Commensal Urinary Lactobacilli Inhibit Major Uropathogens In Vitro With Heterogeneity at Species and Strain Level

Abstract

The human urinary microbiome is thought to affect the development and progression of urinary tract infections (UTI), particularly recurrent UTIs in aging populations of women. To understand the possible interactions of urinary pathogens with commensal bacteria inhabiting the aging bladder, we conducted an initial functional assessment of a representative set of urinary lactobacilli that dominate this niche in postmenopausal women. We created a repository of urinary bladder bacteria isolated via Enhanced Quantitative Urinary Culture (EQUC) from healthy postmenopausal women, as well as those with a culture-proven recurrent UTI (rUTI) diagnosis. This repository contains lactobacilli strains from eight different species. As many other lactobacilli are known to inhibit human pathogens, we hypothesized that some urinary lactobacilli will have similar abilities to inhibit the growth of typical uropathogens and thus, provide a link between the urinary microbiome and the predisposition to the rUTI. Therefore, we screened the urinary lactobacilli in our repository for their ability to inhibit model uropathogens in vitro. We observed that many urinary isolates strongly inhibit model strains of gram-negative Escherichia coli and Klebsiella pneumoniae but demonstrate less inhibition of gram-positive Enterococcus faecalis. The observed inhibition affected model strains of uropathogens as well as clinical and multidrug-resistant isolates of those species. Our preliminary analysis of inhibition modes suggests a combination of pH-dependent and cell-dependent inhibition. Overall, inhibition strongly varies among species and strains of urinary lactobacilli. While the strength of the inhibition is not predictive of health outcomes in this limited repository, there is a high level of species and strain diversity that warrants future detailed investigations.

Keywords: commensal lactobacilli; urinary lactobacilli; urinary microbiome; urinary tract infection; uropathogens.

Figure 1

Urinary lactobacilli L. gasseri 5006-2 and L. delbrueckii 5010-2 inhibit a broad range of uropathogens. Representative images of well-diffusion inhibition assays using E. coli, K. pneumoniae, and E. faecalis (A), and zone of inhibition sizes of one-day old (dark blue) or two-day old (light blue) cultures of L. gasseri 5006-2 [Lg, (B)] and L. delbrueckii 5010-2 [Ld, (C)] against 8 pathogenic strains ( Table 2 ). Error bars indicate standard deviation of three biological replicates, *p < 0.05 and **p < 0.01.

Figure 2

Majority of urinary lactobacilli exhibit varying degrees of inhibition of major uropathogens. Twenty strains of urinary commensal lactic acid bacteria (lactobacilli and one strain of Streptococcus anginosus as a control) were tested against model uropathogenic strains: E. coli CFT073, K. pneumoniae TOP52, and E. faecalis OG1RF. Bars indicate the standard deviation of three replicates. The leftmost plot carries identifications of the patient cohorts for each strain ( Table S3 ): empty square – no UTI; black triangle – rUTI; white triangle – rUTI with prophylactic antibiotics. The data are presented by species alphabetically and sorted by the inhibition against CFT073 within species set.

Figure 3

Lactobacilli cohabiting the same bladder with a pathogen still can inhibit that pathogen in vitro. (A) Table showing average zones of inhibition (mm) by cohabiting E. coli and E. faecalis strains. The comparison indicates that cohabiting pathogen strains (shaded in grey) are inhibited by the respective cohabiting commensal urinary lactobacilli but to lesser degree than other pathogen isolates and the model uropathogen strains (no grey shading). (B) Overall screen of patient isolates shows that ‘better inhibiting’ lactobacilli strains against one pathogen isolate also show ‘better inhibiting’ against another pathogen isolate. In addition, L. rhamnosus strains appear to be better inhibitors, similar to the model screen ( Figure 2 ).

Figure 4

Urinary lactobacillus strains inhibit E. coli growth when co-cultured in liquid medium. Uropathogenic E. coli CFT073 was grown in presence of increasing amounts of initial lactobacilli (L. gasseri 5006-2 and L. delbrueckii 5010-2) with ratios 1:0, 1:1, 1:5, 1:10, and 1:100 estimated by the OD600 measurements. E. coli survival was measured by LB plating of serial dilutions for CFU counting and compared with growth of E. coli only culture (1:0). Survival is calculated as culture density normalized to E. coli only culture. Error bars show only positive arm of standard deviation for three biological replicates. *p < 0.05 and **p < 0.01 and indicate a significant decrease from E. coli grown in the presence of lactobacilli. Over the course of this experiment E. coli for lactobacilli-free condition (1:0) grows from ~ 2.5-3.4×10⁶ to ~ 8.4 - 9.7×10⁸ CFU/mL densities. n/d – the limit of detection of our CFU counting was at about 500 CFU/mL, corresponding to ~ 0.5×10^-6 survival.

Figure 5

Lactobacilli inhibition differs depending on culture treatment. Representative images from well-diffusion assays using whole cell culture, cells resuspended in fresh broth, cell-free filtrate, heated filtrate, and filtrate adjusted to a neutral pH. Images taken for 24 hrs. growth at 37°C.