Type 1 Fimbriae Contribute to Catheter-Associated Urinary TractInfections Caused by Escherichia coli
Andreas Reisner,a,c Mario Maierl,a Michael Jörger,a Robert Krause,b Daniela Berger,a Andrea Haid,a Dijana Tesic,a Ellen L. Zechnerc
University of Applied Sciences, Biomedical Science, Graz, Austriaa; Section of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, MedicalUniversity of Graz, Graz, Austriab; University of Graz, Institute of Molecular Biosciences, Graz, Austriac
Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI),which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, sincetheir role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current studyaimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understandingmolecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physico-chemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not iso-genic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization.The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strainCFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bac-terial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colo-nizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient cathetersrevealed that a median level of �73% of cells from nine samples have switched on type 1 fimbrial expression. This study supportsthe utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1fimbriae during CAUTI.
An enormous number of microbial infections in humans arelinked to medical device interventions, such as urethral and
intravascular catheters, prosthetic grafts, prosthetic joints, andshunts (1). The insertion of medical devices inherently facilitatesaccess of bacterial cells into usually sterile areas of the body andprovides an abiotic surface for biofilm formation. The intrinsicresistance of biofilms against host defense mechanisms and anti-microbials limits efficient treatment of biofilm-associated infec-tions. Consequently, biofilm-associated infections are considereda leading cause of morbidity within hospital environments andnursing homes, causing additional health care costs exceeding $1billion/year in the United States (1).
Catheter-associated urinary tract infections (CAUTI) are themost frequent infections in health care facilities and account formore than one million annual cases of nosocomial bacteriuria inthe United States alone (2). Although most cases of CAUTIs areasymptomatic, many patients are at risk of developing severecomplications, ranging from cystitis and acute pyelonephritis tobacteremia. The abiotic surface of the catheter inserted to enableurine drainage from the bladder provides an additional substra-tum for bacterial biofilm formation that is heavily colonized (3).Although the exact contribution of the catheter biofilm to patho-genicity is unknown, the common recurrence of CAUTI soon af-ter the completion of a course of antibiotic treatment is thought tobe caused by recolonization of the urine by organisms that havesurvived in the catheter biofilm. These properties of attached bac-teria underlie the clinical experience that efficient antibiotic treat-ment of CAUTI requires the replacement of the catheter.
A large number of sophisticated in vitro model systems havebeen applied to understand the basic mechanisms that governmicrobial biofilm formation (4–7). One of the most consistentconclusions from these extensive efforts is that biofilm formationof microbes is dependent on the experimental conditions used (8).
Flow dynamics, nutrient availability and composition, and otherphysicochemical properties have been shown to severely affectmicrobial biofilm formation (see, for example, references 9–11).Thus, although a great deal of knowledge is available based onthese systems, the degree to which the resulting mechanistic mod-els of biofilm development can be extrapolated to complex envi-ronments in vivo is wholly unknown. In addition, conditions pre-vailing during current in vivo models for CAUTI that requiresurgical insertion of catheter pieces (7, 12, 13) differ from thehuman infection with respect to urine composition, the residuallevel of urine in the human bladder, and the urine flow throughthe catheter. This implies that conclusions obtained with CAUTIanimal models alone also need to be interpreted with care. Hence,efforts directed to provide more efficient treatment alternatives orefficient prophylaxis for CAUTIs require the use of in vivo CAUTImodel systems and dynamic in vitro models that are better able toreproduce the physicochemical conditions during infection in hu-mans.
Escherichia coli is responsible for about 80% of uncomplicatedurinary tract infections (UTI) (14) and is isolated from the urineof about 30% of patients experiencing CAUTI (15, 16). Despitethe significant molecular understanding of the pathogenesis ofUTI, research directed to reveal virulence factors that are neces-sary or specific for the onset, persistence, and progression ofCAUTI caused by E. coli is limited (17). Studies using simple in
vitro biofilm models have generated an extensive list of potentialfactors that may assist E. coli in catheter surface colonization andinclude fimbrial adhesins, autotransporter proteins, capsularstructures, flagella, and exopolysaccharides (18–21). However,many of these factors have not been evaluated in, e.g., a clinicalstrain background or in the presence of urine. Interestingly, theonly study reporting gene expression by E. coli UTI isolates duringbiofilm growth in human urine revealed that several biofilm-re-lated factors, including type 1 fimbriae, autotransporter proteinantigen 43 (Ag43), and extracellular matrix components, were notupregulated in the urine biofilms (22). The lack of knowledgeabout bacterial gene expression on catheters derived from patientsexperiencing CAUTI makes it difficult to assess the clinical rele-vance of proposed E. coli biofilm factors.
The current study demonstrates the utility of combining datafrom a dynamic catheterized bladder model in vitro with in vivoexpression analysis for understanding molecular factors relevantfor CAUTI pathogenesis. The role of known E. coli virulence fac-tors for UTI and for biofilm formation in simple in vitro biofilmmodels was evaluated in a dynamic catheterized bladder model.Our results support the conclusion that type 1 fimbriae are re-quired for efficient catheter colonization of E. coli under physico-chemical conditions closely mimicking the human infection.Expression analysis of bacterial cells from a clinical catheter spec-imen underlines the role of type 1 fimbriae for catheter coloniza-tion by E. coli.
MATERIALS AND METHODSStrains, plasmids, oligonucleotides, and media. E. coli strains, plasmids,and oligonucleotides used in this study are listed in Table 1. Bacteria wereroutinely grown in Luria-Bertani (LB) medium or agar containing 5 gNaCl per liter (23) at 37°C. Selective media contained antibiotics at thefollowing concentrations: kanamycin (Km), 50 �g ml�1; chlorampheni-col (Cm), 10 �g ml�1; ampicillin (Amp), 100 �g ml�1; and streptomycin(Sm), 100 �g ml�1.
Artificial urine was prepared and filter sterilized as described previ-ously (24). Human urine was collected anonymously from 5 to 15 healthyvolunteers (both men and women) who had no history of UTI prior tocollection. The urine was pooled, sterilized through a filter cartridge (Sar-tobran P; 0.2 �m; Sartorius, United Kingdom), and kept at 4°C until usewithin the following 48 h. Pooled urine was routinely analyzed semiquan-titatively using Combur10 Test UX (Roche) to avoid using urine withabnormal properties (pH, leukocytes, nitrite, urobilinogen, protein, he-moglobin, bilirubin, glucose, ketone, and specific gravity).
In vitro model of a catheterized bladder. The dynamic catheterizedbladder model originally developed in the David Stickler laboratory wasperformed as previously described (24), with minor modifications. Inbrief, two-compartment glass chambers were maintained at 37°C by cir-culation of water through the outer compartment. A size 14 sterile Foleyall-silicone catheter (Bard, GA) was inserted into the inner compartmentof the glass chambers, followed by inflation of the catheter retention bal-loon with 10 ml sterile water. In contrast to the original model used byStickler and coworkers, the outer glass compartment was 5 cm longer thanthe inner compartment, ensuring temperature maintenance of the top 10cm of the catheter. After connection of catheters to standard drainagebags, sterile urine (�15 ml) was pumped (Watson-Marlow 205S) into theinner chambers to a level just below the eyeholes of the catheter tips.
For inoculation, 0.5 ml of bacterial overnight cultures grown in LB at37°C and 250 rpm for 16 h was transferred to 20 ml of sterile urine (37°C)and incubated for 3 h at 37°C and 250 rpm. For inoculation of the cathe-terized bladder models, an appropriate aliquot of the preculture repre-senting 5 ml of an optical density at 600 nm (OD600) of 0.2 (�0.5 � 108
CFU) was added to the urine in the inner chambers. After 1 h, the urinesupply was resumed for 72 h at a constant rate of 30 ml h�1.
For quantification of biofilm on catheters, the urine flow was stoppedand a sample of the bladder (inner compartment) suspension was col-lected without mixing. The inserted catheters were carefully removedfrom the bladder and cut aseptically. If subject to analysis, the tip of thecatheter (including the eyeholes) was transferred to 1 ml saline (0.9%NaCl solution). Following a rinse of the remaining catheter with 2 ml ofsaline to remove traces of bladder content, the retention balloon sectionwas removed. The next 5-cm catheter section was transferred to 5 mlsaline solution and served as the catheter sample.
Catheter samples and samples taken from the bladder and the tip ofthe catheter were sonicated for 5 min (Sonorex RK100H; Bandelin), vor-texed for an additional 2 min, and serially diluted. Aliquots were plated onLB agar plates to determine CFU/ml bladder content and CFU/cm cath-eter. In cochallenge experiments, bacterial counts of E. coli MG1655Strand CFT073fim were additionally determined on appropriate selectiveplates and subtracted from the total CFU count to reveal relative fitness ofthe MG1655 mutants and CFT073, respectively.
Complementation of MG1655fim. To confirm the effect of fimoperon deletion in MG1655fim on catheter colonization in trans, the en-tire fim operon of MG1655, including the recombinase genes fimB andfimE (sequence positions 4538201 to 4548230; GenBank accession num-ber U00096) was amplified with Phusion high-fidelity polymerase(Finnzymes) using primers op-K-12-fim01 and op-K-12-fim02. After di-
TABLE 1 E. coli strains, plasmids, and oligonucleotides used in thisstudy
StrainMG1655 ilvG, rfb-50, thi 59MG1655Str Smr; ilvG, rfb-50, thi 60MG1655flhDC MS426, flhDC deletion in MG1655 61MG1655flu MS427, flu deletion in MG1655 61MG1655fim MS428, fim operon deletion in MG1655 29CAUTI1-9 E. coli isolates from catheterized of
patients with CAUTIThis study
CAUTI5fim CAUTI5 with TargeTron insertion infimA, Kmr
This study
CFT073 Uropathogenic E. coli 62CFT073fim CFT073 with TargeTron insertion in
fimA, KmrThis study
PlasmidpACD4K-C Cmr, TargeTron plasmid Sigma AldrichpACD4K-Cfim pACD4K-C with intron retargeted for
gestion with the restriction enzyme NotI, the resulting fragment was in-serted in the fosmid vector pEpiFos5 (Epicentre). Following introductionof the resulting plasmid pEpiFosfim into MG1655fim, mannose-sensitiveagglutination of yeast cells (25) was restored to wild-type levels.
Construction and characterization of type 1 fimbrial mutations inclinical E. coli isolates. Insertional gene disruption of fimA in CFT073and CAUTI5 was achieved using a TargeTron gene knockout system(Sigma) according to the manufacturer’s instructions. In brief, oligonu-cleotides for mutant design were created using the TargeTron design site(Sigma) and are listed in Table 1. PCR was used to retarget a group IIintron encoded by pACD4K-C, resulting in pACD4K-Cfim. After intro-duction of pACD4K-Cfim into the target E. coli strain carrying helperplasmid pAR1219, induction of proteins required for intron insertion wasperformed according to the manufacturer’s instructions. Insertion of theintron-containing kanamycin resistance cassette (�1.9 kb) in sense ori-entation after position 10 of the fimA coding region in the resulting plas-mid-free mutants was confirmed by an overlapping PCR strategy. All fimAknockout mutations abolished mannose-sensitive agglutination of yeastcells (25).
Growth rates of CFT073 and its isogenic mutant CFT073fim in artifi-cial and pooled human urine under aerobic conditions were determinedafter inoculation of prewarmed urine to an initial OD600 of 0.001. TheOD600 was measured over 8 h under rigorous shaking (220 rpm) at 37°C.Data points of the exponential growth phase were included for linearregression (lnOD600 versus t) if an R2 of �0.98 was achieved. To detectpotential minor growth differences between CAUTI5 and CAUTI5fim inhuman urine, the wild-type and mutant strains were cocultivated in batchculture for 72 h under aerobic conditions (37°C, 200 rpm). For this pur-pose, cultures initially inoculated with an equal number of wild-type andmutant cells were diluted 1:100 in sterile human urine every 24 h. After 72h, numbers of wild-type and mutant cells were determined by selectiveplating.
IE orientation assay. Swimming and catheter-attached bacteria werecollected from the in vitro model. To stabilize nucleic acids in these sam-ples, 5 ml of bladder content was combined with 10 ml of RNAprotectbacteria reagent (Qiagen). The 5-cm Foley catheter segment underneaththe retention ball was submerged in 5 ml RNAprotect bacterial reagent.Bacteria were separated from the catheter piece by sonication for 5 min(Sonorex RK100H; Bandelin) and vortexing for an additional 2 min. Cellswere then harvested by centrifugation and resuspended in 50 �l of deion-ized water. Quantification of orientation of the fim invertible element (IE)was performed as previously described (26). The same amount of the PCRproducts (50 to 100 ng) was subject to restriction digestion with 5 USnaBI. Products were separated electrophoretically on 3% agarose gelscontaining ethidium bromide. Band intensities of fragments representingON and OFF switches, were quantified using a gel documentation andimage analysis system (AlphaImager and AlphaEaseFC) after backgroundsubtraction.
Catheter samples from hospitalized patients were included if patienturine was positive for a monoculture of E. coli during routine microbio-logical analysis (�105 CFU/ml). After the routine catheter removal orreplacement procedure, approved by the Ethical Review Board of MedicalUniversity of Graz, catheter tips were submerged in RNAprotect bacterialreagent within 5 min and frozen at �80°C. The E. coli isolates causing thepositive urine culture were tested for type 1 fimbrial expression via astandard assay for mannose-sensitive agglutination of yeast cells usingcells cultured on LB agar plates (25). IE orientation in the bacterial pop-ulation was determined as described above. No patient identifier datawere included.
Statistical analysis. The majority of data sets generated during thisstudy passed the test for normal distribution; thus, data are presented asmean values � standard errors (SE) if not stated otherwise. Statisticallysignificant differences, defined as a P value of �0.05, were determinedusing SIGMAPLOT software, version 12.0 (SyStat Software Inc.). Inde-pendent challenges and cochallenge experiments were routinely analyzed
using paired t test. If a data set did not fulfill necessary criteria for aparametric test, the nonparametric Wilcoxon matched-pair test was ap-plied.
RESULTSAn isogenic E. coli K-12 mutant in type 1 fimbriae but not fla-gella or antigen 43 is outcompeted by the wild-type strain dur-ing catheter colonization in vitro. Various genetic studies haveshown that type 1 fimbriae, flagella, and autotransporter proteinAg43 support biofilm formation of E. coli laboratory K-12 strainsin static in vitro model systems using polystyrene, glass, or polyvi-nylchloride surfaces (27–29). To date, only the role of type 1 fim-briae was confirmed during static biofilm formation of clinical E.coli isolates (19) and during initial adhesion to silicone tubingirrigated with human urine for 24 h (30). To determine the rele-vance of these findings for the physicochemical conditions andtime periods characteristic for CAUTI, we tested the ability of E.coli K-12 strains mutated in these potential virulence factors tocolonize an in vitro catheterized bladder model in cochallengecompetition assays.
In brief, the utilized dynamic model for the catheterized blad-der consists of a complete Foley catheter inserted in a tempera-ture-controlled glass vessel representing the human bladder. Ster-ile urine is supplied into the bladder with a peristaltic pumpthroughout the experiment at a physiologically relevant rate. Thissystem was used extensively to study catheter encrustation andblockage in vitro (31) and mirrors the physicochemical parame-ters of CAUTIs after bladder infection, including the flushing ofurine through the catheter, albeit in the absence of a bladder mu-cosa. For each potential virulence factor, the catheterized bladdermodel was inoculated with a 1:1 ratio of wild-type to mutant cells.After 72 h of irrigation with artificial urine, the bacterial popula-tions in the bladder suspension and on the internal all-siliconeFoley catheter surface were analyzed to reveal the competitive fit-ness of the mutant strains (Fig. 1A).
Lack of motility due to the absence of flagella did not affectsurvival in the bladder suspension, since equal numbers of mutantand wild-type strains were isolated (Fig. 1B). Although the non-motile mutant tended to be less competitive during colonizationor propagation on catheter surfaces (only 29.8% � 5.9% of iso-lated cells were mutants), the difference from the wild-type strainpopulation was not significant (P 0.084). Surprisingly, the flumutation resulted in reduced fitness in the bladder suspension(13.6% � 5.2% mutant versus 86.4% � 5.2% parental strain; P 0.02), whereas catheter colonization did not lead to an alteredpopulation structure (P 0.124). Although the mutant deficientin type 1 fimbriae was equally competitive in the urine present inthe bladder, only 1.6% � 0.7% of the cells harvested from thecatheter biofilms were identified as the mutant strain (P 0.03).These results suggested that of the three analyzed factors, type 1fimbriae are most important for mature biofilm formation oncatheters using experimental conditions mimicking the physico-chemical conditions prevailing during CAUTI.
To confirm the attenuation of E. coli biofilm formation on theurethral catheter in the absence of type 1 fimbriae, wild-typeMG1655 and the MG1655fim mutant carrying a deletion of theentire fim operon were inoculated in equal amounts in separatecatheterized bladders in vitro. Quantification of colonization inthe bladder suspension, on the catheter tip, and on the internalcatheter surface was performed 96 h after inoculation (Fig. 2). In
good agreement with the results of the cochallenge experiment, wefound that bladder colonization was not affected by the mutation(Fig. 2A). In contrast, the numbers of mutant cells recovered fromthe catheter tip and the internal catheter surface were 17-fold and39-fold reduced, respectively, compared to the model system thatwas inoculated with the parental strain (P � 0.01). A similar 20-fold attenuation of biofilm formation was observed for an E. colifimH mutant after initial colonization on silicone tubing for 24 h(30).
To confirm that attenuated catheter colonization byMG1655fim can be complemented in trans and is not dependenton constituents not well represented in the artificial urine, a coch-allenge experiment was also performed in pooled human urineusing wild-type MG1655Str and mutant MG1655fim carrying afunctional fim operon on fosmid vector pEpiFosfim (Fig. 2B). Inagreement with our initial results, 72 h after coinoculation ofMG1655Str and MG1655fim carrying the empty fosmid pEpi-Fos5, only 2.1% � 0.8% of the cells harvested from the catheterbiofilms were identified as the mutant strain (P 0.04). In con-trast, carriage of a complementing fim operon on pEpiFosfim inMG1655fim resulted in competiveness equal to that of wild-typeMG1655Str[pEpiFos5] in the bladder urine (P 0.14) and cath-eter surface populations (P 0.35). We concluded that type 1fimbriae are required for efficient colonization of a catheter by thelaboratory E. coli strain under conditions prevailing in a catheter-ized bladder.
Type 1 fimbriae are required for efficient catheter coloniza-tion in vitro by uropathogenic E. coli. Since the laboratory E. colistrain K-12 that was used in our initial experiment may lackrelevant virulence factors advantageous for efficient survival inthe urinary tract, we extended our analysis with the well-char-acterized uropathogenic E. coli (UPEC) strain CFT073. A knock-out mutation in the fimA gene encoding the major fimbrial sub-unit was constructed. Absence of type 1 fimbria expression wasconfirmed in the resulting CFT073fim mutant by diminished type1 fimbria-mediated mannose-sensitive agglutination of yeast cells(data not shown). Cochallenge competition experiments with the
FIG 1 Absence of type 1 fimbriae but not flagella or Ag43 attenuates catheter colonization during CAUTI infection in vitro. (A) Following cochallenge of individualmutant derivatives of E. coli K-12 MG1655 and the wild-type strain MG1655Str in the dynamic catheterized bladder model for 72 h, the population distributionin the bladder suspension (compartment B) and on the biofilm formed on the internal surface of the all-silicone Foley catheter (compartment C) was quantified.(B to D) Stacked bars represent population structure in bladder and on catheter after competitive cochallenge of E. coli K-12 MG1655Str and a flagellar mutantderivative (MG1655flhDC) (B), an Ag43 mutant derivative (MG1655flu) (C), and a fim mutant derivative (MG1655fim) (D). Data are means � SE (n 3).
FIG 2 Absence of type 1 fimbriae attenuates catheter colonization and can becomplemented in trans. (A) Bars represent colonization efficiencies of E. coliK-12 MG1655 and a mutant derivative, MG1655fim, harvested from differentcompartments of the model system 96 h after inoculation: bladder suspension(CFU/ml), catheter tip, and internal catheter surface (CFU/cm). Data aremeans � SE (n 3). The dynamic catheterized bladder model system wasirrigated using artificial urine. (B) Stacked bars represent population struc-tures in artificial bladder suspensions and on internal surfaces of Foley cathe-ters after 72 h of competitive cochallenge of E. coli MG1655 carrying vectorcontrol pEpiFos5 and mutant derivative MG1655fim carrying vector controlpEpiFos5 and type 1 fimbrial complementation vector pEpiFosfim, respec-tively, using pooled human urine. Data are means � SE (n 3).
parental UPEC strain CFT073 using artificial urine confirmed theimportance of type 1 fimbriae for catheter colonization (Fig. 3A).Whereas the initial 1:1 distribution of the mutant and wild-typestrains in the bladder urine was unaffected after 72 h of com-petition (P � 0.05), only 7.2% � 1.8% and 6.0% � 1.1% of thecultivable population on the catheter tip and the internal cath-eter surface, respectively, were found to be mutant cells (P �0.05).
To confirm that this attenuation in catheter colonization byCFT073fim is not dependent on matrix proteins such as Tamm-Horsfall protein and constituents that are not well represented inthe artificial urine, the cochallenge experiment was also per-formed in pooled human urine (Fig. 3B). Again, CFT073fim wassignificantly outcompeted by the parental strain in both popula-tions on the Foley catheter, confirming our previous results; how-ever, we also noted that only 0.5% � 0.1% of the population in thebladder urine suspension was mutant cells (P � 0.01). Becausegrowth rates of CFT073 in artificial urine (1.02 � 0.04 h�1) andCFT073 and CFT073fim in human urine under aerobic conditionsdid not differ significantly (0.99 � 0.02 h�1 versus 1.01 � 0.01h�1), we can exclude two unlikely explanations: (i) that reducedgrowth in human urine causes the loss of competiveness ofCFT073fim, and (ii) that increased generation times in human
urine create the requirement for type 1 fimbriae to avoid wash-outfrom the artificial bladder. Instead, we propose that the humanurine constituents not present in the artificial urine create a dif-ferent conditioning film on the glass surface of the bladder thatleads to an increased requirement for type 1 fimbriae for bacteriato survive in the bladder urine. While the underlying mechanismremains undetermined, the results confirmed a role of type 1 fim-briae for efficient catheter colonization during CAUTI.
Expression of type 1 fimbriae is induced during catheter col-onization in vitro. The attenuated colonization of mutants defi-cient for type 1 fimbriae in the dynamic catheterized bladdermodel and on silicone tubing in vitro (30) suggested that type 1fimbriae need to be expressed during colonization. The majorregulatory mechanism allowing switching between fimbriated andnonfimbriated states is phase variation characterized by a revers-ible inversion of a 314-bp invertible element (IE) containing thepromoter controlling transcription of the type 1 fimbria geneoperon (32, 33). Accordingly, determination of the proportion ofcells carrying the promoter fragment in the ON orientation is agood indication for type 1 fimbria expression in the population(26).
To determine this proportion in the populations colonizingthe catheterized bladder model, samples taken from a model col-onized for 72 h with E. coli MG1655 and CFT073 were conservedusing a nucleic acid stabilizing reagent and then analyzed to de-termine IE orientation (Fig. 4). In the aerated batch culture usedto inoculate the bladders, only 24% � 1% of the MG1655 cellscarried the IE in the ON orientation. This proportion was onlymodestly increased in the bladder population (31% � 3%); how-ever, 67% � 11% of the cells in the catheter sample appeared to bein a fimbriated state. A similar distribution was observed forUPEC CFT073 in artificial urine and human urine (Fig. 4).Whereas the aerated culture revealed a modest proportion of cellsin ON position ranging, from 14% to 18%, the majority of thebladder population appeared to be fimbriated (59% to 63%). Inagreement with our hypothesis, cells from colonized catheterswere found to have a significantly increased proportion of cellscarrying the IE in the ON orientation with a maximum meanvalue in human urine of 88% � 1%. We concluded that type 1fimbriae not only are important for catheter colonization in vitrobut also are expressed at high levels in catheter biofilms.
E. coli strains isolated from catheterized patients predomi-nantly exhibit type 1 fimbrial IE orientation in the ON positionin vivo and require type 1 fimbriae for efficient catheter coloni-zation in vitro. Although the dynamic catheterized bladdermodel mimics physicochemical parameters prevalent duringCAUTI, caution is still warranted about the relevance of thesefindings for the human urinary tract. Many in vitro and in vivostudies have indicated a crucial role for type 1 fimbriae duringbladder colonization in the absence of a catheter; however,expression of type 1 fimbriae in bacteria isolated from the urineof women experiencing symptomatic urinary tract infectionwas low (26, 34).
Therefore, we wanted to confirm our expression results fromthe in vitro model with clinical samples. Foley catheters removedfrom hospital patients that had given a positive urine sample for E.coli were conserved and analyzed for the IE orientation of coloniz-ing bacteria (Fig. 5A). For nine samples analyzed, a median valueof ON orientation was found to be 73% (first quartile, 20%; thirdquartile, 80%). For five of nine samples (56%), IE orientation
FIG 3 Type 1 fimbriae are important for dynamic catheterized bladder colo-nization in UPEC strain CFT073. Stacked bars represent population structuresin inocula, in artificial bladder suspensions, and on the tip and internal surfaceof Foley catheters after competitive cochallenge of E. coli CFT073 and a fimAmutant derivative (CFT073fim) using artificial urine (A) and pooled humanurine (B) for 72 h. Data are means � SE (n 3).
indicated expression of type 1 fimbriae in more than 73% of thecells. However, the small data set suggests quite a variation in thepercentage of fimbriated cells in the catheter samples obtainedfrom different patients, possibly reflecting various catheter mate-rials, different durations of infection at the time of catheter re-moval, or the presence of nonadherent cells in the catheter. Nev-ertheless, the clinical samples analyzed here support theconclusion that type 1 fimbriae are expressed in significantamounts during catheter colonization, possibly enabling betteradherence in vivo.
To assess the importance of type 1 fimbriae in an E. coli isolatefrom patients experiencing CAUTI, a fimA knockout mutation
was created in a randomly chosen E. coli isolate originating fromthe clinical catheter samples (CAUTI5). Cochallenge experimentsof mutant strains with corresponding wild-type strains were per-formed in pooled human urine. Again, the fim mutant strain wassignificantly outcompeted by the parental strain in populations inthe bladder urine and on the Foley catheter, confirming our pre-vious results (P � 0.05) (Fig. 5B). Control cochallenge experi-ments in aerated batch cultures performed over 72 h in humanurine in parallel to the cochallenge experiments in the catheterizedbladder did not result in significant outcompeting of the mutantstrain (P � 0.05). Thus, fitness defects of the fim mutant in thecatheterized bladder are not due to minor growth deficiencies.
FIG 4 Type 1 fimbrial expression is turned on upon catheterized bladder colonization. Stacked bars represent proportions of cells carrying the IE in orientationsON and OFF in populations used for inoculation and isolated from the dynamic catheterized bladder model, which was colonized in vitro with E. coli MG1655and UPEC CFT073 using artificial and/or human urine for 72 h, as indicated. Data are means � SE (n 3).
FIG 5 E. coli colonizing urethral catheters of patients experiencing CAUTI express type 1 fimbriae in vivo and require type 1 fimbriae for efficient cathetercolonization in vitro. (A) Adherent bacteria from catheterized patients reveal a predominant orientation of IE in position ON. Catheter samples removed frompatients experiencing CAUTI were analyzed for IE orientation. Stacked bars represent proportions of cells carrying ON and OFF orientations of IE in catheterpopulations isolated from clinical catheter samples 1 to 9. (B) E. coli isolate CAUTI5 requires type 1 fimbriae for efficient catheterized bladder colonization invitro. Stacked bars represent population structures in inocula, in artificial bladder suspensions, and on the tip and internal surface of Foley catheters aftercompetitive cochallenge of E. coli CAUTI5 and a fimA mutant derivative using pooled human urine for 72 h (B). Data are means � SE (n 3).
Together, this data set underscores the importance of type 1 fim-briae as key virulence factors in CAUTI.
DISCUSSION
In the presence of a Foley catheter in the human urinary tract, thenormal defense mechanism maintained by frequent urine flush-ing and emptying of the bladder is undermined (35). Although theurine volume typically flowing through the bladder is high enoughto wash out bacteria replicating with generation times observed inhuman urine in vitro, persistent bacteria may depend less on effi-cient attachment to the bladder. This resource, combined with thepresence of an additional abiotic colonization surface that lacksthe protective features of the bladder mucosa, support the pro-posal that development and persistence of CAUTI requires anarsenal of virulence factors distinct from those contributing toUTI (36). In this study, we applied a dynamic catheterized bladdermodel to investigate the contribution to catheter colonization ofthree factors encoded by most E. coli strains: flagella, Ag43, andtype 1 fimbriae. We found that only the mutant deficient in as-sembly of type 1 fimbriae exhibited a competitive disadvantageagainst the parental strain during catheter colonization in vitro.Since catheter colonization was sampled 72 h after inoculation,when mature biofilms are already developed, we currently cannotdifferentiate a role of type 1 fimbriae during early attachment aspreviously indicated (30) or at a later stage of biofilm develop-ment. Despite reduced adherence to the catheter, all fim mutantsstill were able to colonize the catheters at up to 5 � 104 and 2 � 106
CFU/cm of catheter in cochallenge and separate inoculation ex-periments, respectively. Thus, additional factors are likely to be ofimportance for efficient biofilm formation on catheter surfacesirrigated with urine.
Ag43 does not appear to be important in this dynamic process,since an isogenic mutant was outcompeted in the bladder suspen-sion but not in the population colonizing the silicone catheter.Since type 1 fimbriation is known to block Ag43-mediated aggre-gation (37), the strong prevalence of type 1 fimbria expression incatheter populations is likely to attenuate positive selection forAg43-expressing cells. Thus, we cannot exclude that Ag43 or otherautotransporter proteins associated with UPEC virulence (38–41)contribute to catheter colonization in strains unable to expresstype 1 fimbriae. In addition, our data suggest that flagellum-me-diated motility or a proposed architectural function (42) is dis-pensable for CAUTI persistence once bacteria have colonized thebladder urine. The role of flagella during CAUTI pathogenicity ismore likely to be relevant during onset of infection when bacteriaascend through the urethra to reach the bladder or up the urethersinto the kidneys (43).
In support of our findings obtained using knockout mutants,type 1 fimbria expression, indicated by monitoring the orientationof the IE element, was found to be most prevalent in cell popula-tions harvested from catheter biofilms. Two mechanisms mightcontribute to this observation: (i) superior catheter attachment oftype 1 fimbriated cells and (ii) upregulation of type 1 fimbriaexpression in cells located in the catheter biofilms. The lattermechanism may reflect the requirement of type 1 fimbria expres-sion to stick to the silicone surface and/or the surrounding cellsbut also may be a response to environmental changes. Indeed,oxygen depletion that is likely to occur in mature biofilms hasbeen shown to select for increased type 1 fimbrial expression (44).Notably, our results are in contrast to a global transcriptome anal-
ysis of UPEC strains from patients experiencing asymptomaticUTI that revealed low type 1 fimbria expression in biofilms grownin the presence of human urine (22). Besides strain background,the use of a static petri dish biofilm model in the transcriptomestudy, in contrast to the dynamic catheterized bladder model inthis study, is likely to account for these different observations.
The phase variation that occurs during bladder and kidneycolonization during experimental murine UTI has been charac-terized extensively (26, 33, 45, 46); however, the expression level oftype 1 fimbriae during human UTI and CAUTI remains equivo-cal. Studies based on immunostaining of E. coli in the urine ofadults experiencing lower UTI suggested that a significant fractionof samples contained type 1 fimbriated cells ranging from 38% to76% of the specimen (47–49), whereas the percentage of fimbri-ated cells in individual samples was not quantified. Other studiesinvestigated fim operon transcription in urine specimens fromwomen with cystitis. One study revealed that an average of only4% of cells in urine specimens from 11 patients carried the type 1fimbrial IE in the ON orientation (26). Whole-transcriptomeanalysis reported detectable levels of fimA and fimH transcripts inonly two out of eight patients (34). In this study, we attempted toanalyze IE orientation in catheter samples from human patients. Amedian level of �73% of cells over all nine samples was found tohave switched on type 1 fimbrial expression based on promoterorientation. Thus, our study supports the conclusion that if E. colistrains infecting the lower urinary tract of catheterized patients arecapable of expressing type 1 fimbriae, type 1 fimbriated cells arepredominant in catheter biofilms. This is in accordance with anearly observation suggesting that long-term catheterization selectsfor E. coli strains capable of type 1 fimbria-mediated mannose-sensitive hemagglutination (50). However, a more sophisticatedcomparative analysis of clinical samples from catheter and voidedurine that takes into account duration of catheterization, cathetermaterial, patient data, and history will be necessary to support thisconclusion.
The confirmation of expression results from in vitro and in vivosamples and the generation of reproducible, normally distributedcolonization data suggest that the dynamic catheterized bladdermodel utilized in this study proves useful for gaining a better un-derstanding of catheter colonization during CAUTI. The differentfates of type 1 fimbria mutant CFT073fim during cochallenge ex-periments with the parental strain in the bladder population de-pending on the use of artificial and pooled human urine mayreflect differences in the nutrient concentration and the absence oforganic matrix proteins in the chemically defined artificial urine(51). Tamm-Horsfall protein (THP), the most abundant proteinin human urine, might contribute to this observation due to itswell-characterized binding to type 1 fimbriated cells (52, 53) aswell as adsorption to abiotic surfaces (54). Although THP concen-tration might be reduced in our experiments due to filter steriliza-tion of the urine, we propose that UPEC cells expressing type 1fimbriae are enriched in vitro in the bladder compared to fimcounterparts by fimbria-mediated binding to THP present on theglass or catheter surface. Thus, for future applications using thedynamic catheterized bladder model, it appears suitable to per-form initial experiments requiring large quantities of uniformurine utilizing artificial urine and to confirm important data setsusing pooled human urine.
Taken together, the results of this study contribute compel-lingly to the growing body of evidence derived from various mod-
els that type 1 fimbriae are key virulence factors in UTI andCAUTI. This underscores the relevance of a recent study demon-strating the utility of small molecules interfering with type 1 fim-bria-mediated adhesion to prevent infection in a murine model ofCAUTI (30). However, UPEC strains express a diversity of fim-briae and adhesins (17, 55, 56); thus, we believe that other fimbrialtypes and proteins also contribute to catheter colonization by E.coli, particularly for strains unable to express type 1 fimbriae. Thedynamic catheterized bladder model will prove useful for analysisof these factors in combination with experiments in in vivoCAUTI models and in parallel to analyses of clinical samples. De-termining the overlap of virulence factors necessary for initiationand persistence in the noncatheterized and catheterized urinarytract will be important to ensure applicability of vaccines devel-oped against UTI (57, 58) for CAUTI. In addition, identificationof unique factors required for catheter colonization will acceleratedevelopment of efficient prevention or treatment options ofCAUTI.
ACKNOWLEDGMENTS
We thank David J. Stickler and Sheridan D. Morgan for discussions andtechnical support for setting up the catheterized bladder model in ourlaboratory and Mark A. Schembri and Carsten Struve for providingstrains and plasmids. We thank anonymous volunteers (students andstaff) from the Biomedical Science Institute, University of Applied Sci-ences, for providing human urine samples.
This work was supported by the NAWI Graz fund (to E.Z.) and theAustrian Science Fund (FWF) under P13277-Gen (to E.Z.) and P21434-B18 (to A.R.).
REFERENCES1. Shirtliff ME, Leid J. 2009. The role of biofilms in device-related infec-
tions. In Costerton JW (ed), Springer series on biofilms, vol 3. Springer,Berlin, Germany.
2. Tambyah PA, Maki DG. 2000. Catheter-associated urinary tract infectionis rarely symptomatic: a prospective study of 1,497 catheterized patients.Arch. Intern. Med. 160:678 – 682.
3. Stickler DJ. 2008. Bacterial biofilms in patients with indwelling urinarycatheters. Nat. Clin. Pract. Urol. 5:598 – 608. http://dx.doi.org/10.1038/ncpuro1231.
4. McBain AJ. 2009. In vitro biofilm models: an overview. Adv. Appl. Mi-crobiol. 69:99 –132. http://dx.doi.org/10.1016/S0065-2164(09)69004-3.
5. Tolker-Nielsen T, Sternberg C. 2005. Growing and analyzing biofilms inflow chambers. Curr. Protoc. Microbiol. Chapter 1:Unit 1B.2. http://dx.doi.org/10.1002/9780471729259.mc01b02s21.
6. Merritt JH, Kadouri DE, O’Toole GA. 2005. Growing and analyzingstatic biofilms. Curr. Protoc. Microbiol. Chapter 1:Unit 1B.1. http://dx.doi.org/10.1002/9780471729259.mc01b01s00.
7. Coenye T, Nelis HJ. 2010. In vitro and in vivo model systems to studymicrobial biofilm formation. J. Microbiol. Methods 83:89 –105. http://dx.doi.org/10.1016/j.mimet.2010.08.018.
8. Bjarnsholt T, Alhede M, Alhede M, Eickhardt-Sørensen SR, Moser C,Kühl M, Jensen PØ, Høiby N. 2013. The in vivo biofilm. Trends Micro-biol. 21:466 – 474. http://dx.doi.org/10.1016/j.tim.2013.06.002.
9. Stewart PS. 2012. Mini-review: convection around biofilms. Biofouling28:187–198. http://dx.doi.org/10.1080/08927014.2012.662641.
10. Reisner A, Krogfelt KA, Klein B, Zechner EL, Molin S. 2006. In vitrobiofilm formation of commensal and pathogenic E. coli strains: impact ofenvironmental and genetic factors. J. Bacteriol. 188:3572–3581. http://dx.doi.org/10.1128/JB.188.10.3572-3581.2006.
11. Hancock V, Witsø IL, Klemm P. 2011. Biofilm formation as a function ofadhesin, growth medium, substratum and strain type. Int. J. Med. Micro-biol. 301:570 –576. http://dx.doi.org/10.1016/j.ijmm.2011.04.018.
12. Guiton PS, Hung CS, Hancock LE, Caparon MG, Hultgren SJ. 2010.Enterococcal biofilm formation and virulence in an optimized murinemodel of foreign body-associated urinary tract infections. Infect. Immun.78:4166 – 4175. http://dx.doi.org/10.1128/IAI.00711-10.
13. Murphy CN, Mortensen MS, Krogfelt KA, Clegg S. 2013. Role of Kleb-siella pneumoniae type 1 and type 3 fimbriae in colonizing silicone tubesimplanted into the bladder of mice as a model of catheter-associated uri-nary tract infections. Infect. Immun. 81:3009 –3017. http://dx.doi.org/10.1128/IAI.00348-13.
14. Mühldorfer I, Ziebuhr W, Hacker J. 2001. Escherichia coli in urinary tractinfections, p 1515–1540. In Sussman M (ed), Molecular medical microbi-ology, vol 2. Academic Press, London, United Kingdom.
15. Macleod SM, Stickler DJ. 2007. Species interactions in mixed-community crystalline biofilms on urinary catheters. J. Med. Microbiol.56:1549 –1557. http://dx.doi.org/10.1099/jmm.0.47395-0.
17. Jacobsen SM, Stickler DJ, Mobley HL, Shirtliff ME. 2008. Complicatedcatheter-associated urinary tract infections due to Escherichia coli and Pro-teus mirabilis. Clin. Microbiol. Rev. 21:26 –59. http://dx.doi.org/10.1128/CMR.00019-07.
18. Beloin C, Roux A, Ghigo JM. 2008. Escherichia coli biofilms. Curr. Top.Microbiol. Immunol. 322:249 –289.
19. Hadjifrangiskou M, Gu AP, Pinkner JS, Kostakioti M, Zhang EW,Greene SE, Hultgren SJ. 2012. Transposon mutagenesis identifies uro-pathogenic Escherichia coli biofilm factors. J. Bacteriol. 194:6195– 6205.http://dx.doi.org/10.1128/JB.01012-12.
20. Wood TK. 2009. Insights on Escherichia coli biofilm formation and inhi-bition from whole-transcriptome profiling. Environ. Microbiol. 11:1–15.http://dx.doi.org/10.1111/j.1462-2920.2008.01768.x.
21. Puttamreddy S, Cornick NA, Minion FC. 2010. Genome-wide trans-poson mutagenesis reveals a role for pO157 genes in biofilm developmentin Escherichia coli O157:H7 EDL933. Infect. Immun. 78:2377–2384. http://dx.doi.org/10.1128/IAI.00156-10.
22. Hancock V, Klemm P. 2007. Global gene expression profiling of asymp-tomatic bacteriuria Escherichia coli during biofilm growth in human urine.Infect. Immun. 75:966 –976. http://dx.doi.org/10.1128/IAI.01748-06.
23. Bertani G. 1951. Studies on lysogenesis. I. The mode of phage liberationby lysogenic Escherichia coli. J. Bacteriol. 62:293–300.
24. Stickler DJ, Morris NS, Winters C. 1999. Simple physical model to studyformation and physiology of biofilms on urethral catheters. Methods En-zymol. 310:494 –501.
25. Sokurenko EV, Courtney HS, Ohman DE, Klemm P, Hasty DL. 1994.FimH family of type 1 fimbrial adhesins: functional heterogeneity due tominor sequence variations among fimH genes. J. Bacteriol. 176:748 –755.
26. Lim JK, Gunther NW, Zhao H, Johnson DE, Keay SK, Mobley HL.1998. In vivo phase variation of Escherichia coli type 1 fimbrial genes inwomen with urinary tract infection. Infect. Immun. 66:3303–3310.
27. Pratt LA, Kolter R. 1998. Genetic analysis of Escherichia coli biofilm forma-tion: roles of flagella, motility, chemotaxis and type I pili. Mol. Microbiol.30:285–293. http://dx.doi.org/10.1046/j.1365-2958.1998.01061.x.
28. Beloin C, Michaelis K, Lindner K, Landini P, Hacker J, Ghigo JM,Dobrindt U. 2006. The transcriptional antiterminator RfaH repressesbiofilm formation in Escherichia coli. J. Bacteriol. 188:1316 –1331. http://dx.doi.org/10.1128/JB.188.4.1316-1331.2006.
31. Stickler DJ, Feneley RC. 2010. The encrustation and blockage of long-term indwelling bladder catheters: a way forward in prevention and con-trol. Spinal Cord. 48:784 –790. http://dx.doi.org/10.1038/sc.2010.32.
32. Abraham JM, Freitag CS, Clements JR, Eisenstein BI. 1985. An invert-ible element of DNA controls phase variation of type 1 fimbriae of Esche-richia coli. Proc. Natl. Acad. Sci. U. S. A. 82:5724 –5727. http://dx.doi.org/10.1073/pnas.82.17.5724.
33. Schwan WR. 2011. Regulation of fim genes in uropathogenic Escherichiacoli. World J. Clin. Infect. Dis. 1:17–25. http://dx.doi.org/10.5495/wjcid.v1.i1.17.
34. Hagan EC, Lloyd AL, Rasko DA, Faerber GJ, Mobley HL. 2010. Esch-erichia coli global gene expression in urine from women with urinary tractinfection. PLoS Pathog. 6:e1001187. http://dx.doi.org/10.1371/journal.ppat.1001187.
35. Feneley RCL, Kunin CM, Stickler DJ. 2012. An indwelling urinary cath-eter for the 21st century. BJU Int. 109:1746 –1749. http://dx.doi.org/10.1111/j.1464-410X.2011.10753.x.
36. Benton J, Chawla J, Parry S, Stickler D. 1992. Virulence factors inEscherichia coli from urinary tract infections in patients with spinal inju-ries. J. Hosp. Infect. 22:117–127. http://dx.doi.org/10.1016/0195-6701(92)90095-4.
37. Hasman H, Chakraborty T, Klemm P. 1999. Antigen-43-mediated au-toaggregation of Escherichia coli is blocked by fimbriation. J. Bacteriol.181:4834 – 4841.
38. Allsopp LP, Beloin C, Ulett GC, Valle J, Totsika M, Sherlock O, GhigoJ-M, Schembri MA. 2012. Molecular characterization of UpaB and UpaC,two new autotransporter proteins of uropathogenic Escherichia coliCFT073. Infect. Immun. 80:321–332. http://dx.doi.org/10.1128/IAI.05322-11.
39. Guyer DM, Radulovic S, Jones FE, Mobley HL. 2002. Sat, the secretedautotransporter toxin of uropathogenic Escherichia coli, is a vacuolatingcytotoxin for bladder and kidney epithelial cells. Infect. Immun. 70:4539 –4546. http://dx.doi.org/10.1128/IAI.70.8.4539-4546.2002.
40. Allsopp LP, Totsika M, Tree JJ, Ulett GC, Mabbett AN, Wells TJ, KobeB, Beatson SA, Schembri MA. 2010. UpaH is a newly identified auto-transporter protein that contributes to biofilm formation and bladdercolonization by uropathogenic Escherichia coli CFT073. Infect. Immun.78:1659 –1669. http://dx.doi.org/10.1128/IAI.01010-09.
41. Valle J, Mabbett AN, Ulett GC, Toledo-Arana A, Wecker K, Totsika M,Schembri MA, Ghigo JM, Beloin C. 2008. UpaG, a new member of thetrimeric autotransporter family of adhesins in uropathogenic Escherichiacoli. J. Bacteriol. 190:4147– 4161. http://dx.doi.org/10.1128/JB.00122-08.
42. Serra DO, Richter AM, Klauck G, Mika F, Hengge R. 2013. Microanat-omy at cellular resolution and spatial order of physiological differentiationin a bacterial biofilm. mBio. 4:e00103–13. http://dx.doi.org/10.1128/mBio.00103-13.
44. Lane MC, Li X, Pearson MM, Simms AN, Mobley HL. 2009. Oxygen-limiting conditions enrich for fimbriate cells of uropathogenic Proteusmirabilis and Escherichia coli. J. Bacteriol. 191:1382–1392. http://dx.doi.org/10.1128/JB.01550-08.
45. Snyder JA, Haugen BJ, Buckles EL, Lockatell CV, Johnson DE, Don-nenberg MS, Welch RA, Mobley HLT. 2004. Transcriptome of uro-pathogenic Escherichia coli during urinary tract infection. Infect. Immun.72:6373– 6381. http://dx.doi.org/10.1128/IAI.72.11.6373-6381.2004.
46. Struve C, Krogfelt KA. 1999. In vivo detection of Escherichia coli type 1fimbrial expression and phase variation during experimental urinary tractinfection. Microbiology 145:2683–2690.
47. Pere A, Nowicki B, Saxen H, Siitonen A, Korbonen TK. 1987. Expres-sion of P, type-I, and type-1C fimbriae of Escherichia coli in the urine ofpatients with acute urinary tract infection. J. Infect. Dis. 156:567–574.http://dx.doi.org/10.1093/infdis/156.4.567.
48. Kisielius PV, Schwan WR, Amundsen SK, Duncan JL, Schaeffer AJ.1989. In vivo expression and variation of Escherichia coli type 1 and P pili
in the urine of adults with acute urinary tract infections. Infect. Immun.57:1656 –1662.
49. Lichodziejewska M, Topley N, Steadman R, Mackenzie RK, Jones KV,Williams JD. 1989. Variable expression of P fimbriae in Escherichia coliurinary tract infection. Lancet i:1414 –1418.
50. Mobley HL, Chippendale GR, Tenney JH, Hull RA, Warren JW. 1987.Expression of type 1 fimbriae may be required for persistence of Esche-richia coli in the catheterized urinary tract. J. Clin. Microbiol. 25:2253–2257.
51. Griffith DP, Musher DM, Itin C. 1976. Urease. The primary cause ofinfection-induced urinary stones. Investig. Urol. 13:346 –350.
52. Orskov I, Ferencz A, Orskov F. 1980. Tamm-Horsfall protein or uro-mucoid is the normal urinary slime that traps type 1 fimbriated Escherichiacoli. Lancet i:887.
53. Pak J, Pu Y, Zhang ZT, Hasty DL, Wu XR. 2001. Tamm-Horsfall proteinbinds to type 1 fimbriated Escherichia coli and prevents E. coli from bind-ing to uroplakin Ia and Ib receptors. J. Biol. Chem. 276:9924 –9930. http://dx.doi.org/10.1074/jbc.M008610200.
55. Spurbeck RR, Stapleton AE, Johnson JR, Walk ST, Hooton TM, MobleyHLT. 2011. Fimbrial profiles predict virulence of uropathogenic Esche-richia coli strains: contribution of Ygi and Yad fimbriae. Infect. Immun.79:4753– 4763. http://dx.doi.org/10.1128/IAI.05621-11.
56. Wurpel DJ, Beatson SA, Totsika M, Petty NK, Schembri MA. 2013.Chaperone-usher fimbriae of Escherichia coli. PLoS One 8:e52835. http://dx.doi.org/10.1371/journal.pone.0052835.
57. Sivick KE, Mobley HL. 2010. Waging war against uropathogenic Esche-richia coli: winning back the urinary tract. Infect. Immun. 78:568 –585.http://dx.doi.org/10.1128/IAI.01000-09.
59. Blattner FR, Plunkett G, III, Bloch CA, Perna NT, Burland V, Riley M,Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, DavisNW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y. 1997. Thecomplete genome sequence of Escherichia coli K-12. Science 277:1453–1474. http://dx.doi.org/10.1126/science.277.5331.1453.
60. Miranda RL, Conway T, Leatham MP, Chang DE, Norris WE, Allen JH,Stevenson SJ, Laux DC, Cohen PS. 2004. Glycolytic and gluconeogenicgrowth of Escherichia coli O157:H7 (EDL933) and E. coli K-12 (MG1655)in the mouse intestine. Infect. Immun. 72:1666 –1676. http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004.
61. Reisner A, Haagensen JA, Schembri MA, Zechner EL, Molin S. 2003.Development and maturation of Escherichia coli K-12 biofilms. Mol. Micro-biol. 48:933–946. http://dx.doi.org/10.1046/j.1365-2958.2003.03490.x.
62. Welch RA, Burland V, Plunkett G, III, Redford P, Roesch P, Rasko D,Buckles EL, Liou SR, Boutin A, Hackett J, Stroud D, Mayhew GF, RoseDJ, Zhou S, Schwartz DC, Perna NT, Mobley HL, Donnenberg MS,Blattner FR. 2002. Extensive mosaic structure revealed by the completegenome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci.U. S. A. 99:17020 –17024. http://dx.doi.org/10.1073/pnas.252529799.
