S. intermedius MIC90 0.063 µg/ml MPC90 0.5 µg/ml AUC/MIC = 166 AUC/MPC = 21 5 4 3 Elimination half-life = 6 h 5 mg/kg PO OD Mutant Selection Window MPC90 0.25 µg/ml 1 MIC 90 0.016 µg/ml 0 0.5 Figure 8 Enrofloxacin – E. coli/S. intermedius. 96 2 µg/ml MAX SUSCEPTIBILITY 2 1 2 3 4 5 6 Hours 7 8 9 10 11 12 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr posing ~ 105 cfu/ml of bacteria to varying drug concentrations, i.e., 2x, 4x, 10x the MIC. Such studies have been used to categorize antibacterial agent as either bacteriostatic or bactericidal based on a log10 reduction in viable cells of ≤ 2 log10 versus ≥ 3 log10, respectively.40 We have previously argued that kill studies might be more meaningful if higher bacterial densities were used, as such burdens were more indicative of bacterial infections. Additionally, we also argued that drug concentrations utilized in kill studies should be based on the measured MIC, MPC, maximum Seite 97 serum and maximum tissue drug concentrations.26,40-43 Figure 9 is a schematic representation of the modified in vitro kill method for determining killing of bacterial inocula of 106 cfu/ml, 107 cfu/ml, 108 cfu/ml, and 109 cfu/ml. Briefly, bacteria are sub-cultured to 3–7 (varies based on organism) agar plates and incubated under optimal conditions for 18–24 hours and, following this, the bacterial cells are transferred to liquid media, incubated for ~ 2 hours and then diluted to yield • inoculate bacteria to three agar plates to produce confluent growth • incubate 18–24 h at 35–37 °C in O2 • Transfer content of plates to broth media • Incubate 2 h at 35–37 °C in O2* • Cell density approximately 109 cfu/ml PERFORMED IN TRIPLICATE • Dilute to give bacterial inocula ranging from 106–109 cfu/ml • Add drug and sample at predetermined times • Dilute to broth to facilitate counting and inoculate agar plates, incubate as described • Determine colony counts and calculate log10 and % reduction (kill) in viable cells • inoculate to plates • incubate 18–24 h at 35–37 °C in O2 * 5% CO2 depending on organism being tested Figure 9 A new approach to in vitro kill measurements. Traditional kill studies utilize 105 colony forming units per milliliter (cfu/ml) of bacteria exposed to varying drug concentrations – usually as multiples of the MIC – i.e., MIC, 5 times (x) MIC, 10 x MIC, etc. The new method involves testing of higher bacteria inocula – 106 (1 million) to 109 (1 billion) cfu/ml against drugs as shown schematically above. 97 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 98 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau bacterial densities ranging from 106–109 cfu/ml. Drug is added to the bacteria and then bacteria are sampled at predetermined intervals and inoculated to the surface of drug-free agar plates and incubated for 18–24 hours under optimal conditions. Colony counts are performed and the log10 reduction and percentage kill of viable cells recorded. Each assay is performed in triplicate and the results averaged. Where multiple strains are tested, the results are averaged. As previously stated, the MIC drug concentration may be ineffective in inhibiting the growth of high density bacterial populations, as less susceptible cells may be present in this population. To inhibit these less susceptible or resistant cells, the MPC drug concentration is necessary. In kill studies conducted in our laboratory with enrofloxacin and E. coli, the killing was slow and incomplete when high density inocula (106–109 cfu/ml) were exposed to the measured MIC drug concentrations for the organisms tested (Figs. 10, 11). For example, 6–25 % of bacterial cells were killed following 1–2 hours of drug exposure. These kill values did not increase substantially after 12–24 hours of drug exposure. These observations are consistent with previous reports from our laboratory for human pathogens exposed to fluoroquinolones in identical kill studies.26,39,43 This observation is also consistent with observation with Mannheimia haemolytica recovered from cattle with bovine respiratory disease and enrofloxacin.44–46 Exposure of high density bacterial populations of E. coli to enrofloxacin at the measured MPC drug concentration yielded more rapid and complete killing (Figs. 10, 11). For example for 106 cfu/ml, a 2.3–3.4 log10 reduction in viable cells (98–> 99 % kill) was seen after 1–2 hours of drug exposure; for 107 cfu/ml, 2.2–2.9 log10 reduction (98–>99 % kill); for 108 cfu/ml, 1.1–1.3 log10 reduction (90–92 % kill); 109 cfu/ml, 0.1 log10 reduction (10–19 % kill). Killing rates exceeded 99 % following 12–24 hours of drug exposure. This data clearly indicates that against high density bacterial populations – such as those potentially present during acute infection – enrofloxacin was rapidly bactericidal with 90–>99 % of viable cells being killed in the presence of the 2 MIC 10^6 1 MIC 10^7 0 MIC 10^8 –1 MIC 10^9 –2 MPC 10^6 –3 MPC 10^7 –4 MPC 10^8 –5 MPC 10^9 –6 –7 0.5 1 2 4 6 Time Figure 10 The killing of E. coli at the MIC and MPC enrofloxacin concentration. 98 12 24 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr drug. At present, kill studies are not yet completed utilizing the maximum serum and timedrug concentrations against bacterial inocula ranging from 106–109 cfu/ml. However, from studies completed with M. haemolytica, killing using maximum serum or tissue drug concentration (1.9–2 µg/ml in cattle; higher than MPC drug concentrations), killing was more rapid and complete – i.e., > 99 % kill for 106–108 cfu/ml following 1 hour of drug exposure and ≥ 96–98 % kill for a 109 cfu/ml inocula following 1 hour of drug exposure. These observations with enrofloxacin testing against E. coli and M. haemolytica indicate rapid in vitro bactericidal activity. In summary, antimicrobial susceptibility/resistance is defined by testing a known density of bacterial cells to varying drug concentrations in vitro and the MIC results are compared to susceptibility breakpoints of an established base of microbiological and pharmacological parameters. An organism with a MIC at or below the susceptibility breakpoint is considered susceptible, whereas an organism with a MIC above the sus- Seite 99 ceptibility breakpoint is considered non-susceptible or resistant. More recently, mutant pre- vention concentration testing has been proposed as an alternative form of susceptibility testing and takes into account higher bacterial densities such as those seen associated with acute infection. In addition to providing a level of susceptibility or resistance to a higher density of bacterial cells, MPC also provides a means to predict the likelihood of the selection of resistant subpopulations during therapy when antimicrobial agents are dosed to be above the mutant selection window or within the mutant selection window. Clearly, the MPC approach to antimicrobial susceptibility testing is still in its infancy and numerous other investigations need to be completed with various bug/drug combinations and in some instances, combinations of antimicrobial agents together. Utilization of strategies such as MPC may provide guidance, optimal therapy of infectious diseases and reduce the rate at which resistance will escalate antimicrobial compounds. 3,500 MIC 10^6 3,000 MIC 10^7 MIC 10^8 2,500 MIC 10^9 2,000 MPC 10^6 1,500 MPC 10^7 1,000 MPC 10^8 500 MPC 10^9 0 – 500 0.5 1 2 4 6 12 24 Time Figure 11 The percent killing of E. coli at the MIC and MPC enrofloxacin concentration. 99 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Seite 100 Proceedings of the 4th International Baytril® Symposium Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril® Joseph M. Blondeau Infection Number Clinical diagnosis 25 dogs Dose (mg/kg BW/day) Duration of treatment Clinical response rate Reference Complicated upper and lower respiratory infections 6–12 96 % (48) 30 cats Feline rhinotracheitis Bronchopneumonia 6–12 96 % (48) 178 dogs Upper and lower respiratory infections 6–10 89.9 % (49) 87 cats Feline rhinotracheitis Bronchopneumonia 6–10 91.9 % (49) Respiratory 20 dogs 5 90 % (48) 30 cats 5 90 % (48) 14 dogs 5 100 % (50) 49 cats 5 89.5 % (50) 129 dogs 5 89.9 % (51) 68 cats 5 88.2 % (51) 48 dogs 5 89.6 % (52) 50 dogs 5 93.3 % (53) 30 dogs 5 93.3 % (54) 100 dogs 10 90 % (55) 16 dogs 5 94 % (56) 25 cats 10 88 % (55) Urinary Pyoderma Table 3 Clinical studies investigating the efficacy of enrofloxacin (Baytril) in companion animals. The 1, 2, 3 punch of Baytril Clinical efficacy Enrofloxacin (Baytril) is approved for the treatment of infectious diseases in companion animals (dogs and cats) and the clinical efficacy has been established and reported by numerous investigators (Tab. 3). Specifically, enrofloxacin is indicated for the treatment of skin, respiratory and urinary tract infections. For pyoderma, clinical efficacy rates for enrofloxacin were reported to range from 89.6–94 % in digs and 88 % in cats. For respiratory tract infections, enrofloxacin clinical efficacy rates ranged from 91.9–96 % in cats and 89.9–96 % in dogs. For urinary tract infections, 100 clinical efficacy rates ranged from 88.2–90 % for cats and from 89.9–100 % for dogs. Rapid bactericidal action As summarized in this report, in vitro measurements showing killing of high density populations of E. coli yielded reductions of viable cells of 90–> 99 % by 1–2 hours against 1 million to 100 million cells/ml by 1–2 hours of drug exposure at the MPC drug concentrations. Dagan et al. previously indicated that eradication of bacterial cells correlated with clinical outcome in human patients with respiratory tract infections.47 As shown, enrofloxacin demonstrates rapid bactericidal activity against the organisms reported in this manuscript. 904091_TRI_Symp_86_103_Blondeau.qxp 03.06.2009 17:32 Uhr Low potential for resistance selection As summarized, traditional measurements of antimicrobial susceptibility or resistance, while useful, may not fully measure the true dynamics of the interaction of an antimicrobial agent and bacteria in vivo, where the number of bacteria associated with infection may exceed the number of cells utilized by MIC testing. As such, MPC measurements have evolved to determine the drug concentration required to block the growth of the least susceptible cells present in high density bacterial populations – such as those seen during acute infection. Suboptimal dosing may lead to the selective amplification of the mutant Seite 101 subpopulations present in high density bacterial populations. Ideally, antimicrobial therapy would eliminate both susceptible and resistant cells present in the population. E. coli, S. intermedius, and P. multocida are amongst the most common pathogens associated with infections in dogs and cats. Both MIC and MPC values were below the achievable serum drug concentrations with approved dosing of enrofloxacin in dogs and cats; enrofloxacin serum drug values remain in excess of the MSW for no less than 7 hours (lowest dosage) and for higher dosages, longer. As such, in the MPC/MSW model, enrofloxacin demonstrates a low potential for resistance selection. References 1. 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Amsterdam, The Netherlands. 31. Blondeau JM, Brunner LS, Norton SE (2006). In vitro activity against Gram-positive pathogens of SS734: a new 4th generation fluoroquinolone (FQ) for the treatment of bacterial conjunctivitis (BC). 46th Interscience Conference on Antmicrobial Agents and Chemotherapy (ICAAC). San Francisco, CA: American Society of Microbiology, Washington, DC. 32. Blondeau JM, Metzler KL (2005). Application of the resistance prevention concentration (RPC) to oxacillin (O), cefazolin (C) and vancomycin (C) against methicillin-susceptible (MS) and resistant (MR) Staphylococcus aureus (SA). 45th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, DC: American Society of Microbiology, Washington, DC. 33. Blondeau JM, Hesje C, Borsos S (2007). 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International Meeting of Emerging Diseases and Surveillance (IMED).Vienna, Austria. 43. Hansen G, Blondeau JM (2002). Continuous bactericidal activity of moxifloxacin against Streptococcus pneumonia. In: Novel Perspectives iin Antibacterial Action (Eds: Gillespie SH,Tillotson GS), Royal Society of Medicine Press Ltd, London; pp. 27–34. 44. Blondeau JM, Borsos S, Blondeau LD et al. (2007). Concentration-dependent killing of Mannheimia haemolytica (MH) by enrofloxacin at the minimum inhibitory, mutant prevention, maximum serum and tissue drug concentra- Seite 103 tions. International Meeting of Emerging Diseases and Surveillance (IMED).Vienna, Austria. 45. Blondeau JM, Borsos S, Hesje C et al. (2007). Comparative killing of bovine isolates of Mannheimia haemolytica (MH) by enrofloxacin (ENR), florfenicol (FL), tilmicosin (TIL) and tulathromycin (TUL) using the measured minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) drug values. International Meeting on Emerging Diseases and Surveillance (IMED). Vienna, Austria. 46. Blondeau J, Borsos S, Hesje C et al. (2009). Comparative killing of bovine isolates of Mannheimia haemolytica (MH) by enrofloxacin (ENR), florfenicol (FL), tilmicosin (TIL) and tulathromycin (TUL) using the measured minimum inhibitory (MIC) and mutant prevention concentration (MPC) and maximum serum (Cmax) and Tissue (Tmax) drug concentration values. International Meeting of Emerging Diseases and Surveillance (IMED).Vienna, Austria. 47. Dagan R, Klugman KP, Craig WA et al. (2001). Evidence to support the rationale that bacterial eradication in respiratory tract infection is an important aim of antimicrobial therapy. J Antimicrob Chemother; 47: 129–140. 48. Hackman F, Schierz G (1989). Die Anwendung des Antiinfectivums Baytril® in der Kleintierpraxis. Veterinarian; 10: 26–29. 49. Cardini G, Ambrogi C, Cerri D (1996). Enrofloxacin: use in infections of the respiratory tract in dogs and cats. 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DeManuelle TC, Ihrke PJ,Vulliet PR (1999). Comparison of enrofloxacin skin concentrations in normal dogs and in dogs with pyoderma. Comp Cont Educ Pract Vet; 21: 49–56. 103 904091_TRI_Symp_Notes.qxp 03.06.2009 14:06 Uhr Seite 104 Notes 904091_TRI_Symp_Notes.qxp 03.06.2009 14:06 Uhr Seite 105 Notes 904091_TRI_Symp_Notes.qxp 03.06.2009 14:06 Uhr Seite 106 Notes
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