special focus review: bacterial biofilms
review
Virulence 2:5, 1-6; September/October 2011; © 2011 Landes Bioscience
Proteus mirabilis biofilms and catheter-associated
urinary tract infections
Sandra M. Jacobsen1 and Mark E. Shirtliff2,3,*
Department of Cell Biology & Molecular Genetics; University of Maryland-College Park; 2Department of Microbial Pathogenesis; Dental School;
3
Department of Microbiology and Immunology; School of Medicine; University of Maryland; Baltimore, MD USA
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
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Key words: Proteus mirabilis, CAUTIs, biofilms, virulence, catheter
Abbreviations: CAUTIs, catheter-associated UTIs; CPSs, capsule polysaccharides; EDTA, tetra sodium ethylenediaminetetraacetic
acid; EPS, extracellular polysaccharide; IgA, immunoglobulin A; LPS, lipopolysaccharide; MR/K, mannose resistant/Klebsiellalike fimbriae; MR/P, mannose resistant/Proteus-like fimbriae; P. mirabilis, Proteus mirabilis; PMF, P. mirabilis fimbriae; RsmA,
repressor of secondary metabolites; UTIs, urinary tract infections; UCA/NAF, uroepithelial cell adhesion/nonagglutinating
fimbriae
Proteus mirabilis inhabits the environment and causes a number
of infections including those of the skin, respiratory tract,
wounds and urinary tract. These organisms express virulence
factors associated with adhesion, motility, immunoavoidance,
nutrient acquisition, host damage, as well as biofilm formation.
P. mirabilis produces biofilms in diverse habitats with those
formed in the human host playing a key role in indwelling
device infections. The most studied P. mirabilis biofilms are
those formed when the organism is grown in urine, resulting
in unique features including the presence of swarmer cells
and struvite and hydroxyapatite crystals upon growth in
urine. Factors relevant to P. mirabilis biofilm formation include
adhesion factors, proteins involved in LPS production,
transporters, transcription factors, two component systems,
communication factors and enzymes. P. mirabilis biofilm
research will lead to a better understanding of the disease
process and will subsequently lead to the development of new
prevention, and treatment options.
are capable of colonizing and causing disease due to their arsenal
of virulence factors including fimbriae [mannose resistant/
Proteus-like fimbriae (MR/P), mannose resistant/Klebsiella-like
fimbriae (MR/K), uroepithelial cell adhesion/nonagglutinating
fimbriae (UCA/NAF) and P. mirabilis fimbriae (PMF)], flagellar
motility, immunoavoidance factors [antigenic variation, capsules,
IgA proteases, lipopolysaccharide (LPS), ZapA], host damaging
factors (degradative enzymes such as proteases, ureases, hemolysins), struvite and hydroxyapatite crystal formation, iron acquisition protein homologs and the ability to form biofilms.5
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Introduction
Proteus, Gram-negative bacilli that thrive in soil, water and the
intestinal tracts of mammals, are capable of swarming or swimming in a coordinated manner, on solid surfaces. Several species
of Proteus bacteria are known to colonize and infect the human
host but the one most frequently linked with causing human disease is Proteus mirabilis. These bacteria are the causative agents of
a variety of opportunistic nosocomial infections including those
of the respiratory tract, eye, ear, nose, skin, burns, throat and
wounds.1,2 P. mirabilis are more commonly associated with urinary tract infections (UTIs) in those individuals with structural
or functional abnormalities, especially ascending infections in
patients undergoing urinary catheterization.3,4 These organisms
*Correspondence to: Mark E. Shirtliff; Email: [email protected]
Submitted: 05/31/11; Revised: 08/16/11; Accepted: 08/17/11
DOI:
Proteus mirabilis in vitro Biofilms
Proteus has been shown to produce biofilms in diverse environments from aquatic conditions6 to indwelling devices [vascular
access ports/hemasites, scleral buckles, ureteral stents, urethral
catheters and tracheesophageal voice prostheses (Provox2)].7-11
P. mirabilis can produce biofilms on nonliving surfaces12,13
including polystyrene, glass, latex and silicone and on biological surfaces. These organisms have been found in both pure and
polymicrobial biofilms.10 The structure of P. mirabilis biofilms
grown in laboratory broth (Luria-Bertani) and urine (artificial
and pooled human urine) has been analyzed using confocal scanning laser microscopy and 3-D imaging.14,15 Differences observed
in the basic structure of these biofilms were media-dependent.
Proteus mirabilis biofilms that developed in Luria-Bertani broth
and pooled human urine appeared as typical mushroom-like
structures with nutrient channels forming 21 to 24 h post-inoculation.14,15 However, P. mirabilis biofilms formed in artificial
urine appeared as flat structures without channels after 24 h postinoculation with swarmer cells observed protruding out of these
biofilms.14 Jones et al. suggest that the formation of swarmer cells
could represent a method by which P. mirabilis can disperse from
the mature biofilm to seed new surfaces.14 In addition, when
grown in artificial and pooled human urine, struvite crystals have
been observed in biofilms produced by these organisms.14
Flagellar motility also has a role in surface perception and,
as a result, biofilm formation. A functioning flagellum is critical
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Table 1. Virulence factors of Proteus mirabilis that have a potential role in biofilm formation
Virulence Factors
Proposed Role
Reference
Mannose-resistant Proteus-like (MR/P)
fimbriae
Adhesion, mutants defective in biofilm formation
25
UDP-glucuronic acid decarboxylase PmrI
LPS modification, mutants defective in biofilm formation
27
Inner-core LPS biosynthetic WaaE
Inner-core LPS biosynthetic protein, mutants decrease in biofilm formation
37
Pst transporter
High-affinity phosphate transporter, mutants defective in biofilm formation
15
RsbA
Membrane sensor of a two component system that enhance EPS* production in the
presence of certain fats
25
Cis-2-decenoic acid
Homolog of Pseudomonas cell communication factor, role in dispersal and inhibit
biofilm development
30
RsmA
RNA binding protein, possible regulation of biofilm formation
63
Urease
Nickel metalloenzyme, Local increase in pH to facilitate crystal formation, Mutants
attenuated in CBA mouse model
36
Capsule
Aggregate precipitating components into stones
39
*EPS: Extracellular polysaccharide.
for sensing and/or responding to the surface signal and flagellar
mutants (including various flhD, fliFGLMPQ, flhA and flgKLB
genes) involved in surface perception that may cause defects in
biofilm formation as with other bacterial species. In addition, the
status of flagellar motors acts as a sensor for P. mirabilis to signal
to the cell to undergo swarmer cell differentiation at the appropriate time, where inhibition of rotation promotes differentiation.16
association with this crystal layer, protected from the antimicrobial effects of impregnated compounds.20,21
Both physical and chemical factors play a role in the initiation and development of the crystalline biofilms observed during
P. mirabilis colonization. The pH of urine can be essential for
bacteria attachment to polymer surfaces as macroscopic aggregates of cells and crystals of calcium and magnesium phosphate
form in alkaline urine, settle on the polymer surface and initiate
crystalline biofilm development.22 Therefore, impeding the rise
of urinary pH and subsequent crystallization could be critical in
preventing biofilm formation on indwelling devices inserted in
the urinary tracts of patients infected with P. mirabilis.
Besides the importance of urinary pH for attachment of
Proteus to catheter surfaces, the rough surfaces on the rims of
the catheter eyeholes, as observed on Foley catheters, are prone
to colonization and crystallization by P. mirabilis due to their
extreme irregularity.23 These irregularities lead to blockages
that occur generally at the eyelet or in the balloon region of the
lumen. Scanning electron microscopy revealed that within two
hours post-inoculation, P. mirabilis cells were trapped within the
crevices in the jagged eyelet surfaces.23 At four hours, microcolonies had established in the surface depressions, and by six
hours, with the increase in urinary pH, crystals had started to
develop in the biofilm.23 After 20 h, an extensive mature crystalline biofilm was formed and was dispersed down the lumen of
the catheter.23
Research has potentially linked a number of virulence factors
to P. mirabilis biofilm formation (Table 1) and a summary of
the events that may occur during biofilm formation are found in
Figure 1. These factors include adhesion factors, proteins involved
in LPS production, transporters, transcription factors, two
component systems, cell communication factors and enzymes.
MR/P fimbriae, whose expression undergoes phase variation,
have been shown to play a role in colonization of these organisms in the mouse model.24 These fimbriae have been observed
to be produced by the majority of cells in the CBA mouse model
of ascending UTI.25 Mutants that constitutively expressed
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Proteus mirabilis in vivo Biofilms:
Pathogenesis from Colonization to Mature Biofilm
Once P. mirabilis comes in contact with either a nonliving or
host surface, the process of colonization begins. After the initial colonization, P. mirabilis form distinctive biofilm structures
during the pathogenesis process. These structures assist in the
persistence of P. mirabilis in the host by protecting these organisms from the host immune system and treatment by antibiotics.17 One of the more frequently studied biofilms produced by
P. mirabilis are those biofilms that form within the urinary
tract, in particular those initiated on urinary catheter surfaces.
Bacterially derived stones, a characteristic of the biofilms developed during P. mirabilis-associated UTIs, account for up to
30% of all urinary tract stones.18 Crystalline biofilms are especially problematic during catheter-associated UTIs (CAUTIs)
due to the blockage of catheters caused by biofilm encrustation.
Also, the majority of patients with recurrent P. mirabilis catheter
encrustation (62%) developed bladder stones, which led to the
colonization of replacement catheters with these organisms.19
The crystalline biofilms associated with these catheters in vivo
show the presence of two main types of crystals, struvite and apatite. Long rectangular crystals made of magnesium ammonium
phosphate are termed struvite while microcrystalline structures
made of a hydroxylated calcium phosphate (where the phosphate
is often replaced by carbonate) are termed apatite.20 In addition,
a conditioning layer of crystal formation rich in calcium and
phosphate is found on catheters, even those impregnated with
antimicrobial agents such as silver.20,21 Bacteria are in intimate
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Figure 1. Proposed mechanism of biofilm formation during P. mirabilis catheter- associated urinary tract infections. In order to establish an infection in
the urinary tract, P. mirabilis must be capable of indwelling catheter attachment to microcrystals and microcolony formation. In conjugation to bacterial cell attachment, the increase in local pH due to urease production causes the formation of struvate crystal formation. To perpetuate the infection,
these organisms must detach or disperse from the initial site of infection to seed adjacent sites of the urinary tract.
MR/P fimbriae (MR/P ON), produced significantly more and
earlier biofilms (18 h) than the wild-type P. mirabilis HI4320
(p = 0.03) or a HI4320 mutant unable to produce these fimbriae
(MR/P OFF, p = 0.05) as examined by spectrophotometry and
microscopy.25 However, after seven days, the wild-type strain produced thicker biofilms (65 μm) than either mutant (12 μm).25
Transposon mutants in the Pst transporter, a phosphate-specific
transport system, have been shown to be defective in biofilm formation when grown in pooled human urine as compared with
the wild-type HI4320 strain as determined by confocal microscopy and 2-D gel electrophoresis.15 RsbA, a histidine-containing
phosphotransmitter of a two-component regulatory system, was
determined to repress swarming and enhance extracellular polysaccharide (EPS) production, thus, augmenting biofilm formation in the presence of certain saturated fats, such as myristic
acid, lauric acid and palmitic acid.26 A homolog of the pmrI gene,
which encodes for a UDP-glucuronic acid decarboxylase involved
in LPS modification, has decrease biofilm formation.27 A P. mirabilis mutant in the homolog of inner-core LPS biosynthetic waaE
gene was found to be defective in swarming and biofilm formation.28 A homolog of the RsmA protein (repressor of secondary
metabolites) was discovered in P. mirabilis and was shown to
suppress swarming and may regulate biofilm formation.29 Cis-2decenoic acid, an organic cell-to-cell communication compound
produced by Pseudomonas aeruginosa, has been shown to disperse established microcolonies and inhibit the development of
biofilms for P. mirabilis.30 The role of swarming during P. mirabilis biofilm formation is still not completely understood. As mentioned previously, research has shown that swarming is repressed
as extracellular matrix production is increased.26 Also, Jones
et al. demonstrated that mutants deficient in swarming and swimming were capable of forming biofilms and blocked catheters
more quickly than the wild-type strain in the catheterized bladder model.31 It is conceivable that swarming must be repressed
in order for these organisms to remain attached to surfaces to
initiate formation of the biofilm.
Two major factors known to be involved in urinary crystal
formation and hence, crystalline biofilm formation by P. mirabilis are bacterial urease and capsule polysaccharides or CPSs.32
Urease, a multimeric nickel metalloenzyme,33 contributes to the
development of urinary stones due to urease-mediated hydrolysis of urea to ammonia and carbon dioxide that alkalinizes
the local environment. This increase in urinary pH causes the
local supersaturation and precipitation of calcium phosphate
and magnesium-ammonium phosphate from urine to form
crystals of carbonate apatite [Ca10 (PO4) 6CO3] and struvite
(MgNH4PO4 ·6H2O), respectively.34 These crystals build up in
the biofilms of the urinary epithelium and indwelling catheters
and eventually block the flow of urine through the catheter and
from the bladder or kidney. Mutants in P. mirabilis urease are
attenuated in the CBA mouse model35 and colonize the bladder
and kidneys in 100-fold-fewer bacteria than the urease-positive
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strain after two days post-inoculation and caused no urolithiasis
during infection.36
Besides bacterial urease, capsules are thought to hasten struvite
crystal growth12,37 observed during UTIs and CAUTIs associated
with P. mirabilis by aggregating precipitated urinary components
into crystalline stones.38 Proteus CPSs tend to be acidic due to
the presence of uronic acid, pyruvate or phosphate groups, thus
enabling this structure to bind to metal cations such as Ca 2+
and Mg2+.1 Purified partially anionic CPS of P. mirabilis ATCC
49565 added to artificial urine at a pH of 7.5 to 8.0 induced more
struvite formation than other CPS types, as examined by particle
counting (Coulter counter) and by phase-contrast microscopy.39
Anti-Biofilm Therapies and Prevention Strategies
Currently, much of the research on P. mirabilis biofilms focuses
on the formation of and the potential methods of eradication of
these biofilms on various indwelling device surface materials (silicone, plastics). Of the 18 currently available urethral catheters
tested, all were susceptible to P. mirabilis biofilm encrustation
and blockage.40 Encrustation occurred in indwelling urethral
catheters by a clinical strain of P. mirabilis ranging from 17.7
h (silver-coated latex) to 47 h (all silicone) when studied in
the laboratory model of the catheterized bladder using pooled
human urine.41 Swarming organisms, such as P. mirabilis, have
been shown experimentally to be better able to migrate over
Foley catheter surfaces, in particular those with hydrogel coatings.42 In addition, P. mirabilis is capable to assisting nonmotile
bacteria, such as Staphylococcus aureus, to be transported over
catheters.42 Based on the study by Chang et al. adherence and
biofilm formation of P. mirabilis to surfaces may be facilitated
by the presence of pre-existing biofilms as biofilm formation on
gentamicin-containing polymethyl methacrylate increases upon
preincubation with autoclaved killed or live Staphylococcus epidermidis biofilms.43
A number of compounds have been tested for their effectiveness against P. mirabilis crystalline biofilms on catheter materials including triclosan,44-46 nalidixic acid,47 tetra sodium EDTA
(EDTA),48 urease inhibitors,41 quorum sensing inhibitors,49 and
Ibicella lutea extract.50 Several groups have examined the effect
of the commonly used antimicrobial compound triclosan on
P. mirabilis biofilm development. It has been shown that triclosan impregnated silicone and latex-based catheters were free or
reduced of crystalline biofilms from P. mirabilis as compared with
a water control (blocked ranging from 18 to 27 h).45,46 In a study
by Chew et al. ureteral stents eluted with triclosan had reduced
P. mirabilis biofilm formation when suspended in artificial urine.44
However, it must be noted that P. mirabilis strains have varying
susceptibility to triclosan and thus can be selected for upon the
use of this biocide.51 This is also true of antibiotic impregnated
catheters. While the use of antibiotics have shown improved efficacy, the widespread use of antibiotics and the issues of resistance
development cannot be ignored.
Silicone catheter retention balloons treated with a solution of nalidixic acid has been shown to significantly extend
the lifespan of the catheter by reducing catheter encrustation.47
Alternatively, EDTA-treated silicone catheters took significantly
longer to cause blockages by encrustation (45 h in saline vs.
67 h in daily EDTA treatments, p = 0.047) as examined scanning
electron microscopy.48 Urease inhibitors, such as fluorofamide,
have also shown some efficacy since they reduce pH and reduce
calcium and magnesium salt deposits on silicone catheters and
the resulting biofilm formation as examined by scanning electron microscopy.41 Quorum sensing antagonists, p-nitrophenyl
glycerol and tannic acid, have been shown to inhibit the quorum sensing system and subsequently inhibit P. mirabilis biofilm
formation in artificial urine.49 Although not specifically tested
in human urinary catheters, other quorum sensing antagonists
such as synthetic furanone compounds like (5Z)-4-bromo-5(bromomethylene)-3-butyl-2(5H)-furanone that can be derivatives of those produced by marine alga Delisea pulchra have
shown excellent efficacy.52 Encrustation and catheter blockage
can also be reduced by increasing a patient’s fluid intake with
citrate-containing drinks.22 Novel anti-biofilm natural products
can result in the reduced ability of P. mirabilis to develop biofilms, such as one study where glass and polystyrene surfaces were
treated with Ibicella lutea extract, a South American indigenous
plant, as demonstrated by spectrophotometry.50 In the many
studies using silver as an antimicrobial coating in clinical studies,
a moderate benefit has been noted,53 most likely due to the low
rate of silver ions elution and the protective effects of crystal layer
development over the catheter luminal surface that may shield
bacteria from the silver antimicrobial effects.20,21 However, this
limited benefit may be improved by the incorporation of nanosilver, nanometer sized silver particles, to increase the surface area
and silver dissolution in the surrounding liquid.54 As with other
modalities, large scale clinical testing is somewhat limited so the
efficacy is unknown.
Catheter materials with various coatings have been examined
for the development of P. mirabilis biofilms. Hydrogel coating
(based on polyvinylpyrrolidone) has been shown to accelerate biofilm formation on silicone as compared with uncoated
silicone via the laboratory model of catheterized bladder.55 An
anti-fouling coating of marine mussel adhesive protein, which
resembles polyethylene glycol [mPEG-DOPA(3)], has potential
to resist conditioning film formation on silicone disks incubated
in pooled human urine and uropathogen attachment in human
urine as examined by scanning electron microscopy.56 However,
the efficacy of these coating would need to be tested in randomized and large scale clinical studies to judge the validity of these
surfaces.57
In a study by Stickler et al. test drainage and artificial catheterized bladder systems remained sterile for 10 d with the use
of a silver-releasing device located in the draining tube.58 This
demonstrates that urinary catheters were protected from ascending bacteria, including P. mirabilis, from contaminated urinedrainage bags as shown by scanning electron microscopy and
chemical analysis.58 Introducing an electric current through silver
electrodes attached to catheters was demonstrated to significantly
decrease encrustation by P. mirabilis biofilms.59 Cellulose acetate/
bromothymol blue sensors in urine collection bags detected the
presence of P. mirabilis biofilm encrustation approximately 12 d
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prior to catheter blockages.60,61 With the addition of lytic bacteriophages, P. mirabilis biofilm formation on Foley catheter
sections impregnated with a hydrogel coating was reduced by
90% as compared with untreated controls.62 For a recent review
of novel biomaterials, incorporation of antimicrobial agents and
other modern modalities aimed at reducing infectious episodes
with urinary catheters, please refer to Hamill et al.
Summary
Acknowledgments
There are still many things that are unknown concerning
P. mirabilis biofilm formation including the method by which
these organisms produce these structures. Since crystalline
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Virulence
Volume 2 Issue 5