1. health and fitness
2. disease

Linköping University medical dissertations, No. 1300
Extended-spectrum beta-lactamase
producing Enterobacteriaceae:
aspects on detection, epidemiology and multi-drug resistance
Maria Tärnberg
Printed by LiU-Tryck, Linköping, Sweden, 2012
Linköping University medical
dissertations, No. 1300
ISBN 978-91-7519-938-2
ISSN 0345-0082
What goes around, comes around…
Abstract
Beta-lactam antibiotics are the largest and most commonly used group of antimicrobial agents
in Sweden as well as world-wide. They show very good tolerability and many of the drugs can
be administrated orally. Bacteria expressing extended-spectrum beta-lactamases (ESBLs),
enzymes hydrolysing penicillins and cephalosporins, may not respond to therapy using some
of these antibiotics. The isolates are also often co-resistant to other antimicrobial agents, thus
further limiting treatment options. Often parenterally administrated carbapenems is one of few
safe treatment options left.
In this thesis we have investigated the occurrence of ESBL producing Enterobacteriaceae in
clinical isolates from Östergötland, Sweden, from 2002 until end of 2007 and the occurrence
of multiresistance among ESBL producing E. coli. During these investigations we developed a
simple method well suited for high-throughput analysis, for detection and sub typing of
common ESBL genes.
During the six year period, the prevalence of ESBL producing Enterobacteriaceae in
Östergötland was very low, <1%, but increasing. The number of patients with ESBL
producing E. coli increased significantly from 5 to 47 per year; K. pneumoniae remained
between one and four per year. The genes found were dominated by CTX-M group 1 (67%),
followed by group 9 (27%). There has been no reason to suspect an outbreak of nosocomial
origin. The total consumption of antimicrobial agents was 10.7-12.1 DID per year in primary
care; 1.14-1.30 DID per year in hospital care.
Of eight oral agents tested, only three showed a generally high susceptibility; mecillinam
(91%), nitrofurantoin (96%) and fosfomycin (99%). The corresponding figures for the fifteen
tested parenterally administrated drugs were; amikacin (96%), tigecycline (99%), colistin
(99%) and ≥99% susceptibility for the carbapenems.
Sixty eight percent of the isolates were multiresistant. The most common multiresistance
pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprimsulfamethoxazole, ciprofloxacin, gentamicin and tobramycin. A significant difference in
susceptibility between CTX-M groups, in favor of group 9 over group 1, was seen for many of
the antibiotics tested; amoxicillin-clavulanic acid, aztreonam, cafepime, ceftibuten,
ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam, temocillin, and tobramycin.
In conclusion this thesis shows that the prevalence of ESBL producing Enterobacteriaceae in
Östergötland was very low but increasing, and the total consumption of antimicrobial agents
was stable. A majority of the isolates were multiresistant and a significant difference in
susceptibility between CTX-M groups, in favor of group 9 over group 1, was seen for many of
the antimicrobial agents tested.
Table of contents
1
1.1
1.2
1.2.1
1.2.2
1.3
1.4
1.5
1.6
1.6.1
1.6.2
2
3
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
4
4.1
4.2
4.3
4.4
4.5
5
5.1
6
7
8
9
Introduction
Enterobacteriaceae
Antimicrobial agents
Beta-lactam antibiotics
Other antimicrobial agents
Extended spectrum beta-lactamases
Susceptibility testing
Molecular detection
Epidemiology of antibiotic resistance
Extended-spectrum beta-lactamases
Resistance surveillance
Aims
Material and methods
Setting
Antibiotic consumption
Bacterial isolates
Susceptibility testing
Preparation of DNA
PCR
Nucleotide sequencing
Cloning
Bioinformatics tools
Statistics
Ethical clearance
Results
Paper I
Paper II
Paper III
Paper IV
Paper V
Discussion
Conclusion
Acknowledgements – Tack
Images and copyright permissions
Disclaimer
References
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List of papers
Paper I
Monstein H-J, Tärnberg M & Nilsson LE. 2009. Molecular identification of CTX-M and
blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13sequence tagged PCR-amplicons. BMC Infect Dis. 9:7.
Paper II
Tärnberg M, Nilsson LE & Monstein H-J. 2009. Molecular identification of blaSHV,
blaLEN and blaOKP beta-lactamase genes in Klebsiella pneumoniae by bi-directional
sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons. Mol Cell
Probes. 23:195-200.
Paper III
Östholm-Balkhed Å, Tärnberg M, Nilsson M, Johansson AV, Hanberger H, Monstein H-J &
Nilsson LE. 2010. Prevalence of extended-spectrum beta-lactamase-producing
Enterobacteriaceae and trends in antibiotic consumption in a county of Sweden. Scand J Infect
Dis. 42:831-8.
Paper IV
Tärnberg M, Östholm Balkhed Å, Monstein H-J, Hällgren A, Hanberger H & Nilsson LE.
2011. In vitro activity of beta-lactam antibiotics against CTX-M-producing Escherichia coli.
Eur J Clin Microbiol Infect Dis. 30:981-7.
Paper V
Östholm Balkhed Å*, Tärnberg M*, Monstein H-J, Hällgren A, Hanberger H & Nilsson LE.
In vitro susceptibility of CTX-M-producing Escherichia coli to non-beta-lactam agents.
Submitted.
*Å Östholm Balkhed and M Tärnberg contributed equally to this work.
Abbreviations
BSAC
cfu
CLSI
DDD
DID
ESBL
EUCAST
LPS
MIC
MDA
MPA
NCBI
PCR
SMI
SRGA
Strama
UTI
British Society for Antimicrobial Chemotherapy
colony forming unit
Clinical and Laboratory Standards Institute
Defined Daily Dose
DDD per 1 000 inhabitants and day
extended spectrum beta-lactamase
European Committee on Antimicrobial Susceptibility Testing
lipopolysaccharide
minimal inhibitory concentration
multiple displacement amplification
Medical Products Agency
National Center for Biotechnology Information
polymerase chain reaction
Swedish Institute for Infectious Disease Control
Swedish Reference Group of Antibiotics
Swedish Strategic Programme against Antibiotic Resistance
urinary tract infection
1
Introduction
1.1
Enterobacteriaceae
Members of the bacterial family Enterobacteriaceae are found in the environment but also
make up part of the normal microbiota of the intestine in humans and other animals. They are
rod-shaped and stain Gram-negative, non-sporulating, facultative anaerobes that ferment
different carbohydrates to obtain carbon. [1] They may grow as mucoid colonies when
cultivated on agar plates, but only Klebsiella spp. are truly encapsulated [2]. The
Enterobacteriaceae can be divided in 51 genera [3] and the number of species is continuously
increasing.
Members of the Enterobacteriaceae can cause many different kinds of infections. Urinary
tract infections (UTIs) are the most common, followed by pneumonia, wound infections and
infections of the bloodstream and central nervous system, see table 1. Some genera are
common causes of intestinal infections such as enteritis and diarrhoea. They also make up an
essential part of nosocomial infections, especially catheter related UTIs and ventilator
associated pneumonia. [1, 2, 4, 5]
Table 1. Clinically important members of the family Enterobacteriaceae commonly causing infections [1, 2, 4, 5].
Genus
Clinically important species
Citrobacter
C. freundii
Common type of infections
UTIs, pneumonia, meningitis, septicaemia, wound infections
Enterobacter
E. aerogenes, E. cloacae
UTIs, pneumonia, septicaemia, wound infections
Escherichia
E. coli
UTIs, diarrhoea, septicaemia, meningitis
Klebsiella
K. pneumoniae, K. oxytoca
UTIs, pneumonia, septicaemia
Morganella
M. morganii
UTIs, septicaemia
Plesiomonas
P. shigelloides
diarrhoea, septicaemia
Proteus
P. mirabilis, P. vulgaris
UTIs, pneumonia, septicaemia, meningitis, wound infections
Providencia
P. rettgeri, P. stuartii
UTIs
Salmonella
S. enteritica
diarrhoea, typhoid fever, septicaemia, UTIs, osteomylitis
Serratia
S. marcescens, S. liquefaciens
UTIs, pneumonia, wound infections, septicaemia
Shigella
S. sonnei, S. flexneri
diarrhoea, dysentery
Yersinia
Y. pestis, Y. enterocolitica
plague, enteritis, diarrhoea, septicaemia
Escherichia coli are important inhabitants of the human intestine – one of the most frequently
found species of facultative anaerobes in this environment. Although they constitute less than
one percent of the total microbiota, they are found in the faecal flora of almost all healthy
adults [6]. There are several toxin producing strains of E. coli causing diarrhoea, but most
commonly these bacteria cause community onset UTIs [4, 5], especially among women and
adolescent girls [7]. Other types of infections that can involve E. coli are meningitis,
septicaemia, pneumonia, intra-abdominal and gynaecological infections etc [4].
Klebsiella spp. are found in human faeces, and sometimes pharynx, but are also frequently
found in the environment. They are common causes of nosocomial infections such as
catheter related UTIs and ventilator associated pneumonia, and the mortality of these
infections is high. The clinically most important member of the genera is Klebsiella
pneumoniae, followed by Klebsiella oxytoca. [2]
1
For a long time the gold standard method for defining a bacterial species has been DNA-DNA
hybridisation, rendering a taxonomical definition, which is not possible to perform in a routine
clinical microbiological laboratory. At routine laboratories differentiation between species of
the Enterobacteriaceae is usually performed using different biochemical tests [1]. The isolates’
ability to ferment different carbohydrates and inorganic compounds is assessed and interpreted
by laboratory personnel of different experience level. Isolates with fermentation patterns
difficult to interpret, but considered clinically important, may be subjected to full or partial
DNA-sequencing of the 16S rRNA gene as an alternative way to designate a species [8].
Among the Enterobacteriaceae the difference between species is sometimes minute,
phenotypically as well as by 16S rDNA sequencing, thus sometimes only the genus can be
determined in the setting of a clinical routine laboratory.
1.2
Antimicrobial agents
The use and antimicrobial spectrum of a drug varies with geographic location and over time,
and may be stipulated in local policies. The validity of drugs in Sweden are found in FASS,
the Swedish pharma industries’ register of all approved drugs, and assessments are regularly
made by the Swedish Reference Group of Antibiotics (SRGA).
1.2.1 Beta-lactam antibiotics
Beta-lactam antibiotics are the largest and most commonly used group of antimicrobial agents
in Sweden as well as world-wide. The group is distinguished by a chemical structure known
as the beta-lactam ring (figure 1a). Based on their chemical structure they can be divided in
four different groups, depending on the ring structure fused to the beta-lactam ring (figure 1be), but are often divided in the following groups; penicillins, cephalosporins, carbapenems,
monobactam and beta-lactamase inhibitors. [9]
The beta-lactam antibiotics block the transpeptidation of the cell wall component
peptidoglycan, through inhibition of penicillin binding proteins, but exactly how this leads to
cell death is not known [9-11]. Since penicillin binding proteins are not found in cells of the
Animalia the toxicity of beta-lactams is very low, but allergy against penicillins and other
beta-lactams can be very serious [12]. Beta-lactam antibiotics are mainly semi-synthetic
compounds, originating from fungi and bacteria found in the environment [9]. More than 80
different beta-lactams are in clinical use, but in Sweden only 23 are marketed [13]. Some
beta-lactams have a very narrow antimicrobial spectrum, while others have a very broad
spectrum and targets both Gram-positive and Gram-negative bacteria.
Resistance against beta-lactams is primarily mediated by a structural change of the penicillin
binding proteins (leading to lower affinity of the drug) or by bacterial production of enzymes
cleaving the beta-lactam ring. Other mechanisms are decreased permeability or active
transportation via efflux pumps. [11]
Penicillins are also called penams and are bicyclic (figure 1b). Penicillin G was one of the
first antibiotics to be commercialised in the 1940’s. They are divided in sub groups depending
on their antimicrobial spectrum and stability against penicillinases, and may be administrated
orally or parenterally. Penicillins are most often excreted non-metabolised by the kidneys, thus
the concentration in urine can reach high levels. [11]
Mecillinam, also known as amidinocillin, is often given as its pro drug pivmecillinam. In
Sweden only the oral formula is available [14], and it exerts good activity especially against
E. coli, Klebsiella spp., and Proteus mirabilis. The drug is used in the treatment of
uncomplicated UTIs. [15]
2
Temocillin is a narrow-spectrum parenteral drug with very limited availability; it is only
approved in the UK and Belgium. It is stable against extended-spectrum beta-lactamases
(ESBLs) and can be used in the treatment of septicaemia, UTIs and lower respiratory tract
infections caused by Enterobacteriaceae. Clinical use has shown that it can be considered as
an alternative in treating infections caused by ESBL producing Enterobacteriaceae, primarily
UTIs. [16]
Cephalosporins, or cephems, are also bicyclic (figure 1c). Although discovered earlier, they
did not become commercially available until the 1960’s. They all show stability against a
wide range of beta-lactamases. [10] Noteworthy is the ability of emergence of resistance
during treatment of Enterobacter spp., Citrobacter spp., Serratia spp., Morganella spp., and
Providencia spp. This is due to either spontaneous mutation or up-regulation of the
production of naturally occurring beta-lactamases. [17, 18] Cephalosporins can be
administrated orally, intramuscularly or intravenously, depending on the drug. They are often
given empirically in combination with an aminoglycoside or metronidazole (depending on
suspected aetiology) for treatment of serious infections such as severe pneumonia, intraabdominal infections, septicaemia or in patients with febrile neutropenia, but it has been
widely debated if combination therapy is favourable or not [19-22]. In everyday language the
cephalosporins are classified into “generations”, but for clarity this should be avoided
scientifically.
Ceftibuten is an oral cephalosporin approved for the treatment of UTIs and acute
exacerbations of chronic bronchitis [23], but SRGA only promotes its use for treatment of
UTIs. The antimicrobial spectrum includes the urinary tract pathogens E. coli, Klebsiella spp.
and Proteus spp. and some pathogens of the respiratory tract. [24]
Ceftazidime shows good activity against several Gram-negative bacteria, including
Enterobacteriaceae (especially E. coli, Klebsiella spp. and Proteus spp.), and Pseudomonas
aeruginosa. It is administrated parenterally and mainly used for treatment of nosocomial
pneumonia and in patients with cystic fibrosis, and suspected septicaemia in neutropenic
patients. [25, 26]
Cefotaxime is a broad spectrum cephalosporin for parenteral administration, used for the
treatment of serious infections in and from internal organs, skin and soft tissues, including
meningitis. The antimicrobial spectrum covers a large portion of the Enterobacteriaceae
(especially E. coli, Klebsiella spp., Proteus spp., Salmonella spp. and Shigella spp.) and
several skin and respiratory tract pathogens. [27]
Cefepime is a broad spectrum cephalosporin with higher stability against beta-lactamases
than the other approved cephalosporins available in Sweden. Thus its antimicrobial spectrum
almost completely covers the Enterobacteriaceae, including the members naturally producing
beta-lactamases. It is also effective against P. aeruginosa and several other Gram-negative
non-Enterobacteriaceae, and Gram-positive pathogens of the skin and respiratory tract. It is
administrated parenterally and the use is mainly treatment of nosocomial pneumonia and
suspected septicaemia in neutropenic patients. [28]
Carbapenems are bicyclic compounds (figure 1d) that came into use in the 1980’s. They
show very good stability against beta-lactamases, including many of the ESBLs. In
combination with their unique mechanism of outer membrane permeability this results in a
very broad spectrum of activity, including Gram-positive and Gram-negative aerobic and
anaerobic bacteria. All carbapenems are administrated parenterally. [29]
3
Imipenem is always given together with an enzyme inhibitor, cilastatin, preventing its rapid
degradation by the kidneys to inactive metabolite [29]. The antibacterial spectrum is very
broad and includes P. aeruginosa, Acinetobacter spp., most Enterobacteriaceae (not Proteus
spp., Providencia spp. or Morganella spp.), Enterococcus faecalis, pathogens of the skin and
respiratory tract and many anaerobes. The drug is saved for severe infections originating from
internal organs and suspected septicaemia in neutropenic patients, and is effective against
infections caused by ESBL producing bacteria, but not against methicillin-resistant
staphylococci, Enterococcus faecium or Stenotrophomonas spp. [30]
Meropenem has an antibacterial spectrum similar to that of imipenem, also including
Neisseria meningitides, Proteus spp., and Morganella spp. This broadens the use, compared
to imipenem, to also include meningitis. The activity against enterococci and
Stenotrophomonas spp. is insufficient. [31]
Ertapenem is a newer carbapenem, with a narrower antibacterial spectrum than the others. It
is active against common pathogens of the skin and respiratory tract, Enterobacteriaceae and
some anaerobes, but not P. aeruginosa or Acinetobacter spp. [32]. The drug is approved for
the treatment of pneumonia, intra-abdominal, acute gynaecological and diabetic foot
infections [33], but the recommendation of SRGA is to save the drug for treatment of
cephalosporin resistant Enterobacteriaceae in the polyclinic setting, since it only requires
once daily dosing [32].
Monobactams are monocyclic compounds (figure 1e), and aztreonam is the only clinically
available drug of this group [29]. The antibacterial spectrum is narrow; it covers aerobic
Gram-negative bacteria, and the drug is administrated parenterally. Approved indications
include gonorrhoea and complicated infections originating from the urinary or respiratory
tract. Aztreonam seldom give rise to adverse side effects. [34]
Beta-lactamase inhibitors show very weak antibacterial activity and are used in
combinations with penicillins to act as “suicide substrates”. They form stable intermediates,
thus capturing and deactivating the beta-lactamases. They are stable against penicillinases,
some chromosomal cephalosporinases and some ESBLs. [29]
Amoxicillin-clavulanic acid is a combination whose antibacterial activity covers both
Gram-negative and Gram-positive, aerobes and anaerobes, found on various sites such as
skin, respiratory tract and saliva. It also exerts effect on parts of the Enterobacteriaceae. The
drug is used in the treatment of uncomplicated pneumonia and upper respiratory tract
infections, UTIs, and bite wounds. [35]
Piperacillin-tazobactam is a parenteral, broad spectrum drug combination used in serious
infections such as nosocomial pneumonia, intra-abdominal infections or in patients with
febrile neutropenia. The antibacterial spectrum covers pathogens of the skin and respiratory
tract, Enterobacteriaceae, Pseudomonas spp. and many anaerobes. [36]
4
Figure 1. Chemical structures of a) the beta-lactam ring; the core structure of b) penicillins, c) cephalosporins, d)
carbapenems and e) monobactam; and f) an oxyimino group.
1.2.2 Other antimicrobial agents
Aminoglycosides belongs to the oldest commercially available antibacterial drugs; they
were first used in the 1940’s and originate from soil bacteria. They are one of the most
important groups of antibiotics, which exert strong bactericidal effect on various aerobic
Gram-negatives (including all pathogenic Enterobacteriaceae), staphylococci and some
mycobacteria. Aminoglycosides inhibit the prokaryotic protein synthesis by binding the 16S
subunit of the small 30S subunit in ribosomal RNA. Adverse events, such as nephrotoxicity
and ototoxicity, are commonly encountered when dosage is too high [37]. More than ten
different aminoglycosides have been developed, but the Swedish Medical Products Agency
(MPA) has only approved the use of four of them; amikacin, gentamicin, netilmicin, and
tobramycin [38]. Generally, amikacin is regarded as the more effective since its chemical
structure makes it less affected by enzymatic deactivation [37]. Aminoglycosides are
administrated parenterally, often in combination with a beta-lactam antibiotic, in infections
such as endocarditis, septicaemia, severe pneumonia, intra-abdominal infections or in
patients with febrile neutropenia [38]. Tobramycin is also available as inhaler for treatment
of patients with cystic fibrosis [39].
Resistance to aminoglycosides can be mediated by a) bacterial production of aminoglycoside
modifying enzymes, b) decreased uptake due to membrane permeability, transport or efflux,
c) point mutations at the drug target or d) methylation enzymes changing the target [37].
The folic acid inhibitors, trimethoprim and sulfamethoxazole, are often given in
combination, in a ratio of 1:5. The combination is sometimes called co-trimoxazole or
trim-sulfa and abbreviated TMP-SMX. They exert a synergistic effect by inhibiting the
enzymes of two following steps in the synthesis of tetrahydrofolate, a coenzyme involved
in many reactions including the synthesis of DNA. Sulfamethoxazole is a sulfonamide,
5
one of the very first antimicrobial drugs discovered and used in the 1930’s. Trimethoprim
came in use in the 1960’s and is a pyrimidine. [40]
The drug combination can be administrated orally or parenterally and is approved for the
treatment of UTIs and serious infections originating therefrom, prostatitis, acute
exacerbations of chronic bronchitis, pneumocystosis, salmonellosis and shigellosis. The
antibacterial spectrum includes pathogens of the skin and respiratory tracts, most
Enterobacteriaceae and Stenotrophomonas spp. [41] Trimethoprim can be used alone and is
given orally for the treatment and long term prophylaxis of uncomplicated UTIs. The
antibacterial spectrum is narrow and includes common Gram-negative and Gram-positive
pathogens of the urinary tract. [42]
Resistance to both trimethoprim and sulfamethoxazole is common and may be caused by
changes of permeability, overproduction or modifications of target enzymes. Most common
is production of a modified dihydrofolate reductase causing resistance to trimethoprim. [40]
The first quinolone developed in the early 1960’s was nalidixic acid, a fully synthetic drug
[43]. The drug is no longer in clinical use in Sweden, but has been used in vitro as a
screening substance for quinolone resistance [44]. In the 1980’s the flouroquinolones were
developed. Some flouroquinolones have a very broad antimicrobial spectrum and targets both
Gram-positive and Gram-negative bacteria. They can be administrated both orally and
parenterally. They disrupt the DNA synthesis through inhibition of the enzymes DNA-gyrase
and topoisomerase IV. Resistance to the quinolones was for a long time of chromosomal
origin, due to single mutations in the target enzymes or changing permeability. In the last ten
years plasmid mediated quinolone resistance, particularly qnr-genes, has arisen. [43]
Ciprofloxacin is a very broad spectrum fluoroquinolone targeting respiratory tract
pathogens, Pseudomonas spp., Acinetobacter spp., most Enterobacteriaceae, and also
Bacillus anthracis [45]. Acquired resistance is common, and the approved indications include
Gram-negative respiratory tract infections, UTIs and prostatitis, gastrointestinal, urogenital
and intra-abdominal infections and other infections caused by susceptible pathogens [46].
Nitrofurantoin is a narrow spectrum drug, covering Staphylococcus saprophyticus,
enterococci and E. coli. It came into use as early as the 1950’s, but resistance is unusual.
Nitrofurantoin is given orally in combination with food and adverse effects are unusual. It
is used solely for treatment and long term prophylaxis of UTIs. The mechanism of action is
unclear but seems to involve damaging of bacterial DNA. [47]
Colistin, or polymyxin E, originates from Bacillus colistinus and was discovered in the late
1940’s. It was in clinical use during the 60’s and 70’s, but due to problems with
nephrotoxicity it was then abandoned. The reputation as a drug with severe side effects has
been questioned; the nephrotoxicity has been shown to be reversible, and can be minimised
using careful monitoring and selection of patients. Colistin can be administrated orally,
topically, intravenously or via inhalation, and is mainly used in the treatment of P. aeruginosa.
[48] In Sweden only the inhaler is available, for use in patients with cystic fibrosis [49]. The
intravenously administrated formula is available for the infectious disease departments through
a special license issued by the MPA [50]. The drug is a large polypeptide, and interacts
electrostatically with the outer membrane and neutralises LPS. The bactericidal effect is
extremely rapid. The antibacterial spectrum is narrow but covers several Enterobacteriaceae
(E. coli, Enterobacter spp., Klebsiella spp., Salmonella spp., and Shigella spp.), the non-
6
fermenters P. aeruginosa, Acinetobacter spp. and Stenotrophomonas spp., and Gramnegatives of the respiratory tract. Resistance is unusual. Lately colistin has emerged as an
alternative treatment for infections caused by multiresistant Gram-negatives. [48]
Fosfomycin is a small epoxide molecule discovered in the late 1960’s, originating from soil
bacteria. It is inhibiting the synthesis of peptidoglycan, thus blocking the formation of the
cell wall. [51] The drug can be administrated orally or intravenously, but is currently
unavailable in Sweden. A special licence for use is available from the MPA [50]. The
antibacterial spectrum covers some Enterobacteriaceae (especially E. coli and P. mirabilis)
and some pathogens of the skin and respiratory tracts. It is used in serious infections of
internal organs including meningitis, soft tissue infections and UTIs. [51]
Tigecycline is one of few newer drugs (launched in the mid 00’s) covering Gram-negative
bacteria. The antibacterial spectrum includes enterococci, staphylococci and streptococci,
some Enterobacteriaceae, and a few anaerobes. [52] It is administrated intravenously for the
treatment of complicated skin, soft tissue and intra-abdominal infections and is considered
a last line treatment option [53]. Tigecycline is the only commercially available drug of the
glycylcyclines, a group of antimicrobial agents structurally related to the tetracyclines; the
core structure is minocycline. Tigecycline inhibits the prokaryotic protein synthesis by
binding ribosomal 30S, in the same manner as tetracyclines. Resistance is rare, the size and
shape of the drug prevents the common resistance traits inhibiting tetracyclines. [52]
1.3
Extended-spectrum beta-lactamases
Beta-lactamases are enzymes that hydrolyse beta-lactam antibiotics, and the most common
mode of action for beta-lactam resistance in Gram-negative bacteria. They do this by
breaking up the nitrogen-carbonyl bond in the beta-lactam ring. [9]
The classification of beta-lactamases is complicated and based on similarities at the amino
acid level (the Ambler classification [54]) and by function (the Bush/Jacoby classification
[55]). The issue of an exact definition of extended-spectrum beta-lactamases had been under
debate [56-59].
In this thesis an ESBL is defined as an enzyme found in the Bush/Jacoby functional group
2be. They belong to Ambler group A, hydrolyses penicillins, early cephalosporins and at
least one of the oxyimino (figure 1f) beta-lactams (aztreonam, cefepime, cefotaxime,
ceftazidime and ceftriaxone), and also exhibit in vitro inhibition by clavulanic acid. [55]
Unfortunately, not all ESBLs are typical. There are enzymes with extended cephalosporinase
activity belonging to Ambler group D, and others that are not inhibited by clavulanic acid. In
the Bush/Jacoby system these unusual enzymes can be found in groups 2ber, 2ce, 2de and 2e.
[55] An “e” in the classification indicates “extended-spectrum”, an “r” indicates “inhibitor
resistant”.
Beta-lactamases of TEM- and SHV-type are found in the functional groups 2b, 2be and 2br;
some TEM can also be found in group 2ber [55]. The parental enzyme TEM-1 was
discovered in the mid-1960’s from a Greek patient, a few years later the SHV-1 was
described. They show roughly two-thirds likeliness at the amino acid level and both enzymes
have by point mutations evolved to new penicillinases but also extended-spectrum
cephalosporinases. In particular, mutations at Ambler position (see ref [60] for Ambler
positions) 238 and 240 seems to confer ESBL phenotype. [61] The first ESBL encountered
7
was SHV-2, in the mid 1980’s [62]. More than 180 TEM and 120 SHV variants are known
today [63].
CTX-M beta-lactamases are found exclusively in functional group 2be [55]. They are thought
to originate from chromosomal ESBL genes found in Kluyvera spp. [64], an opportunistic
pathogen of the Enterobacteriaceae found in the environment. The first CTX-M proteins were
discovered in the late 1980’s [64], and today more than 100 variants have been sequenced
[63]. Based on their amino acid sequences, they can be divided in five groups (CTX-M group
1, 2, 8, 9, and 25) [64].
ESBL, and ESBL-like, genes can be found both chromosomally or on transferable genetic
elements such as plasmids. They are predominantly found in Enterobacteriaceae but are not
uncommon among non-fermentative Gram-negative rods such as P. aeruginosa or
Acinetobacter spp. TEM genes are primarily found on mobile elements, as are CTX-M genes.
SHV genes on the other hand are often found chromosomally in K. pneumoniae but almost
always are mobile in E. coli. [65]
Chromosomal ESBL/ESBL-like genes are commonly found in clinically encountered
Enterobacteriaceae such as K. oxytoca (K1/KOXY/OXY), Proteus vulgaris (CumA) and
Proteus penneri (HUGA). They are also found in a variety of more uncommon species,
occasionally involved in infections of particularly the immunocompromised (e.g. Citrobacter
spp. (CdiA, CKO, and SED-1), Kluyvera spp. (KLUA, KLUC, and KLUG), Serratia
fonticola (FONA) or Rahnella aquatilis (RAHN-1)). [61, 64, 66] Apart from SHV genes,
LEN genes in K. pneumoniae are also chromosomally encoded non-extended-spectrum betalactamases with amino acid sequences similar to ESBLs [67, 68]. It is not always easy to
distinguish between all these different enzymes of different origin, and this constitutes a
problem at clinical laboratories.
Bacterial isolates expressing CTX-M enzymes are often co-resistant to several other
antimicrobial agents. It has been shown that the large plasmids harbouring CTX-M also cocarries other resistance denominators. The CTX-M containing plasmid involved in a Swedish
outbreak has been sequenced and it also carries TEM-1 and OXA-1 beta-lactamases, and
resistance genes to antimicrobial agents such as aminoglycosides, quinolones, tetracyclines,
and trimethoprim-sulfamethoxazole [69].
1.4
Susceptibility testing
The MIC – minimal inhibitory concentration – is the lowest concentration of a drug where
the growth of a bacterial isolate is inhibited, in other words, the concentration where no
visible growth can be seen when tested in vitro. This is not to be confused with MBC –
minimal bactericidal concentration – the lowest concentration where the bacterial isolate is
99.9% killed. In “MICological” papers whole populations of bacterial isolates are examined,
and often described using the terms MIC50; the concentration where half the population is
inhibited, or MIC90; the concentration where 90 percent of the population is inhibited.
Sometimes the mode (i.e. the value that occurs most frequently) is given. MIC is measured
in mg/L. [70, 71]
MIC-values can be transformed into an ordinal scale, where S stands for sensitive, I for
intermediate and R for resistant. The breakpoints for where a certain bacterial species is
considered to be S, I or R is set by committees and organizations, and aims at classifying the
bacterium as treatable or not. Internationally, the American CLSI (Clinical and Laboratory
8
Standards Institute, formerly known as the NCCLS) breakpoints and guidelines dominates. In
Europe, several national breakpoint-committees have joined forces under the EUCAST, the
European Committee on Antimicrobial Susceptibility Testing. In Sweden the Swedish
Reference Group of Antibiotics, SRGA, has for a long time set up breakpoints and guidelines
for clinical laboratories. Recently they joined with the other Scandinavian dittos to form the
Nordic-AST, and regarding breakpoints and susceptibility testing, the EUCAST
methodology is now implemented in Sweden.
When establishing breakpoints many factors have to be taken under consideration; the MIC
of the bacterium and the pharmacokinetics of the antibacterial agent in relation to the clinical
outcome. How to do this is under debate [72].
The gold standard methods for measurement of MICs are broth or agar dilutions,
cumbersome methods usually not performed by clinical routine laboratories. Etest
(BioMérieux, Marcy L’Etoile, France) is a commercially available gradient strip that
correlates well with the dilution methods, and is more convenient to handle. A fixed gradient
concentration of drug has been applied on the back of a plastic strip; the front is printed with
a reading scale. The strip is placed on an agar dish inoculated with a standardized amount of
bacteria, and then incubated for a certain time, according to the manufacturer’s instructions.
After incubation the MIC-value can be determined by reading the lowest concentration on the
strip where no growth can be observed.
The disk diffusion method was standardised in 1966 by Bauer and co-workers [73] but has
since been developed and adapted. A paper disc containing a fixed amount of the drug is
placed on an agar dish inoculated with a standardized amount of bacteria, and then
incubated for a certain time. Several discs are placed on the same agar dish. After
incubation, the zone diameters around the discs are measured. This method gives no actual
MIC-value, but the measurement can be interpreted and the bacterial isolate classified as
sensitive, intermediate or resistant.
1.5
Molecular identification
For use in downstream molecular methods, DNA needs to be purified from the sample (i.e.
bacterial isolate). DNA extraction used to be cumbersome but today automated systems have
made this a routine procedure. When samples contain minute amounts of DNA multiple
displacement amplification (MDA) can be used as a method to amplify entire genomes,
chromosomes and plasmids. This can be done with many different sample sources such as
forensic evidence, tissue samples, long time stored DNA, or frozen bacterial cultures. MDA
was described in the early 2000’s and is not based on PCR, but instead relies on an
isothermal reaction using random hexamer primers and the bacteriophage ɸ 29 DNA
polymerase [74]. This polymerase “pushes away” any 5’-end of double stranded DNA
coming in its way by creating a new replication fork, and continues to extend the
complementary DNA strand, thus creating a hyper branched structure (figure 2). MDA has
been shown to exert many advantages over other whole genome amplification methods; it
requires very small amounts of starting material, is very accurate, generates very long DNAstrands, and outcompetes Taq polymerase inhibitors (useful in downstream applications).
[75]
PCR is one of the basic tools in the molecular biologist’s toolbox; it is a method for
amplification of specific DNA fragments. PCR was described in the early 1980’s and uses
two specific primers complementary to the DNA strand and a heat-stable DNA polymerase
9
(often Taq polymerase). Using cycling temperatures the double stranded DNA denatures,
the primers anneal to the now single stranded DNA, and finally the polymerase incorporate
nucleotides and extend the complementary DNA strand. This is repeated for 25-40 times,
generating (theoretically) twice as many DNA fragments each cycle (figure 3). [76]
Figure 2. MDA reaction. Illustration by Per Lagman.
To ensure high specificity, primers should be carefully designed. Not only should they fulfill
the physical requirements, they should also make a base pair match only to the desired DNA
target sequence. Factors to take into consideration include that the primers should a) include
an area of interest of the DNA strand to be examined, b) be of equal length (18-25
nucleotides is usually appropriate), c) have an equal melting temperature, Tm, in both
members of the pair, that should be as high as possible (≥55°C is favourable), d) have a GC
content around 50%, e) not have too many GC’s in a row, f) be perfectly matched at the 3’end, and g) not make base pair matching within themselves or each other [76, 77]. This
explorative work can be done manually but is often aided by computer programs and/or free
internet resources such as Primer3 [78] or PrimerQuest [79]/OligoAnalyzer [80].
To ensure that a PCR-product of the right size has been amplified, amplicons are usually
visualised by electrophoresis before downstream applications. Amplicons are separated based
on its size, and then made visible by ethidium bromide (or other DNA binding dyes). To
make certain the amplicon is the desired one, it has to be sequenced. DNA sequencing was
described in 1977 by Sanger and co-workers [81], and has since been developed. The basis of
this procedure is that dideoxynucleotides inhibits DNA polymerase. By adding both
“normal” nucleotides and labelled dideoxynucleotides to a cocktail containing polymerase
and single stranded DNA fragments, occasionally a dideoxynucleotide will be incorporated,
terminating the elongation. Fragments of all sizes will be synthesised and by determine the
size and label at the 3’-end of each fragment, it is possible to “read” the sequence (figure 4).
[76] This used to be very cumbersome, involving radioactively labelled nucleotides,
acrylamide gel electrophoresis, x-ray film and manual interpretation, but thanks to
technology this is today a routine procedure that can be carried out in a high-throughput
manner. The raw data from the sequencing contains a chromatogram (figure 5) that has to be
interpreted, either manually or computer assisted.
10
Figure 3. PCR reaction. Illustration by Per Lagman.
11
Figure 4. Sanger sequencing. Illustration by Per Lagman.
12
Figure 5. Sequencing chromatogram. Double peaks, indicating the presence of more than one allelic variant, are
marked by arrows.
Bioinformatics tools are used to analyse the obtained sequence data. A sequence is usually
compared with other sequences in the same set of samples and with the content of free online
databases such as GenBank [82] using the Blast tool [83]. Sequences are aligned with each
other, by free online resources such as ClustalW [84] or freeware/software, to visualise if
and where differences are found (figure 6a). This may render a re-evaluation of
chromatograms. When large numbers of sequences are dealt with it may be easier to
visualise likeliness by a phenetic tree/dendrogram; a graphic representation of affinity
(figure 6b).
Figure 6. Alignment of six DNA sequences; three unique and three similar. a) Identical nucleotides indicated by
dots, b) identical sequences grouped together.
1.6
Epidemiology of antimicrobial resistance
It is well accepted that the use of antimicrobial agents, by selective pressure, is what
primarily drives resistance. For Europe, a relationship between antibiotic consumption and
resistance among clinical isolates, on a national level, has been shown [85]. Reducing the
pressure will not make the resistance go away instantly, as has been shown for vancomycin
resistant enterococci after the ban of avoparcin in animal husbandry [86, 87], or in
interventions reducing the use of trimethoprim-sulfamethoxazole [88-90]. It is speculated
that if no fitness cost is present for bacteria carrying resistance genes, these genes will not
be lost when the selective pressure is gone [91].
Selection of resistant subpopulations can occur within a patient during antimicrobial
treatment. This is frequent for certain “drug-bug” combinations such as cephalosporins with
Enterobacter spp. and Citrobacter spp. [18]. More often development of resistance is a rare
event that can take place in the environment, in healthy or ill animals or humans, by point
mutations or direct uptake of genetic material from other sources. These resistant bacteria
13
are spread by clonal expansion (vertical transfer), i.e. the mother cell passes the genes on
during cell division, generation after generation. Occasionally resistance genes are relocated
from the bacterial chromosome to transferrable elements such as plasmids, and can then be
spread by horizontal transfer, between bacteria within and between species. [9]
1.6.1 Extended-spectrum beta-lactamases
ESBLs of the TEM and SHV types began to spread in the 1980´s. For almost two decades
these enzymes were most commonly encountered in nosocomial infections due to ESBL
producing K. pneumoniae. In the late 1990´s, early 2000´s, the enzymatic background in
infections caused by ESBL producing bacteria began to change; the CTX-M enzymes has
since come to dominate. What is worrisome is the spread of CTX-M to E. coli and
community onset UTIs. Today CTX-M enzymes are the dominating ESBLs world-wide, but
the dominating enzyme differs somewhat between countries. The huge success of these
genes is thought to involve clonal spread and the fact that humans, animals and foodstuff
travel across continents on a prevalent basis. [92, 93]
Scandinavia is known as a low-consumption area regarding antimicrobial agents, and this is
reflected in the generally low levels of antimicrobial resistance. Despite this we are not
spared when it comes to ESBLs, but, when compared to other parts of Europe, cephalosporin
resistance is low (figure 7). [94]
The first documented outbreak of ESBL producing bacteria in Sweden, in 2002, comprised of
CTX-M group 1 producing E. coli [95]. In the past four years, papers communicating
nosocomial outbreaks and clinical infections due to CTX-M producing E. coli and K.
pneumoniae in Sweden have increased (figure 8). The outbreaks have been due to CTX-M 15
(or very similar) producing bacteria. [96-98] Regarding ESBL producers causing infections in
the community and hospital environment, the pattern is very similar regardless of
investigator; CTX-M group 1 and particularly CTX-M 15 is dominating, with CTX-M group
9 and CTX-M 14 as the runner up. CTX-M group 2 is found sporadically, as are TEM and
SHV. [99-103] This is in concordance with the situation in Denmark [104-106], Finland
[107] and Norway [108, 109].
1.6.2 Resistance surveillance
Every year (since 2001) the Swedish Institute for Infectious Disease Control (SMI), the
National Veterinary Institute (SVA) and the Swedish Strategic Programme against
Antibiotic Resistance (Strama, until 2009) publishes a report, SWEDRES [110]/SVARM
[111], on antimicrobial consumption and antimicrobial resistance in human and veterinary
medicine, on a national level. Similar resistance surveillance reports are conducted in
Denmark [112] and Norway [113].
All Swedish routine clinical laboratories take part in the annual “100-study”, the Resistance
Surveillance and Quality Control Programme coordinated at SMI. Resistance patterns of
certain “drug-bug” combinations (100 of each species), obtained in the daily work are
reported. About three quarters of the Swedish laboratories also participate in the European
Antimicrobial Resistance Surveillance Network (EARS-Net, formerly EARSS). Resistance
patterns of certain “drug-bug” combinations from all invasive infections are reported. For both
programmes E. coli have been reported from 2001, K. pneumoniae since 2005.
14
15
Figure 7. Maps showing the proportion of E. coli with reduced susceptibility to third generation cephalosporins for the years 2002, 2007 and 2010 in Europe. Data
provided by ECDC, extracted from The European Surveillance System – TESSy. Maps were modified by the author to better fit the size of printing.
Figure 8. Map of Scandinavia with reported outbreaks and findings of ESBL. Outbreaks; Stockholm 2002 [95],
Stavanger 2004 [108], Uppsala 2005 [96], Kristianstad 2005-2006 [97], and Kalmar 2008 [98]. Reports at the
national level; Denmark 2008 [105], Finland 2002-2004 [107], Norway 2003 [109], and Sweden 2007 [103]; and
at a local level; Copenhagen 1998-2003 [104], 2008 [106], Stockholm 2001-2006 [99], Stockholm County 2005
[102], Örebro County 1999-2008 [101], and Östergotland County 2002-2007 (this thesis) [100].
16
The national results from these two surveillance programs are presented in SWEDRES.
They are also available as interactive databases; ResNet [114] and EARS-Net [94]. ESBL
producing Enterobacteriaceae were made obliged by law for registration by the laboratories
to SMI, on 1 February, 2007.
Strama is active on the national and local level [115]. In Östergötland, Strama is organised by
the county council and is comprised of infectious disease specialists, infection control
specialists, primary care specialists, microbiologists, pharmacists, and the medical directors at
the local hospitals. The overall mission is to preserve the usefulness of antimicrobial therapy
in human infections by:
•
monitoring and analysing antimicrobial resistance in hospitals and primary care
•
monitoring and analysing consumption of antimicrobial agents in hospitals and primary
care
•
providing guidelines for treatment and prophylaxis
•
giving feed-back to care givers on the adherence to guidelines
This is accomplished by active participation of the Strama-group, its working committees and
care givers, through collection, communication and intervention in the area of antimicrobial
use, resistance and antimicrobial spread. This thesis has its origin in this important work
against antibacterial resistance at the local level.
17
2
Aims
The aims of this thesis were to
•
investigate the occurrence and genotypes of ESBL producing Enterobacteriaceae in
clinical isolates and hygiene screenings from Östergötland, Sweden, in relation to the
consumption of antimicrobial agents
•
determine MICs of beta-lactams and other antimicrobial agents to establish the
occurrence of multiresistance among ESBL producing E. coli and to explore the
potential role of some unorthodox antimicrobial agents as treatments of infections
caused by these bacteria
•
develop simple, accurate and cost-effective methods, well suited for highthroughput analysis, for detection and sub typing of ESBL genes of Bush/Jacoby
group 2be in bacteria belonging to the family Enterobacteriaceae
18
3
Material and methods
Papers I & II: Methodological papers describing an approach of high-throughput PCR
followed by amplicon sequencing, exemplified with CTX-M and SHV-like genes.
Paper III: A retrospective and descriptive epidemiological study on ESBL producing
Enterobacteriaceae in Östergötland County, Sweden.
Papers IV & V: Descriptive studies presenting data on antimicrobial resistance in a
population of ESBL producing E. coli.
3.1
Setting
Östergötland is a rural county in the south east of Sweden, surrounded by mainland, lake
Vättern and the Baltic Sea. The population density is low, around 43 inhabitants per square
kilometre. The population is continuously increasing, and was around 421 000 at the end of
the study period (2007) [116]. There are three hospitals serving the region: one university
hospital (~600 beds) in the municipality, and two smaller secondary care hospitals (~300
and ~200 beds respectively) in the second and third biggest towns, around 40 primary care
centres and 40 private practitioners. The health care system in the entire region is served by
one clinical microbiology laboratory located at the University Hospital in Linköping. The
laboratory is accredited by the Swedish Board for Accreditation and Conformity Assessment
(SWEDAC).
3.2
Consumption of antimicrobial agents
Sales data provided by the National Corporation of Swedish Pharmacies (Apoteket AB, the
sole pharmaceutical distributor in Sweden until 2010), regarding antimicrobial agents were
presented in paper III. The ATC classification and DDD measurement unit were used to
record drug consumption [117]. Data were presented as DID; DDD per 1000 inhabitants and
day.
3.3
Bacterial isolates
All clinical isolates in this thesis were collected at the Clinical Microbiology Laboratory,
Linköping University Hospital during the years 2001 until end of 2007. All isolates were
subjected to biochemical tests to identify them to the species level [1]. All sample sources
were included and all isolates showed ESBL phenotype. Information on gender, age, sample
source and ward level was extracted from the laboratory data journals. The isolates were
stored in Nutrient Broth No 2 (Lab M, Bury, UK) containing 15% glycerol at -70°C until
further analysed.
Paper I: 20 K. pneumoniae and 34 K. oxytoca of clinical origin, collected 2001-2007.
Paper II: 20 K. pneumoniae of clinical origin, 19 of them were also in paper I, collected
2002-2007.
Paper III: All initial and repeat clinical isolates of Enterobacteriaceae with ESBL phenotype
collected 1 January 2002 until 31 December 2007; 208 E. coli, 18 K. pneumoniae, one
Citrobacter koseri and one Shigella sonnei. Isolates of K. oxytoca and P. vulgaris were
excluded due to their carriage of intrinsic beta-lactamase genes.
Papers IV and V: 198 isolates of E. coli, from paper III, repeat isolates excluded.
The control strains used were purchased or received as gifts, see table 2. For MICdeterminations E. coli ATCC 25922 was used as a control.
19
Table 2. Control strains used for PCR-optimisation.
Source
Strain
Culture Collection University of Gothenburg
K. pneumoniae CCUG 54718CTX-M-15
Paper
I
T
American Type Culture Collection
K. oxytoca CCUG 15717
I
K. pneumoniae CCUG 59349qnrB1/CTX-M-15
V
K. pneumoniae CCUG 59358qnrS1/aac(6’)-Ib-cr/CTX-M-15/28
V
K. pneumoniae ATCC 700603SHV-18
I + II
K. pneumoniae ATCC 11296SHV-11
II
K. pneumoniae ATCC 13883SHV-1
II
Dr D. Livermore
K. oxytoca 1980K1
I
Health Protection Agency, UK
E. coli 1204SHV-2
II
E. coli J53SHV-2
II
E. coli J53SHV-1
II
Dr L. Cavaco
E. coli DH10BqnrC
V
National Food Institute, Denmark
E. coli TG1qnrD
V
Prof P. Nordmann
E. coli LoqnrA
V
Hopital de Bicetre, France
3.4
Susceptibility testing
Disc diffusion was performed by the clinical routine laboratory on all clinical isolates,
according to the guidelines of the SRGA valid during the period 2002-2007. Disc diffusion
data on ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole, meropenem and
imipenem was presented in paper III.
ESBL screening (paper I-III) was performed by the clinical routine laboratory on all clinical
isolates, according to the guidelines of the SRGA. Isolates with decreased susceptibility (i.e. I
or R) to cefadroxil was further tested with cefotaxime and ceftazidime, but more often
cefotaxime and ceftazidime were tested directly. Isolates with reduced susceptibility for these
cephalosporins, as deduced by disc diffusion, were further analysed by a confirmatory Etest
(BioMérieux, Marcy L’Etoile, France) with cefotaxime and ceftazidime with and without
clavulanic acid (CT/CTL and TZ/TZL, respectively). An ESBL phenotype was considered
confirmed when
a) an at least 8-fold reduction (≥ 3 twofold dilutions) of the minimum inhibitory
concentration (MIC) in the presence of clavulanic acid was seen, or
b) when there were signs of phantom zones or deformation zones (manufacturer’s
instruction)
Isolates with inconclusive results were re-analysed by the research laboratory.
Etest was performed by the Clinical Microbiology Research Laboratory at Linköping
University, according to the manufacturer’s instructions (BioMérieux). In paper I MICdistributions on cefotaxime, ceftazidime, and piperacillin-tazobactam were presented. A
complete set of MIC data was presented in papers IV and V, for E. coli only. The
antimicrobial agents tested against, and their breakpoints are given in table 3. Breakpoints
according to the EUCAST Clinical Breakpoints v.1.3 [44], except for temocillin where BSAC
breakpoints v.10.2 [118] were used.
20
Table 3. EUCAST breakpoints (*BSAC breakpoint) used in paper IV and V, S≤/R>.
beta-lactams (paper IV)
breakpoint
other antimicrobial drugs (paper V) breakpoint
amoxicillin-clavulanic acid
-/8
amikacin
aztreonam
1/8
chloramphenicol
8/16
cefepime
1/8
ciprofloxacin
cefotaxime
1/2
colistin
ceftazidime
1/8
fosfomycin
32/32
ceftibuten
1/1
gentamicin
2/4
ertapenem
0.5/1
nalidixic acid
NA
imipenem
2/8
nitrofurantoin
64/64
mecillinam
8/8
tigecycline
1/2
meropenem
2/8
tobramycin
2/4
piperacillin-tazobactam
8/16
trimethoprim
2/4
temocillin
8/8*
trimethoprim-sulfamethoxazole
2/4
8/8
0.5/1
2/2
Multiresistance (in papers III and V) was defined in isolates with an ESBL phenotype and
decreased susceptibility (I or R) for a minimum of two additional antimicrobial agents with
different mode of action. This is in accordance to the definition proposed by Magiorakos et
al. [119].
3.5
Preparation of DNA
Due to capsule production in some of the isolates, DNA-extraction was performed on all K.
pneumoniae in paper II. One loop full (1 µL, or approximately five cfu) were extracted using a
BioRobot EZ1 and the DNA Tissue Kit and DNA Bacteria card (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions.
In papers I and III whole genome amplification by MDA was performed directly from the
frozen isolates. One loop full (1 µL) of frozen material was added to the Illustra GenomiPhi
V2 amplification kit, as recommended by the manufacturer (GE Healthcare Life Sciences,
Uppsala, Sweden). In paper II, 1 µL of purified DNA was used as a template in the MDA
reaction. Finally 80 µL ultra-pure water (Eppendorf, Hamburg, Germany) was added and the
MDA-DNA was divided in aliquots, stored at -20°C until further analysed. MDA-DNA from
the isolates in paper III was used in paper V.
3.6
PCR
Primers (table 4) were designed manually with the aid of OligoAnalyzer [80], except for the
universal CTX-M primer pair, first described by Mulvey and co-workers [120].
All primers used in this thesis were tagged with standard vector sequences, for use as
sequencing primers. The M13 uni (-21) sequence; (CGT) TGT AAA ACG ACG GCC AGT, was
used as a tag on the forward primer for CTX-M, qnrA, B, C, D and S and aac(6’)-Ib-cr
genes. SP6; CA TTT AGG TGA CAC TAT AG, was used on the forward primer for SHV-like
genes and T7; TAA TAC GAC TCA CTA TAG GG, on the reverse primer. For TEM-genes the
forward primer was tagged with 3AOX; GCA AAT GGC ATT CTG ACA TCC. See table 4.
Primers were tested with control-strains using gradient annealing temperatures from 46-60ºC
to obtain the optimal temperature for each gene. PCR cycling conditions are shown in table 5.
21
aac6Ib-rev
348 bp
67
2 - 26
419 - 399
484 - 460
87 - 111
416 bp
65
M13-aac6Ib-for
427 bp
473 - 454
489 bp
36
qnrS-rev
M13-qnrS-for
qnrD-rev
235 bp
141 - 163
297 bp
M13-qnrD-for
44
516 - 494
290 bp
182 - 203
353 bp
M13-qnrC-for
qnrC-rev
386 - 364
29
qnrB-rev
197 bp
24 - 47
845 - 821
145 - 166
260 bp
74
M13-qnrB-for
483 bp
554 - 531
549 bp
qnrA-rev
M13-qnrA-for
tem-as
91
13 - 38
782 bp
3AOX-tem-se
874 bp
854 - 836
95
211 - 236
T7-shv-us-as
815 bp
61
1 - 20
893 bp
% of gene
22
GTG TTT GAA CCA TGT ACA CGG CTG G
TGT AAA ACG ACG GCC AGT GAC ACT TGC TGA CGT ACA GGA ACA G
TCT GAC TCT TTC AGT GAT GC
TGT AAA ACG ACG GCC AGT GGA AAC CTA CMR TCA TAC ATA TCG
CGA TTT TCC CAC AGT TCG CAC
TGT AAA ACG ACG GCC AGT CAG GAA TAG CTT GGA AGG GTG TG
GCA TTG CTC CCA AAA GTC ATC AG
TGT AAA ACG ACG GCC AGT ATG CAG ACC TAC GAG ATG CTT C
GTG ATA TAK GCR CTR CAA AAC CA
TGT AAA ACG ACG GCC AGT GGY ACT GAA TTT ATY GGC TGY C
TCC AGA TCG GCA AAG GTA AGG TCA
TGT AAA ACG ACG GCC AGT TCA GCA AGA GGA TTT CTC ACG CCA
AGT GAG GCA CCT ATC TCA GCG ATC T
GCA AAT GGC ATT CTG ACA TCC CAT TCC CGT GTC GCC CTT ATT CCC TT
TA ATA CGA CTC ACT ATA GGG TGC CAG TGC TCG ATC AGC G
C ATT TAG GTG ACA CTA TAG ATG CGT TAT DTT CGC CTG TG
TGG GTR AAR TAR GTS ACC AGA AYC AGC GG
CG TTG TAA AAC GAC GGC CAG TGA ATG TGC AGY ACC AGT AAR GTK ATG GC
sequenced Target position Sequence
SP6-shv-se
538 bp
size
803 - 775
613 bp
size
Product Sequenced
ctxm-U2-as
M13-ctxm-U1-se
Primer
Table 4. Primers used and designed/modified in this thesis. Nucleotides in italics are vector sequences, nucleotides in red are degenerated; Y=C/T; R=A/G; K=G/T; S=C/G;
D=A/G/T; M=A/C. Target positions refer to the position in the CDCs of the following sequences in GenBank: CTX-M-1 accession No. X92506; SHV-1 AF148850; TEM-1
J01749; qnrA1 AY070235; qnrB1 DQ351241; qnrC EU917444; qnrD FJ228229; qnrS1 AB187515 and aac-(6’)-Ib L25666.
Table 5. PCR programs used.
CTX-M
paper I & III
x30
Initial denaturation Denaturation Annealing Extension Final extension
Time
Temperature
15 min
30 sec
30 sec
2 min
10 min
95°C
95°C
55°C
72°C
72°C
SHV
paper II & III
x30
Initial denaturation Denaturation Annealing Extension Final extension
Time
Temperature
15 min
30 sec
30 sec
1 min
10 min
95°C
94°C
58°C
72°C
72°C
TEM
x30
paper III
Initial denaturation Denaturation Annealing Extension Final extension
Time
Temperature
15 min
30 sec
1 min
10 min
95°C
94°C
66°C
72°C
qnrA, B & S, aac-(6')-Ib
x30
paper V
Initial denaturation Denaturation Annealing Extension Final extension
Time
Temperature
15 min
20 sec
20 sec
30 sec
8 min
95°C
94°C
55°C
72°C
72°C
qnrC & D
x30
paper V
Initial denaturation Denaturation Annealing Extension Final extension
Time
Temperature
15 min
20 sec
20 sec
30 sec
8 min
95°C
94°C
58°C
72°C
72°C
All PCR reactions were carried out using block thermal cyclers. For CTX-M and TEM genes
thermo cycler ABI 2720 (Life Technologies Ltd, Paisley, UK) was used; for SHV, qnr and
aac-(6´)-Ib genes a Mastercycler gradient thermo cycler (Eppendorf). The reaction mix was
made up of HotStarTaq Master Mix (Qiagen) and 10 pmol of each primer in a final volume of
25 µ L. Amplicons were visualised using pre-cast 2% agarose gel (E-gel, Life Technologies)
(papers I, II and III) or the QIAxcel capillary electrophoresis system using the QIAxcel DNA
Screening kit (Qiagen) (papers III and V).
3.7
Nucleotide sequencing
In all papers nucleotide sequencing was performed by a customer DNA sequencing service
(Eurofins MWG Operon GmbH, Ebersberg, Germany). The samples were pre-treated
according to the guidelines by Eurofins valid during the period. SHV-amplicons were
sequenced in both directions, the others only in the forward direction. This procedure was to
ensure that double peaks in sequences of K. pneumoniae carrying two SHV or SHV-like
genes (chromosomally and/or plasmid mediated) were true and not artefacts.
23
3.8
Cloning
In some isolates in paper II, nucleotide sequencing revealed the presence of more than one
allelic variant. These isolates were subjected to cloning using a TOPO-TA cloning kit with
chemical competent TOP10 cells, according to the manufacturer’s instructions (Life
Technologies).
3.9
Bioinformatics tools
All generated DNA sequences (papers I-III and V) were aligned, edited and compared using
the bioinformatics freeware CLC (v. 3.2.3 to v. 6.5) [121]. Initial alignments were also done
using the free internet resource ClustalW2 [84]. Sequences of clinical isolates and control
strains were compared to sequences deposited in NCBI GenBank using the BlastN tool [122].
Dendrograms were constructed with the CLC freeware, using bootstrap (100) analysis and
UPGMA-clustering.
3.10
Statistics
In paper III linear regression was used to evaluate changes in antimicrobial consumption.
Binary logistic regression was performed to evaluate changes in the incidence of ESBL
producing bacteria. In papers IV and V the Mann–Whitney test was used to analyse
differences in antimicrobial susceptibility between CTX-M groups 1 and 9. A p-value of
≤0.05 was considered statistically significant.
3.11
Ethical clearance
Ethical clearance was not considered necessary in this investigation on archival isolates. The
bacterial isolates were obtained from samples taken in routine diagnosis of the customary
health care service. No sensitive information regarding patients was extracted from the
laboratory data journal system and upon publication all samples were decoded. This work was
a part of the local Strama mission.
24
4
4.1
Results
Paper I
K. oxytoca, with K1/OXY but without CTX-M genes, can be amplified in PCRs targeting
CTX-M genes, depending on the primer used. Primer alignment with the used universal CTXM primer pair and type sequences retrieved from GenBank is shown in table 6. All K. oxytoca
in this material carried an OXY-2 genotype, see figure 9.
Compared to K. pneumoniae with CTX-M genes, K. oxytoca express lower levels of
resistance to cefotaxime (MIC 0.5-8 vs. 64-256 mg/L) and ceftazidime (MIC 0.125-4 vs. 16256 mg/L) but higher to piperacillin-tacobactam (MIC ≥128 vs. 4-64 mg/L). When
phenotypically tested for ESBL with Etest, all K. oxytoca were positive in the test with
cefotaxime ± clavulanic acid, but negative in the corresponding test with ceftazidime. K.
pneumoniae were more often positive using both Etests (80%).
Note that the following CTX-M genes are indistinguishable from each other using this primer
pair: CTX-M 1 and 61; CTX-M 2, 20, 44, 56, 75 and 97; CTX-M 3, 10, 22, 66 and 80; CTXM 14, 17, 18, 21, 24, 46-50, 83 and 104, CTX-M 15, 28, 82, 88 and 109; CTX-M 19 and 99,
CTX-M 30 and 37; CTX-M 53, 55, 57, 69, 79 and 114; and CTX-M 65 and 90.
25
ATGTGCAGYACCAGTAARGTKATGGC
ATGTGCAGCACCAGTAAAGTGATGGC
********T********G**G*****
********C********G**G*****
********T********A**T*****
********C********A**G*****
********C***G****A**G*****
********C********A**G*****
********T***G****A**G*****
********C***G****A**G*****
********C***G****A**G*****
********C***G****A**G*****
********C********G**C***A*
********T********GACG****T
CGCGAAGTCGA***G**G*CGC****
***GCA**T**A*****G**T*****
***GCA**T**A*****A**T*****
********T********G**G*****
*****T**T*****C**A**G*****
********C*****T**G**G*****
********C*****C**G**G*****
********C********GT*C*****
Gene
CTX-M 1
CTX-M 2
CTX-M 8
CTX-M 9
CTX-M 25
OXY 1
OXY 2
OXY 3
OXY 4
OXY 5
OXY 6
Sed 1
CdiA
CKO 1
CumA
HugA
KluA 1
KluC 1
KluG 1
FONA 1
RAHN 1
Accession No.
X92506
X92507
AF189721
AF174129
AF518567
Z30177
Z49084
AF491278
AY077481
AJ871872
AJ871879
AF321608
X62610
AF477396
X80128
AF324468
AJ272538
AY026417
AF501233
AJ251239
GU584929
ctxm-U1-se
CTX-M universal primer:
3’ to 5’-direction (reverse):
5’ to 3’-direction (forward):
CCGCTGATTCTGGTCACTTACTTCACCCA
******G*******G**C**C**T*****
*****TA*******C**T**C**C*****
******G*******G**C**T**T*****
******G*******G**T**C**C*****
******G*G*****G**C**T**T*****
******G*AT*A**C**C**C**T*****
******G*AT*A**C**T**T**C*****
******G*G**A**G**C**T**T*****
******G*G*****G**C**T**T*****
******G*GT*A**G**C**T**T*****
******G*G*****A**C**T**C**A**
******A*C**C**C**C**C**T**A**
**TT**C*CG****T*TC**TA*T**A**
**AT*AA**T*A**GGTC**T**C**A**
**AT*AA**T*A**CGTT**T**T**A**
******G*******G*****C**T*****
**AT**G***T***T*****C**C**G**
******A*******C**T**C**C*****
***T**G*GT****G**C**T**C**G**
******G*G*****G**G**T**C*****
26
Pos.
211-236
211-236
211-236
211-236
211-236
211-236
208-233
202-227
208-233
208-233
211-236
223-248
220-245
265-290
217-242
211-236
211-236
211-236
211-236
223-248
41-66
ctxm-U2-as
GGCGACYAAGACCASTGRATRAARTGGGT
CCGCTGRTTCTGGTSACYTAYTTYACCCA
Pos.
775-803
775-803
775-803
775-803
775-803
775-803
772-800
766-794
772-800
772-800
775-803
787-815
784-812
790-818
781-809
775-803
775-803
775-803
775-803
786-814
605-633
Table 6. Partial DNA sequence alignment of some CTX-M and CTX-M-like genes, and the universal degenerated primers; ctxm-U1-se and ctxm-U2-as. Asterisks indicate
sequence homologies. The degenerated nucleotide sequence positions in the primers and its corresponding nucleotides in the aligned genes are indicated in red; Y=C/T;
R=A/G; K=G/T; S=C/G; D=A/G/T; M=A/C. For clarity, sense and antisense DNA sequences of the universal degenerated ctxm-U2-as (reverse) primer are given.
Figure 9. Phenetic tree of K. oxytoca, K. pneumoniae and type sequences.
27
4.2
Paper II
All K. pneumoniae carried SHV or SHV-like genes. More than one allelic variant was found
in six (of 20) isolates, confirmed by bi-directional sequencing, see table 7. Of the 14 isolates
with only one gene present, five were SHV-11-like, four were SHV-1-like, two were SHV-5like, two were LEN genes and one isolate carried an OKP gene, see figure 10.
Note that SHV-1 is indistinguishable from SHV-28, -83 and -125; SHV-5 from SHV-55; and
SHV-11 from SHV-36 and -61 using this primer pair.
Table 7. Isolates with more than one allelic variant of SHV-genes. Red and black indicates what gene goes
with what amino acid substitution.
amino acid position with two allelic variants
isolate
35
226
19
Leu35Gln
-
Gly238Ser Glu240Lys SHV-12
238
240
SHV-1-like
33
-
-
Gly238Ser Glu240Lys SHV-5-like
SHV-1-like
110
Leu35Gln
-
Gly238Ser
137
-
Pro226Ser Gly238Ser Glu240Lys SHV-5-like
SHV-33
138
-
Pro226Ser Gly238Ser Glu240Lys -
SHV-5-like + SHV-33
143
-
Pro226Ser Gly238Ser Glu240Lys -
SHV-5-like + SHV-33
-
revealed by cloning
probable other gene
SHV-2-like + SHV-11-like -
Figure 10. Phenetic tree of K. pneumoniae and type sequences, * indicates sequences of cloned isolates.
28
4.3
Paper III
During the six year period studied (2002-2007), ESBL producing Enterobacteriaceae was
detected in 144 patients. This accounted for less than one percent of all positive samples
analysed by the clinical microbiology routine laboratory. The number of patients with
ESBL producing E. coli increased significantly (p <0.001) from 5 to 47 per year; K.
pneumoniae remained between one and four per year. Patients were ranging from new-born
to 90 years old, quartiles 29, 56 and 71 years, respectively. Sixty percent of patients were
female.
ESBL production was found in four species; E. coli (224 isolates, 132 patients), K.
pneumoniae (23 isolates, 14 patients), C. koseri (1 isolate, 1 patient) and S. sonnei (1
isolate, 1 patient). Four patients had both ESBL producing E. coli and K. pneumoniae.
Thirty four percent of all samples came from urine in hospital care, 28% from urine
in primary care; followed by hygiene screenings (15%), wounds (15%), and blood
(5%). Eighteen patients (13%) had ESBL isolated at two different locations; 13 of
them in urine plus one other location. Ten patients had repeat isolates over a period of
more than six months, all of urinary origin.
Multiresistance was seen in 58% of E. coli; 40% showed reduced susceptibility to
gentamicin, 63% to trimethoprim-sulfamethoxazole and 66% to ciprofloxacin. No isolates
were resistant to carbapenems. Ten percent were fully susceptible to all five tested drugs.
Fifty percent of K. pneumoniae were multiresistant.
CTX-M genes were detected in 95% of E. coli and 50% of K. pneumoniae. The distribution
of genes is presented in table 8. E. coli most frequently carried CTX-M genes belonging to
group 1 (67%), followed by group 9 (27%). TEM-1 like genes were found in 68% E. coli,
almost all in combination with CTX-M genes. All K. pneumoniae isolates with CTX-M
belonged to group 1. The C. koseri and S. sonnei isolates carried CTX-M group 9 genes.
Consumption of antimicrobial agents was studied for a longer period; nine years (2001-2009).
The use expressed as DID each year is presented in table 9. The overall consumption was
unchanged; around 12.7 DID per year. Ninety percent of all antimicrobial agents were
prescribed in primary care. However, the consumption pattern changed during the period;
cephalosporins and trimethoprim decreased by 50% (p <0.001 and 0.009, respectively),
nitrofurantoin and penicillins with beta-lactamase inhibitors increased with 165% and 60%
(both p <0.001) respectively. Below 30% increase was seen for broad-spectrum penicillins
(p 0.001), pivmecillinam (p 0.030), tetracyclines (p 0.001) and trimethoprimsulfamethoxazole (p <0.001). In hospital care there was an increase in the use of
carbapenems (52%, p <0.001), penicillins with beta-lactamase inhibitors (267%, p <0.001),
pivmecillinam (31%, p 0.030), and trimethoprim-sulfamethoxazole (71%, p 0.010).
29
Table 8. Beta-lactamase genes found in E. coli and K. pneumoniae. *Seven patients with E. coli had two or more
different combinations of gene sets, thus the total number of patients when adding is >128.
Group of genes
E. coli n (%)
isolates
patients
n = 208
n = 128*
CTX-M group 1
K. pneumoniae n (%)
isolates
patients
n = 18
n = 13
140 (67)
86 (67)
9 (50)
8 (62)
CTX-M group 1
42 (20)
28 (22)
-
-
CTX-M group 1 + TEM-1
96 (46)
60 (47)
-
-
2 (1)
2 (2)
9 (50)
8 (62)
CTX-M group 1 + TEM and/or SHV
CTX-M group 2
1 (<1)
1 (<1)
-
-
CTX-M group 9
57 (27)
36 (28)
-
-
CTX-M group 9
18 (9)
15 (12)
-
-
CTX-M group 9 + TEM-1
39 (19)
24 (19)
-
-
TEM and/or SHV
8 (4)
5 (4)
7 (39)
4 (31)
No CTX-M, TEM or SHV
2 (1)
2 (2)
2 (11)
1 (8)
Table 9. Consumption of antimicrobial agents in Östergötland County.
DDD/1000 inhabitants/day and year
Antimicrobial agents
2001
2002
2003
2004
2005
2006
2007
2008
2009
Beta-lactamase sensitive penicillins
4.33
3.99
3.79
3.49
3.49
3.70
4.09
3.86
3.77
Broad-spectrum penicillins
1.11
1.06
1.08
1.11
1.14
1.14
1.21
1.22
1.19
Pivmecillinam
0.48
0.46
0.47
0.49
0.47
0.47
0.50
0.53
0.52
Cephalosporins
0.46
0.45
0.45
0.40
0.37
0.37
0.36
0.32
0.25
Penicillins/beta-lactamase inhibitors
0.23
0.24
0.24
0.22
0.25
0.29
0.31
0.35
0.36
Tetracyclines
2.14
2.00
2.09
2.17
2.25
2.42
2.55
2.49
2.48
Quinolones
0.86
0.86
0.89
0.90
0.92
0.95
0.90
0.83
0.85
Trimethoprim
0.52
0.54
0.55
0.51
0.49
0.51
0.48
0.38
0.28
Nitrofurantoin
0.12
0.14
0.14
0.17
0.21
0.21
0.24
0.25
0.32
Trimethoprim–sulfamethoxazole
0.11
0.11
0.11
0.12
0.12
0.13
0.13
0.13
0.15
Primary care
Total consumption in primary care
11.58 11.08 11.03 10.71 10.94 11.52 12.13 11.74 11.46
Hospital care
Beta-lactamase sensitive penicillins
0.11
0.10
0.10
0.09
0.09
0.10
0.10
0.10
0.10
Broad-spectrum penicillins
0.12
0.13
0.13
0.14
0.15
0.14
0.14
0.12
0.14
Pivmecillinam
0.03
0.03
0.03
0.03
0.03
0.04
0.04
0.04
0.04
Cephalosporins
0.21
0.20
0.21
0.21
0.20
0.20
0.21
0.20
0.20
Penicillins/beta-lactamase inhibitors
0.02
0.02
0.02
0.03
0.03
0.04
0.05
0.07
0.07
Carbapenems
0.05
0.05
0.06
0.06
0.06
0.06
0.07
0.08
0.08
Tetracyclines
0.17
0.16
0.18
0.19
0.21
0.17
0.22
0.22
0.18
Quinolones
0.14
0.16
0.16
0.15
0.15
0.15
0.15
0.14
0.13
Trimethoprim
0.02
0.04
0.02
0.03
0.03
0.03
0.04
0.04
0.02
Trimethoprim–sulfamethoxazole
0.01
0.02
0.02
0.01
0.01
0.02
0.02
0.02
0.02
Aminoglycosides
0.01
0.01
0.01
0.01
0.01
0.01
0.02
0.02
0.01
1.14
1.17
1.17
1.14
1.20
1.20
1.30
1.28
1.26
Total consumption in hospital care
30
4.4
Paper IV
MIC-distributions are presented in table 10 and 11. All isolates were susceptible to imipenem
and meropenem, 99% to ertapenem. More than 90% susceptibility was seen to mecillinam
and between 80 and 90% to temocillin, amoxicillin-clavulanic acid and piperacillintazobactam. Isolates carrying CTX-M group 9 was significantly more susceptible and showed
more than 90% susceptibility to temocillin (p 0.048), ceftibuten (p <0.001), ceftazidime (p
<0.001), amoxicillin-clavulanic acid (p 0.014) and piperacillin-tazobactam (p 0.009).
The overall susceptibility was low (<20%) for aztreonam and cefepime, but CTX-M group 9
was significantly more susceptible (p <0.001 and 0.030, respectivley). For ceftibuten and
ceftazidime the susceptibility of CTX-M group 1 was very low (<10%). No isolates with
CTX-M were susceptible to cefotaxime, and the overall susceptibility was only 3%.
31
1
1
4
2
2
32
9 32 60 42 22 13
2 7 38 37 22 13
5 24 20 3
7
8
3
2
1
4
4
25 26 31 33
4 7 18 33
20 17 13
13 28 22 10 39 40 24
2 9 8 32 40 24
12 25 12 1 2
1
3
2
2
6
6
20
1
22
8 8.1
7 3.0
1 11
34
8.3
95
24
35
3.6
21
31
0
53 2.5 96
50 0 100
3 0
98
63
90
5.5
1/8
All E. coli
CTX-M gr 1
CTX-M gr 9
3
4
3
6
3 37
3 9.8
95
All E. coli
CTX-M gr 1
CTX-M gr 9
Cefepime
8/8
1/8
1
1
8.6
5.3
13
1/2
2
5
5
10 91
5 95
5 87
All E. coli
CTX-M gr 1
CTX-M gr 9
Ceftazidime
1
6
4
2
1/1
8 27 37 14 35 36 24
4 9 12 35 33 24
5 19 27 1
2
2
3
17
21
9.0
2
7
6
83
79
91
All E. coli
CTX-M gr 1
CTX-M gr 9
Cefotaxime
22 32 67 33 12
14 25 45 23 9
8
6 19 8 2
3
3
8
3
5
1 15 67 82 30
1 6 26 71 25
6 34 10 5
Number of isolates with indicated MIC (mg/L)
0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128 %S %R breakpoints
8/8*
All E. coli
CTX-M gr 1
CTX-M gr 9
Ceftibuten
All E. coli n = 198
CTX-M group 1 n = 132
CTX-M group 9 n = 55
Mecillinam
Temocillin
<0.064
Table 10. MIC-distributions. Breakpoints according to EUCAST (S≤/R>), except *BSAC. MIC50 in bold underlined, MIC90 in bold italics.
2
2
2
3
2
2
1
1
26 11
25 11
1
9 28 29
4 7
9 22 21
5
5
13 46 30 16
12 43 29 16
1 1
47 120 31
19 87 26
23 29 3
5 30 72 30
3 13 39 26
2 15 29 4
9
9
5
4
12 7.6
12 0
18
10 84
4 82
2 96
All E. coli
CTX-M gr 1
CTX-M gr 9
Ertapenem
All E. coli
CTX-M gr 1
CTX-M gr 9
Meropenem
All E. coli n = 198
CTX-M group 1 n = 132
CTX-M group 9 n = 55
Imipenem
<0.008
10
4
3
1
1
0.008
1/8
-/8
30
52
21
33
11
16
52
34
15
35
127
87
5
38
46
29
28
1
16
15
6
6
28
20
5
33
8 1
6
2 1
8 11 1 2
8 11 1
2
2
2
161
106
47
99
99
96
100
100
100
100
100
100
1.0
0
3.6
0
0
0
0
0
0
0.5/1
2/8
Number of isolates with indicated MIC (mg/L)
0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 >16 %S %R breakpoints
2/8
57
83
3.6
16
20
5.5
10
9.8
3.6
Number of isolates with indicated MIC (mg/L)
<0.064 0.064 0.125 0.25 0.5 1 2 4
8 16 32 64 128 >128 %S %R breakpoints
8/16
Table 11. MIC-distributions. Breakpoints according to EUCAST (S≤/R>). MIC50 in bold underlined, MIC90 in bold italics.
All E. coli
CTX-M gr 1
CTX-M gr 9
All E. coli
CTX-M gr 1
CTX-M gr 9
Aztreonam
All E. coli
CTX-M gr 1
CTX-M gr 9
Amoxicillin-clavulanic acid
Piperacillin-tazobactam
Table 10. Cont.
4.5
Paper V
MIC-distributions are presented in table 12. A majority of isolates (≥ 95%) were susceptible
to amikacin, colistin, fosfomycin, nitrofurantoin and tigecyclin. Below 80% susceptibility
was seen for chloramphenicol (77%), gentamicin (60%), tobramycin (55%), trimethoprimsulfamethoxazole (35%), ciprofloxacin (34%) and trimethoprim (29%). CTX-M group 9 was
significantly more susceptible, compared to CTX-M group 1, to ciprofloxacin (28% vs. 44%,
p 0.039), gentamicin (53% vs. 76%, p 0.003) and tobramycin (44% vs. 76%, p <0.001).
Sixty eight percent of the isolates were multiresistant. The most common multiresistance
pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprimsulfamethoxazole, ciprofloxacin, gentamicin and tobramycin (16%). This and other
multiresistance patterns are presented in table 13. Among the non-multiresistant isolates the
most common resistance patterns were ESBL phenotype plus resistance to ciprofloxacin
(11%) or resistance to both trimethoprim and trimethoprim-sulfamethoxazole (8%).
Eighteen isolates (9%) showed full susceptibility apart from the ESBL phenotype.
No isolates showed evidence of qnrA, B, C or D genes, but one isolate carried qnrS1. All
isolates in this study with reduced susceptibility to amikacin (n=9) also showed reduced
susceptibility to tobramycin and ciprofloxacin and carried the gene aac(6’)-Ib-cr.
Table 13. The most common resistance patterns of multiresistant isolates. The isolates were susceptible
for the other tested drugs (nalidixic acid not included). S=susceptible, I=intermediate, R=resistant.
TMP-SMX=trimethoprim-sulfamethoxazole.
trimethoprim TMP-SMX ciprofloxacin chloramphenicol gentamicin tobramycin
n=31
n=13
n=11
n=9
n=9
n=8
n=7
n=7
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I/R
R
R
S
S
R
R
S
S
S
R
S
R
R
S
S
34
R
S
I/R
R
S
S
S
R
I/R
S
I/R
I/R
S
S
I/R
S
All E. coli
CTX-M gr 1
CTX-M gr 9
Trimetoprim-sulfamethoxazole
All E. coli
CTX-M gr 1
CTX-M gr 9
Trimetoprim
All E. coli
CTX-M gr 1
CTX-M gr 9
Tigecyclin
All E. coli
CTX-M gr 1
CTX-M gr 9
Gentamicin
All E. coli
CTX-M gr 1
CTX-M gr 9
Tobramycin
All E. coli n=198
CTX-M group 1 n = 132
CTX-M group 9 n = 55
Amikacin
6
6
20
15
5
1
1
17
13
3
2
2
3
1
2
10
5
3
8
7
1
5
3
2
5
4
1
21 20
15 12
4
8
5
5
3
3
24 105 53 10
18
63 40 8
4
36 10 2
6 53 55
5 28 34
1 24 16
2 61 34
2 31 20
28 11
4
4
35
1
1
3
2
4
3
1
2
2
1 10 16
6 14
2
12 11 27 39
5 3 23 36
3 8 3 2
11 118 47 13
8 67 41 8
3 46 4 2
129
81
40
141
91
42
27
5
32
8
8
1
1
2
8
6
2
1
2
2
1
1
11
7
4
4
4
1
1
35
39
27
29
31
24
99
99
100
60
53
76
55
44
76
96
94
100
Number of isolates with indicated MIC (mg/L)
<0.032 0.032 0.064 0.125 0.25 0.5 1
2 4 8 16 32 64 128 >128 %S
Table 12. MIC-distributions. Breakpoints according to EUCAST (S≤/R>). MIC50 in bold underlined, MIC90 in bold italics.
65
61
73
71
67
76
1.0
1.5
0
40
47
24
39
54
9.1
2.5
3.0
0
2/4
2/4
1/2
2/4
2/4
%R breakpoints
8/16
All E. coli
CTX-M gr 1
CTX-M gr 9
Cloramfenicol
All E. coli
CTX-M gr 1
CTX-M gr 9
Nitrofurantoin
All E. coli
CTX-M gr 1
CTX-M gr 9
Colistine
All E. coli
CTX-M gr 1
CTX-M gr 9
Fosfomycin
All E. coli
CTX-M gr 1
CTX-M gr 9
Ciprofloxacin
All E. coli
CTX-M gr 1
CTX-M gr 9
Nalidixic acid
Table 12. Cont.
41
21
13
4
4
1
1
24
13
11
9
3
6
5
3
2
3
1
2
83 82
56 58
23 18
4
4
1
1
1
1
4
5
3
3
36
4
2
2
6
4
1
1
1
6
3
2
6
4
2
29 10
22 6
7 2
4
3
1
1 121
94
1 24
37 70 39
29 43 23
7 22 13
1
1
11
8
3
17
10
6
2 18 112 21
2 14 76 15
4 29 6
2
2
7
4
2
1 23 107 46
1 17 68 31
5 30 14
8
5
3
2 24
16
1 3
1
1
2
7
5
2
2
2
10
25
13
1
1
149
103
42
77
81
71
96
96
96
99
99
98
99
99
100
34
28
44
Number of isolates with indicated MIC (mg/L)
<0.032 0.032 0.064 0.125 0.25 0.5
1 2
4 8 16 32 64 128 >128 %S
23
19
29
3.5
3.8
3.6
1.0
0.8
1.8
0.5
0.8
0
64
71
53
8/8
64/64
2/2
32/32
0.5/1
%R breakpoints
NA
5
Discussion
During the time period studied in this thesis, from 2002 until end of 2007, the prevalence of
ESBL producing Enterobacteriaceae in Östergötland was very low, <1%, but increasing. This
was lower than the national data reported to EARSS (2.3% in 2007) [123], but on the other
hand our collection includes all sample sources and EARSS only comprises of invasive
isolates. When comparing only invasive isolates to the EARSS data during 2002-2007, the
resistance was actually slightly higher than the pooled national data (0.5-2.8% vs. 0.3-2.3%).
Numerically, ESBL was found more often in urine, but calculated as percentage it was more
commonly found among blood isolates, as has also been shown in Norway [124] and Denmark
[125].
ESBL producing Enterobacteriaceae was made obliged to register in February 2007. SMI
presents national data in SWEDRES 2007 concluding that ESBL is more often found in E.
coli (77%) than K. pneumoniae (12%), urine is the most commonly found sample source
(70%), a majority of patients are female (69%), mean age is >55 years, and patients may carry
more than one species producing ESBL [123]. Our material comprises of a higher percentage
of E. coli, a larger proportion of men, and a lower percentage of urine samples, but essentially
these two datasets confirm each other.
Three other Swedish investigator teams have examined ESBL producing Enterobacteriaceae
during the same period of time as us, but they have mainly focused on deducing ESBL genes
and fingerprinting. They have all performed PCR followed by amplicon sequencing, revealing
(for E. coli) CTX-M group 1 as the dominating group of genes (57-80%), followed by CTX-M
group 9 (16-31%). CTX-M group 2 was found sporadically, as were TEM and SHV genes.
[99, 101, 102] Despite the fluctuations in percent this is in concordance with our findings;
CTX-M group 1 is dominating (67%), followed by group 9 (27%). This proportional
relationship is seen throughout most of Europe [92].
Three nosocomial outbreaks due to ESBL producing Enterobacteriaceae during the studied
period have been described in Sweden; a small outbreak of CTX-M group 1 producing E. coli
in Stockholm involving Stockholm South General Hospital and the smaller Rosenlund
Hospital in 2002 [95]; a large outbreak of multiresistant CTX-M 15 producing K. pneumoniae
at Uppsala University Hospital in 2005 [96]; and an outbreak of multiresistant CTX-M 15
producing E. coli at Kristianstad Hospital in 2005-2006 [97]. In 2008 there was an outbreak
in Kalmar County, comprising of CTX-M 15 [98]. In our region there has been no reason to
suspect an outbreak of nosocomial origin.
The national use of antimicrobial agents during 2001-2009 was presented in SWEDRES 2009
[126]. The total consumption in Östergötland was lower both in primary (10.7-12.1 DID) and
hospital care (1.14-1.30 DID) as compared to the entire country (12.8-13.9 DID and 1.30-1.55
DID, respectively). Despite an unchanged total consumption, and a slight increase of drugs
known not to select for ESBL, ESBL increases in our county.
Aggregated data on cases with ESBL producing Enterobacteriaceae from 2007 and forwards
are presented by SMI. When calculated as cases/100 000 inhabitants the number of cases in
Östergötland is lower than the country average, but there is an increase in number of cases in
the period posterior to the scope of this thesis. For 2008, cases reported from Östergötland
increased to 81, a year later it was 113. [127] In 2010, SMI decided to widen their definition
of an ESBL, according to the proposal of Giske et al. [57] to also include plasmid mediated
37
AmpC, ESBLM, and carbapenemases, ESBLCARBA. This extension makes the aggregated
data somewhat difficult to compare with older data. The expanded definition has not (yet?)
been accepted internationally by the scientific community [58], and has not been used in this
thesis. Unpublished data from the laboratory journal data system, covering years 2008-2011,
shows that the number of patients affected by ESBL producing E. coli and K. pneumoniae
increases. E. coli was found in 75, 108, 148 and 181 patients per year, and K. pneumoniae in
11, 16, 17 and 20 patients per year, during the period beyond the scope of this thesis.
A significant difference in susceptibility between CTX-M groups, in favor of group 9 over
group 1, was seen for many of the antimicrobial agents tested; amoxicillin-clavulanic acid,
aztreonam, cafepime, ceftibuten, ceftazidime, ciprofloxacin, gentamicin, piperacillintazobactam, temocillin, and tobramycin. Others confirm this relationship regarding
ceftazidime and aztreonam [109]; gentamycin and tobramycin [128]; and ciprofloxacin,
trimethoprim-sulfametoxazole, and gentamicin [129]. Morosini, on the other hand, found an
association between resistance to ciprofloxacin and trimethoprim-sulfamethoxazole and CTXM group 9 [130]. Sixty eight percent of the isolates were multiresistant, almost equal to the
Norweigan study by Tofteland [109]. Strikingly, there was no difference in multiresistance
between CTX-M groups, as could have been suspected based on the higher susceptibility rates
for CTX-M group 9.
According to the SRGA, ertapenem should be reserved for the treatment of ESBL producing
Enterobacteriaceae in outpatient departments, since it only requires parenterally administration
once daily [32], but in lower UTIs it is more favourable to treat with an oral agent. Of the
eight oral agents tested in this thesis, only three showed a generally high susceptibility;
mecillinam (91%), nitrofurantoin (96%) and fosfomycin (99%). More than 90% susceptibility
in isolates possessing CTX-M group 9 was seen for ceftibuten. Nitrofurantoin and mecillinam
are most easily accessible in our region and can be regarded as primary treatment options for
community onset UTIs, regardless of ESBL production or not.
Among the parenterally administrated drugs ≥95% susceptibility was seen for amikacin,
tigecycline and colistin. In isolates possessing CTX-M group 9 ceftazidime, piperacillintazobactam and temocillin showed ≥90% susceptibility. Carbapenems were >99% susceptible.
For serious infections involving ESBL producing bacteria the carbapenems are regarded as
the first line treatment, but amikacin and tigecyclin can be used as a second line treatment
option in our region.
The reporting of beta-lactam antibiotics as susceptible in ESBL producing isolates is
controversial, but recently the EUCAST abandoned this view and decided that all
cephalosporins in isolates of Enterobacteriaceae should be reported as found [131]. Despite
the high level of multiresistance, there are still treatment options left for all isolates tested in
this study. This may lull us into a false sense of security. More resistant bacteria, such as
isolates expressing NDM-1 [132], emerges and the development of new antimicrobial agents
against Gram-negative bacteria is stagnant. Focus is targeted on combining cephalosporins
with different beta-lactamase inhibitors [133, 134]; promising results in vitro and in murine
studies have been shown for the new beta-lactamase inhibitor NXL104 [135-137].
What is alarming is the continuing increase of ESBL producing Enterobacteriaceae, both at
county and national level. Increasing antimicrobial resistance is often associated with a high
selective pressure, but cannot be accountable in this particular case. Despite a low level of
antimicrobial consumption there is an influx of these resistant bacteria to the society. It has
38
been shown that foreign travel is one route of transmission [138-140] and many researchers
have found ESBL producing bacteria in foodstuff [141-144] and animal husbandry [145-148].
Even more alarming is the persistence of these genes in the environment; in treated
wastewater from hospitals and community [149-152], wastewater sludge [153], river water
and sediment [154-156] and wild animals [157]. ESBL producers have even been found on
the Antarctic [158].
It is difficult to compare studies on antimicrobial susceptibility, mainly because a majority
lacks MIC distributions; they present only SIR data and sometimes MIC50/MIC90. The use of
different breakpoints (CLSI, EUCAST or other), sometimes just referred to but not given in
figures makes comparison difficult, especially over time and between continents. When it
comes to comparing antimicrobial susceptibility among ESBL producing bacteria, different
distributions of ESBL enzymes among the isolates tested make comparisons difficult to
perform. Sometimes investigators aggregate different species or do not perform genotypic
analyses as to determine the enzymes present, also complicating the matter.
One issue rarely discussed is the population of isolates being examined. Many papers on
antimicrobial susceptibility in vitro or studies examining enzyme occurrence are based on
various mixed strain collections that hardly can be described as a representative
(sub)population. One strength of our research is the well characterized and coherent study
population.
Unless sequenced, PCR amplification is merely a screening method. This is pointed out in
every text book concerning the matter, and is common knowledge among molecular
biologists. To really know what is in the test tube one has to deduce the sequence nucleotide
by nucleotide, but for a decent assumption one can rely on amplicon size and the accuracy of
the primers. In the interdisciplinary world of clinical microbiology decent assumptions have
for a long time been regarded as good enough. In an era of escalating resistance against betalactam antibiotics, sequencing of genes is crucial to keep track of resistance development.
When single point mutations change resistance patterns so dramatically, good enough just
isn’t good enough any longer.
We often encounter lack of understanding regarding the M13-tagged primers in our PCRs.
The concept is that using the same tag on each primer, regardless of target, enables highthroughput sequencing, in any size laboratories. Many different amplicons, targeting various
genes, can be pipetted onto the same 96-well plate (or even 384 well plate) and sequenced
simultaneously using the selected tag as a sequencing primer. If multiplex or bi-directional
PCR is desirable, one simply tag each primer with different tags, such as standard vector
primers SP6 or T7, and divide the sample. The idea of tagged primers for PCR is not new; it
was demonstrated as early as 1996 by Frenay and co-workers [159].
The genes encoding ESBLs of Bush/Jacoby group 2be are approximately 900 bp in size, and
contain many positions where point mutations have been found. To distinguish them from
each other and similar non-ESBL genes, sequencing of the entire gene, or as large portion of it
as possible, is necessary. With today’s reagent mixes and technology, sequencing of
amplicons this size is not an issue. Nowadays most authors, if performing genotypic analyses,
sequence amplicons after inhouse performed PCR. There are three commercially available
systems for ESBL detection; two tube microarray systems and one PCR-ELISA system [160].
These screening methods may require further investigation of samples for a thorough analysis.
39
One objection against our approach is the use of very broad range primers, targeting both
ESBL and non-ESBL genes. Since Klebsiella spp. is known to be indistinct in their
biochemical pattern [161, 162], sequencing of their chromosomal ESBL-like genes confirms
the species identity and rules out false positive ESBL phenotypes. Non-ESBL betalactamases, chromosomal or plasmid mediated, may cause resistance profiles similar to
ESBLs due to increased expression and permeability changes [163-165]. In combination with
phenotypic tests sometimes difficult to interpret, misidentification occasionally occurs.
5.1
Conclusion
The main findings in this thesis were that the prevalence of ESBL producing
Enterobacteriaceae in Östergötland was very low but increasing, and the total consumption of
antimicrobial agents was stable. A majority of the isolates were multiresistant and a
significant difference in susceptibility between CTX-M groups, in favor of group 9 over group
1, was seen for many of the antimicrobial agents tested. More than 90% of the isolates were
susceptible to amikacin, the carbapenems, colistin, fosfomycin, mecillinam, nitrofurantoin and
tigecycline.
It is my strong belief that as long as almost one billion of the planet’s human population lack
pure water, 2.5 billion lack basic sanitation and 70% of industrial waste in the developing
world are dumped untreated into water resources [166] the battle against antimicrobial
resistant bacteria will continue. The overuse, misuse and abuse of antimicrobial agents in
human infections and the agricultural sector, previously and present, will affect us for a long
time.
40
6
Acknowledgements – Tack
Att forska är ingenting för ensamseglare, det är i allra högsta grad någonting som blir bäst när
man gör det tillsammans. Jag vill rikta ett ärligt och innerligt tack till min forskargrupp för att
ni har lyssnat, läst, diskuterat, stöttat och funnits där;
•
min huvudhandledare Lennart Nilsson
•
mina bihandledare Hans-Jürg Monstein och Håkan Hanberger
•
och mina medarbetare, medförfattare och doktorandkollegor; Anna Ryberg,
Anita Hällgren, Anita Johansson, Annika Samuelsson, Barbro Isaksson,
Carina Claesson, Hans Gill, Maria Lindqvist, Maud Nilsson och Åse
Östholm Balkhed.
41
7
Images and copyright permissions
Figure 1 was drawn using ChemWriter [167].
Figure 2-4 was drawn by Per Lagman, LiU-Tryck, in co-operation with the author.
Figure 5 is part of a raw-data file, modified by the author.
Figure 6 was drawn using CLC Sequence Viewer [121].
Figure 7 was based on data provided by ECDC, extracted from The European Surveillance
System – TESSy, and modified by the author to better fit the size of printing.
Figure 8 was purchased from Shutterstock Images LLC, New York, USA, and modified by
the author.
Figure 9-10 was drawn using CLC Sequence Viewer [121], based on edited raw data.
Licences for re-printing of the articles were obtained from Elsevier Limited, Oxford, UK
(paper II), Informa Healthcare, London, UK (paper III) and Springer, Heidelberg, Germany
(paper IV). The authors are copyright holders of paper I.
42
8
Disclaimer
“The views and opinions of the authors expressed herein do not necessarily state or reflect those
of the ECDC. The accuracy of the authors’ statistical analysis and the findings they report are not
the responsibility of ECDC. ECDC is not responsible for conclusions or opinions drawn from the
data provided. ECDC is not responsible for the correctness of the data and for data management,
data merging and data collation after provision of the data. ECDC shall not be held liable for
improper or incorrect use of the data.”
43
9
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2.
3.
4.
5.
6.
7.
8.
9.
10.
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12.
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15.
16.
17.
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