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. Author manuscript; available in PMC: 2009 May 11.
Published in final edited form as: J Infect Dis. 2007 Apr 26;195(11):1671–1677. doi: 10.1086/517524

Importance of the Endocarditis and Biofilm-associated Pilus (ebp) Locus in the Pathogenesis of Enterococcus faecalis Ascending Urinary Tract Infection

Kavindra V Singh 1,2, Sreedhar R Nallapareddy 1,2, Barbara E Murray 1,2,3
PMCID: PMC2680192  NIHMSID: NIHMS110086  PMID: 17471437

Abstract

Background

We recently showed that the ubiquitous Enterococcus faecalis ebp (endocarditis and biofilm-associated pilus) operon was important for biofilm and experimental endocarditis. Here we assess its role in murine urinary tract infection using wild type E. faecalis OG1RF and its nonpiliated ebpA allelic replacement mutant (TX5475).

Methods

OG1RF and TX5475 were administered transurethrally in a 1:1 ratio (competition assay) or individually (monoinfection). Kidneys and bladders were cultured 48 h postinfection. These strains were also tested in a peritonitis model.

Results

No differences were observed in the peritonitis model. In mixed urinary infection, OG1RF significantly outnumbered TX5475 in kidneys (P=0.0033) and bladders (P=<0.0001). More OG1RF CFU were also recovered from kidneys of monoinfected mice at the four inocula tested (P=0.015 to 0.049) and ID50s of OG1RF for kidney and bladder (9.1 × 101 and 3.5 × 103 CFU, respectively) were two-three log10 lower than with TX5475. Increased tropism for the kidney relative to the bladder was observed by both OG1RF and TX5475.

Conclusion

The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.

INTRODUCTION

Among the various infections reported with Enterococcus faecalis, urinary tract infections (UTI) are the most common [1-3]. Little is known about the bacterial factors necessary for E. faecalis to cause infections in general and even less has been reported related to the urinary tract [2, 4, 5]. The presence of Esp, encoded by an acquired gene, was shown to increase persistence of bacteria in the urinary bladders of mice, with no histological changes, when a parent and its esp mutant were used in a ascending urinary tract infection model [5]; however, there was no difference found in kidneys. Kau et al. [2], using a similar model, demonstrated that E. faecalis has a preference for the kidney relative to the bladder and concluded that this model can be used to study the factors involved in the pathogenesis of urinary tract infections.

E. faecalis is also known to form biofilm on biomedical devices such as urinary catheters and central venous catheters and several genes [6-9] have been shown to be important for biofilm formation among E. faecalis strains, including fsr via its effect on gelatinase [7, 10, 11]. Biofilm likely contributes to the high occurrence of enterococci in UTIs in the hospital setting, in part because of the large number of catheterized patients, and in part because biofilm formation may promote persistence of bacteria in the urinary tract. Therefore, it is important to understand the relationship between genes involved in biofilm formation and the pathogenicity of E. faecalis in the urinary tract.

We recently identified and characterized the ebp operon (endocarditis and biofilm associated pili) which consists of, ebpA, ebpB, ebpC and an associated srtC (encoding sortase C) gene which also has an independent promoter [12]. These surface pili are immunogenic and pleomorphic in nature and their role in the establishment of biofilms and infective endocarditis in a rat model was shown using the wild type (w.t.) E. faecalis strain OG1RF and its isogenic nonpiliated ΔebpA (TX5475) mutant. Our hypothesis here is that E. faecalis Ebp-associated pili may play a role similar to that of the fimbriae of Escherichia coli and Proteus mirabilis in promoting colonization and adherence to uroepithelium. In this study, using w.t. E. faecalis OG1RF, which expresses sortase C-dependent pleomorphic surface pili [12], and its nonpiliated TX5475, we describe their ability to colonize kidneys and bladders in both monoinfection and a mixed infection competition assay in a murine UTI model. We also tested both strains in a mouse peritonitis model of sepsis.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Bacteria used in the study include wild type OG1RF (resistant to rifampicin 100 μg/ml, fusidic acid 25 μg/ml) (ATCC 47077), a well-known plasmid-free isolate used in many laboratories [13]; OG1RF is a medium biofilm producer [7] and has been shown to be pathogenic in various animal models [12, 14-17]. A recently described nonpiliated, biofilm-deficient ebpA isogenic deletion (polar) mutant of OG1RF, TX5475 with ebpA replaced with kanamycin resistance gene was also used [12]. Brain Heart Infusion broth (BHIB) (Difco Laboratories) was used for routine bacterial growth. Kanamycin (KAN) and heat inactivated horse serum were purchased from Sigma. The concentration of antibiotics used in BHI agar (BHIA) plates for the growth of nonpiliated TX5475 was KAN 2000 μg/ml.

UTI model for competition assay, monoinfections and ID50 determination

Female, 4- to 6-week-old, outbred ICR mice (Harlan Sprague Dawley) with a mean weight of 25 g were used in this study. Pre-approved protocol and guidelines of the Animal Welfare Committee of the University of Texas Health Science Center at Houston were followed throughout the course of the animal experiments. Mice were prescreened for UTI by culturing the urine ∼12 h prior to inoculation. The urinary bladders of mice were emptied by gently pressing the abdomen before infecting them. For preparation for administration to mice, bacteria were grown in 10 ml of BHIB + 40% horse serum for 10 h at 37°C with gentle shaking. Cells were pelleted for 10 minutes (10,000 rpm at 10°C), and resuspended in 10 ml of 0.9% saline. Further dilutions were also prepared in saline, and plated to determine the actual inoculum that had been administered. An inoculation method similar to a recently described mouse UTI model [2] was used. A group of 16 mice was used in the competition assay while groups of 5 mice per inoculum were used in each of two independent monoinfection experiments with combination of the results. In brief, isoflurane anesthetized mice were infected via intraurethral catheterization (polyethylene catheter, ∼2.5 - 3 cm long; outer diameter, 0.61 mm; Clay Adams) with 200 μl of the bacterial suspension consisting of an approximately 1:1 ratio of w.t. E. faecalis OG1RF:nonpiliated TX5475, respectively, in a competition assay; two independent experiments using 10 and 6 mice, respectively, were performed and the results were combined. In two independent monoinfection experiments, w.t. OG1RF was used in the inocula range of 101 - 106 while the nonpiliated TX5475 was tested in the inocula range of 103 - 106 to generate data for comparison of CFU recovered from kidneys and bladder of mice as well as for the calculation of 50% infective dose (ID50). Similar to the method described by Kau et al. [2], we also used an inoculum volume of 200 μl in order to facilitate direct delivery of the bacteria to both bladder and kidneys, simultaneously. The urethral catheter was removed immediately after injecting the bacteria, and all the animals were provided free access to food and water during the course of study. Daily observations were made for any morbidity and mortality in animals. Mice were euthanized by CO2 inhalation at 48 h after transurethral challenge, the urinary bladder and kidney pair were excised, weighed, homogenized in 1 ml and 5 ml of saline, respectively, and dilutions were plated onto BHIA for recovery of bacteria in case of monoinfections and onto BHIA and BHIA+KAN 2000 μg/ml in the case of mixed infection in order to determine the recovered CFUs (%) of w.t. OG1RF versus nonpiliated TX5475. The detection limit of bacteria in this experiment was 10 CFU. Mice with sterile cultures of kidney and urinary bladder homogenates were considered to have no UTI infection. Identity of the recovered test bacteria from infected organs was confirmed by plating them on Bile Esculin Azide agar (BEA) and BHIA + RIF100 plates for w.t. E. faecalis OG1RF and on BHIA + Kan2000, BHIA + RIF100 and BEA plates for the nonpiliated TX5475. Randomly picked nonpiliated TX5475 colonies were also tested by PCR, pulsed-field gel electrophoresis (PFGE) [18] and/or by high stringency hybridization of PFGE gels to reconfirm the identity of test bacteria.

Mouse peritonitis model

E. faecalis strains OG1RF and TX5475 were tested using our previously published method [17]. Mice were injected intraperitoneally with appropriate dilutions of premixed bacteria/sterile rat fecal extract (SRFE) and were observed for 5 days. Two fold dilutions of both test bacteria in a range of ∼1 × 108 – 1 × 109 CFU were used as the inocula for LD50 determination using six mice per group. The LD50 was determined by the method of Reed and Muench [19].

Statistics, ID50s, and mean virulence index

The log10 CFU per gram of TX5475 and w.t. OG1RF in tissue of each animal (kidney or bladder) from mixed infection were analyzed for significance by the paired t test. Differences in the log10 CFU of TX5475 versus w.t. OG1RF in monoinfections were evaluated by the unpaired t test. Fisher's exact test was used to compare the total infection of kidneys and bladders (combining all inocula) in monoinfection with OG1RF versus TX5475. Prism version 4.00 for Windows (GraphPad Software) was used for statistical analysis. The fifty percent infective dose (ID50s) values were calculated by a previously published method [19]. The mean virulence index was calculated using the following equation (as previously described for other organisms in mixed infections [12, 15, 20]):

Mean virulence index=(GM−CFUofOG1RF)T=0∕(GM−CFUof theTX5475mutant)T=0(GM−CFUofOG1RF)T=48∕(GM−CFUofTX5475mutant)T=48

where GM-CFU is the geometric mean expressed as CFU.

RESULTS

Competition assay

In the competition experiment, approximately equal numbers (as judged by OD at 600nm) of w.t. OG1RF and TX5475 mutant were administered transurethrally. The actual CFU of w.t. E. faecalis OG1RF and the TX5475 mutant (combined from two experiments) in the inoculum mix were 1.1 × 107 and 1.2 × 107, respectively; thus, the percent of w.t. E. faecalis OG1RF in the inoculum was 49% (figure 1). W.t. OG1RF outcompeted the TX5475 mutant in all 16 mice at 48 h post infection, increasing significantly from time zero (T = 0) in kidneys and urinary bladder, with mean paired differences of 3.1- fold (P = 0.0033) and 179- fold (P = <0.0001), respectively (figure 1). The mean virulence indices of nonpiliated TX5475 relative to w.t. OG1RF in kidneys and bladders were 0.30 and 0.005, respectively.

Figure 1.

Figure 1

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Mixed infection (competition assay) by w.t. E. faecalis OG1RF and nonpiliated TX5475 in the kidneys and urinary bladder of mice (n=16). Data are expressed as log10CFU of w.t. E. faecalis OG1RF or TX5475. The log10CFU/gm from both kidneys were combined and averaged. Solid diamonds and triangles represent w.t. E. faecalis OG1RF and TX5475, respectively, from kidney homogenates and empty diamonds and triangles represent w.t. E. faecalis OG1RF and TX5475, respectively, from urinary bladder homogenates. Horizontal bars represent geometric mean titer. Mean fold difference in CFU are given. Log10 CFU were compared for statistical significance using a paired t test.

Monoinfection of kidneys and bladders and ID50s

Because of the possibly that nonpiliated TX5475 colonies may have adhered to or been entrapped by the surface pili of OG1RF and, therefore, may have been retained at the infection site/s during the course of experiment, we also performed monoinfections with four equal inocula of each organism. The total number of kidneys infected (38/40 with OG1RF versus 27/40 with TX5475) (P=0.0031) and total urinary bladders infected (30/40 with OG1RF versus 18/40 with TX5475) (P=0.0115), combining from all four monoinfection inoculum groups indicated that TX5475 was significantly attenuated in its ability to infect both kidneys and bladders versus its parent strain w.t. OG1RF (table 1). CFU counts recovered from animals showed that when mice (n=10) were infected with 1-4 × 103, 1-4 × 104, 1-4 × 105, 1-4 × 106 CFU of w.t. OG1RF, the GM-CFU recovered from kidneys at 48 after transurethral challenge were 2.1 × 104, 2.4 × 105, 1.7 × 106, 1.0 × 106, respectively (figure 2). In mice infected with 2-4 × 103, 2-4 × 104, 2-4 × 105, 2-4 × 106 CFU of TX5475, the GM-CFU recovered from kidneys were 6.2 × 101, 3.6 × 103, 1.3 × 104, 8.9 × 103, respectively (figure 2), showing ≥two log10 reduction in GM-CFUs of TX5475 versus GM-CFUs of w.t. OG1RF for the same inoculum (figure 2). The mean CFU differences (log10) seen in all four inocula groups (103, 104, 105 and 106) between w.t OG1RF versus nonpiliated TX5475 were 2.5 ± 1.2 (P=0.0493), 1.8 ± 0.9 (P=0.0490), 2.1 ± 0.8 (P=0.0156) and 2.1 ± 0.9 (P=0.0333), respectively, (figure 2). In bladders after monoinfection, although fewer bladders were infected by TX5475 (table 1), the CFU differences were not significant with mean differences (log10) of 1.2 ± 1.2, 0.9 ± 1.3 and 0.9 ± 1.1 (in the 103, 105 and 106 inocula group, respectively) and 2.7 ± 1.4 (in the 104 inoculum group). The results in table 1 also indicate tropism for the kidney, since 20 – 30% of the urinary bladders were found to be sterile in the same mice whose the corresponding kidneys were infected (OG1RF) and 20 – 50% of the urinary bladders were found to be sterile in the same mice whose kidneys were infected with the nonpiliated TX5475. The ID50s derived from monoinfection experiments using w.t. OG1RF for kidney pairs and urinary bladders were determined to be 9.1 × 101 and 3.5 × 103 CFU, respectively, while the ID50s using TX5475 for kidneys and bladder were determined to be 1.67 × 104 and 1.68 × 105 CFU, respectively, showing that ≥two log10 more cells were required to infect 50% of the mice versus w.t. OG1RF. ID50s also showed that approximately one log10 fewer CFU of either bacteria were needed to infect 50% of the kidney pairs than to infect 50% of the urinary bladders.

Table 1.

Tropism for the kidneys by E. faecalis strains OG1RF and its isogenic nonpiliated ΔebpA (TX5475) mutant in a murine model of ascending urinary tract infection

Infected kidney pair and urinary bladder
Inocula used Kidneys
 Bladder
OG1RF TX5475 OG1RF TX5475
103 8/10 3/10 5/10 3/10
104 10/10 8/10 7/10 3/10
105 10/10 8/10 8/10 6/10
106 10/10 8/10 10/10 6/10
ID50 9.1 × 101 1.6 × 104 3.5 × 103 1.6 × 105
Total infected 38/40 27/40 30/40 18/40
P=0.0031a P=0.0115b
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a

P values, determined by Fisher's exact test, are for total kidneys infected by OG1RF compared with TX5475;

b

P values are for total urinary bladders infected by OG1RF compared with TX5475

Figure 2.

Figure 2

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Monoinfection (106, 105, 104, and 103 inocula) using w.t. E. faecalis OG1RF or nonpiliated TX5475 in the kidneys of mice (n=10). Data are expressed as log10CFU/gm of w.t. E. faecalis OG1RF or TX5475 recovered from kidney homogenates 48 hrs after transurethral challenge. The log10CFU from both kidneys were combined and averaged. Solid and empty triangles represent w.t. E. faecalis OG1RF and TX5475, respectively. Horizontal bars represent geometric mean titer. Mean difference in CFU is given as log10 ± standard deviation (SD) for the respective inocula. TX5475 CFUs (recovered from kidneys) were significantly reduced versus OG1RF with all four inocula using unpaired t test.

Mouse peritonitis model

In an experiment using six mice per inoculum, both test bacteria caused mortality at a similar rate (monitored at 3- 6 h intervals for 96 h) in mice (data not shown) and were not significantly different by statistical analysis using log rank (Mantel-Cox) test, although there was a small (1.9 fold) decrease in LD50 for TX5475.

DISCUSSION

E. faecalis was recognized as an important organism associated with UTIs as long ago as the early 1900s, but much needs to be learned about the pathogenesis of this organism in this site. Among gram negative bacteria, E. coli is the leading cause of community-acquired UTIs [4, 21-25] and E. coli surface fimbriae or pili (type 1, P and related fimbriae, and F1C fimbriae) are known to mediate initial adherence [5, 26-28]. Recently, it has been shown that the flagella of uropathogenic E. coli contribute to the persistence of the organism in bladder and kidney [29]. Among gram positive bacteria, pili have been recognized in the past few years [30-37] but their role in UTI has not yet been reported. Our recently published study [12] showed the presence of pleomorphic endocarditis and biofilm-associated pili on E. faecalis OG1RF and that a nonpiliated ebpA deletion mutant of OG1RF (TX5475) was significantly attenuated in a rat model of infective endocarditis and formed significantly less biofilm in an in vitro assay. Results of the current study using w.t. OG1RF versus TX5475 was an effort to determine if the ebp-locus played a role in colonization of kidneys and urinary bladders in a murine UTI model. The reduced recovery of TX5475 mutant versus w.t. OG1RF from kidneys (≥2 log10) and bladders (≥1 log10), evident with all four inocula used in monoinfection, confirmed this hypothesis. Although our results with the ebpA allelic replacement mutant TX5475 (that produces none of the three Ebp proteins) [12] unambiguously demonstrated a role for the ebp locus in urinary tract infection in this model, we acknowledge that we have not definitively excluded the possibility of an independent contributory effect of its associated sortase C. It is unlikely, however, that srtC played an independent role under the conditions tested, because RT-PCR at these conditions showed elevated levels of the independent srtC transcript (Nallapareddy et al., unpublished data). Furthermore, the complementation of TX5475 with srtC did not restore biofilm formation [12]. Our ongoing studies with different sortase mutants may be helpful in identifying any additional effects of sortase C. Both test bacteria used for infection in the present study were grown in 40% serum + BHIB. Serum was chosen for the growth of test bacteria in the present study as our recently published study [12] showed an enhanced level of Ebp when w.t E. faecalis OG1RF was grown in 40% horse serum + BHIB versus growth in BHIB alone. It has previously been shown that growth in serum increased the adherence of tested strains to urinary tract epithelial cells and that clinical UTI isolates adhered to urinary tract epithelial cells more efficiently than to the cultured cells by at least 1.5- to 3-fold, with the greatest increase being observed in UTI strain adherence to Girardi heart cells (8-fold) [38, 39]. Similarly, others have shown the differential expression of several virulence-related genes in E. faecalis in response to biological cues in serum and urine [40] and it would be of interest to test growth in urine as well.

An E. faecalis protein that has been described previously as influencing bladder colonization is Esp which is reported to be displayed on the cell surface; the esp gene was found significantly more often in strains of human UTI origin than in fecal isolates [5, 41]. A disruption mutation in esp reduced colonization of the urinary bladder in an ascending model of urinary tract infection [5], although this study failed to demonstrate any difference in bacterial counts in kidneys after infection with the esp mutant versus its parent strain, even in mice whose bladders showed differences. Another study which also used the ascending model of mouse urinary tract infections was unable to show a role for aggregation substance (AS) or enterococcal binding substance (EBS), either in competition assay or in mono-infection [4]. Both the esp mutant testing and AS/EBS studies [4, 5] noted inconsistent urinary bladder colonization in mice whose kidneys were infected, as was also observed in the present study with both w.t. OG1RF and TX5475; this is consistent with a previously published study of Kau et al. [2] that E. faecalis has greater tropism for the kidneys. The Kau et al. study [2] showed that when the inoculum volume of E. faecalis was increased from 50 μl to 200 μl, in order to facilitate direct delivery of the bacteria to both the bladder and the kidneys simultaneously, there was more consistent colonization and recovery of bacteria from >90% of kidneys in mice infected with E. faecalis. In preparing for this study, we also found that when 50 μl inocula were used to infect the mice, only one of six mice had infected kidneys with no infected bladders versus using 200 μl inocula volume which showed six of six mice with infected kidneys and three of six mice with infected bladder (data not shown). Kau et al. [2] suggested that many or most episodes of enterococcal cystitis may be due to seeding from an upper tract infection and that asymptomatic bacteriuria with enterococci is often localized to the upper tract rather than the bladder; although we are unaware of data from humans that indicate that upper tract infection with enterococci is more common, this is certainly an interesting hypothesis.

Competition assays in which equal CFUs (in a mixed suspension) are inoculated to infect animals have been successfully used in endocarditis models [15, 16, 42] to differentiate the infective capabilities of w.t. organisms and their isogenic mutants. Kau et al. [2] showed the growth advantage by a pyelonephritis clinical isolate over a laboratory strain of E. faecalis in the kidneys, but not in the bladder, in a mouse UTI model and showed utility of this model to study factors involved in the pathogenesis of urinary tract infections, as it has been with E. coli [29]. In the competition assay of the present study, we found a significant differences between recovered CFUs of w.t. OG1RF versus the TX5475 from kidneys (3.1 fold) and bladders (179 fold). However, in all four inoculum groups of monoinfections, the differences seen in recovered CFUs of w.t. OG1RF versus TX5475 from kidneys were much greater (up to 1000 fold) versus the differences seen in recovered CFUs from kidneys in the competition assay. We suspect that the smaller differences seen between kidney counts of w.t. OG1RF and the TX5475 in the competition assay might be due to some of the nonpiliated TX5475 cells adhering to the Ebp surface pili of w.t. OG1RF and remaining at the infected sites and this hypothesis is partially supported by our earlier observation of frequent association of Ebp pili with more than one cell in immunogold electron micrographs [12]. Our previous study also found that anti-Ebp stained pili were seen in few w.t. OG1RF cells (<20% of TSBG- and <2% of BHIB-grown cells, respectively) and one possibility is that a low % of piliated w.t. OG1RF cells could be immunologically advantageous to the organism [12]. It is also possible that in vivo conditions increase production of pili.

In the mouse peritonitis model, TX5475 did not show significant attenuation versus w.t. OG1RF during the course of infection although the total mortality showed ∼two fold more cells of TX5475 were needed to cause disease in 50% of the animals. The, atmost, very modest attenuation by TX5475 in peritonitis model versus the high attenuation observed in the UTI model when compared with w.t. OG1RF shows the importance of selecting an appropriate animal model in order to assess the effect of adhesin related genes. The Ebp proteins previously have been recognized as the members of the microbial surface component recognizing adhesive matrix molecules (MSCRAMM) family [43] and a UTI murine model appears to provide suitable in vivo conditions to allow ebp-associated pili of w.t. OG1RF to attach and colonize the host renal tissue.

In conclusion, a nonpiliated TX5475 mutant of OG1RF showed marked attenuation versus w.t. OG1RF in terms of ID50, virulence index, CFU in kidneys after monoinfection and total numbers of kidneys and bladders colonized in an ascending mouse UTI model. There was increased tropism of each strain for kidneys versus bladders, and results with bladders also showed attenuation of TX5475 based on ID50s and the competition assays. The degree of attenuation of the nonpiliated TX5475 is impressive and is consistent with our previous report showing significant reduction in biofilm production and attenuation in an infective endocarditis rat model [12]. Our previous finding that 100% of 408 strains tested contain ebp genes using high stringency hybridization indicates that these genes are part of the core E. faecalis genome and suggests that all strains could rely on these pili for colonization and/or infection of tissues. Extrapolating from a report that pilicides target and interfere with pilus assembly function and disrupt the infection process of uropathogenic E. coli [44], these apparently conserved enterococcal surface pili could be a potential target for new and yet to be discovered gram positive specific pilicides as a way to treat or prevent enterococcal urinary tract infections in human.

ACKNOWLEDGEMENT

This work was supported in part by NIH grant R37 AI47923 from the Division of Microbiology and Infectious Diseases, NIAID to Barbara E. Murray. We would like to express our sincere thanks to Richard A. Hull, Baylor College of medicine, Houston, TX for helping us learn the urinary tract catheterization techniques for the experimental UTI model. We would also like to express our sincere thanks to Karen Jacques Palaz and Charlene L. Thomson for their technical help.

Financial support: National Institutes of Health grant R37 AI47923 from the Division of Microbiology and Infectious Diseases, NIAID to B.E.M.

Footnotes

Potential conflict of interest: None reported

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