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. 2017 Mar 22;9(382):eaaf1283.
doi: 10.1126/scitranslmed.aaf1283.

Bacterial Virulence Phenotypes of Escherichia coli and Host Susceptibility Determine Risk for Urinary Tract Infections

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Free PMC article

Bacterial Virulence Phenotypes of Escherichia coli and Host Susceptibility Determine Risk for Urinary Tract Infections

Henry L Schreiber 4th et al. Sci Transl Med. .
Free PMC article

Abstract

Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in their urovirulence, that is, their ability to infect the bladder in a mouse model of cystitis. We found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and metabolism, could be used to predict subsequent colonization of the mouse bladder. Together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

Conflict of interest statement

Competing interests: T.J.H. is a part-time employee at Fimbrion Therapeutics (St. Louis, MO), a company co-founded by T.M.H. and S.J.H. that is developing small molecule therapeutics to treat UTIs caused by UPEC. T.M.H. is an advisory board member with GlaxoSmithKlein (Brentford, United Kingdom), Paratek Pharmaceuticals (Boston, MA), Achaogen Pharmaceuticals (San Francisco, CA), and Ocean Spray (Middleborough, MA). S.J.H. is an advisory board member of Genentech (San Francisco, CA) and Roche Pharmaceuticals (Basel, Switzerland), a consultant for Regeneron (Tarrytown, NY) and Wellspect Healthcare (El Segundo, CA) and a founding member of QureTech Bio (Umea, Sweden).

Figures

Figure 1
Figure 1. Phylogenetic distribution of UAEC strains from rUTI patients
The phylogenetic relatedness of the urine-associated E. coli (UAEC) strains (n=43, taxon labels in red) was contextualized within the broader phylogeny of reference E. coli strains (n=46, taxon labels in black) by comparing the single-copy core genes of the strains using the RAxML algorithm. Reference E. coli strains that were associated with urinary disease (e.g. cystitis, pyelonephritis, or asymptomatic bacteriuria) are in bold. Asterisks indicate UAEC strains chosen as representative isolates for their clonal clusters (Fig. S2B). Bootstrap supports are indicated at internal nodes, and bootstrap values >95 have been removed. UAEC strains were found in four out of five E. coli clades (indicated by red and purple bars on the left). Black arrows indicate model UPEC strains commonly used in UTI research.
Figure 2
Figure 2. Carriage of putative urovirulence factors (PUFs) is enriched in both UAEC and non-UAEC strains from the B2 clade
(A) Comparisons between indicated groups were performed using the Mann-Whitney U test. Significant differences were calculated for each group and significant results are indicated as: ***, P<0.001; ****, P<0.0001. (B) Clinical UAEC strains and reference E. coli strains that were not associated with urinary disease (non-UAEC) (x-axis) were examined for the presence of 31 PUF sequences (y-axis) using BLAST and alignment-based searches. The binary presence of PUFs (indicated by black squares) was tallied for each strain and for each PUF (indicated in parentheses on both axes). Two-dimensional hierarchical clustering identified clusters of PUFs that tended to co-occur in UAEC strains (dendrogram along the y-axis) and showed that PUF carriage was associated with phylogeny (dendrogram along the x-axis, phylogeny indicated in column labeled ‘Clade’).
Figure 3
Figure 3. Increased PUF carriage does not correlate with increased colonization efficiency
(A) The colonization efficiencies of 21 representative UAEC strains and the model strain UTI89 from indicated clades were tested in C3H/HeN mice. Bacteria were enumerated from individual harvested bladders at 24 hours post-infection (hpi) (black boxes). Each UAEC strain was categorized as: “deficient” (n=4, blue stippled outline) at <104 CFU/bladder, “variable” (n=5, orange outline) above and below 104 CFU/bladder, or “robust” (n=12, green outline) at >104 CFU/bladder. The black broken horizontal line represents the limit of detection of bacteria. Data presented represent the median (gray bar) of mouse inoculations for each strain. (B) No enrichment of PUF carriage, as measured by PUF scores, was found in comparisons of robust, variable or deficient colonizer strains. Comparisons were performed with Mann-Whitney U test; horizontal solid bars indicate median values. (C) Using Spearman’s rank correlation (ρ statistic indicated at the top), there was no significant correlation between carriage of PUFs and bladder burden with UAEC strains at 24 hpi. Squares indicate the median bladder colonization of B2 (red) or non-B2 (purple) UAEC strains.
Figure 4
Figure 4. Both B2 and non-B2 UAEC strains cause chronic cystitis in mice
Incidence of chronic cystitis, defined as persistent high-titer bacteriuria (>104 CFU/mL urine), high-titer bladder colonization (>104 CFU/bladder) and chronic inflammation at 28 days post-infection (dpi), was measured for a subset of B2 and non-B2 UAEC strains infecting C3H/HeN mice. Both B2 and non-B2 UAEC strains could cause chronic cystitis in mice. PUF scores and incidence of chronic cystitis are indicated for each strain. Horizontal bars indicate median values.
Figure 5
Figure 5. PUF carriage does not increase competitive advantage during co-infection
UAEC strains isolated from two patients (9 and 41) with different strain-induced rUTIs were differentially marked with antibiotic resistance markers. C3H/HeN mice were then coinfected with the supplanting strain (9.2p or 41.4p) at an equal dose with their enrollment strain from the same patient (9.1a or 41.1a, respectively). A competitive index (CI) was calculated from the ratio of the supplanting strains over their enrollment strains in the urine from each mouse at 24 hpi. Clades and PUF scores are indicated for each strain. Horizontal bars indicate median values.
Figure 6
Figure 6. Differential expression of core genes by UAEC strains distinguishes robust from deficient colonizers
(A) Principle component analysis (PCA) was used to cluster UAEC strains based on their expression of 3,340 core genes under type 1 pili-inducing culture conditions used to culture bacteria before inoculation into mouse bladders. B2 strains separated from non-B2 strains along PC1, whereas robust colonizers separated from deficient colonizers along PC2. Linear regression identified the expression of 42 core genes in type 1-inducing conditions as correlated to bladder bacterial burden at 24hpi in C3H/HeN mice after correction for false discovery (P-adjusted<0.1). These 42 genes included pathways genes mediating nutrient uptake, such as lamB (B) and chemotaxis, such as and tar (C), whose correlations to bladder burden are visualized here as representative examples. Data presented represent averages derived from three independent experiments that were corrected for variance in read depth between samples and gene length.
Figure 7
Figure 7. Colonization efficiency by UAEC strains varies between mouse models
Select UAEC strains were inoculated into C3H/HeN mice (black squares and light gray bars) and C57BL/6 mice (black circles and dark gray bars). Bacteria were enumerated from individual harvested bladders at 24 hpi (black squares and circles). Median values for each infection are given (gray bars). Each UAEC infection of the two mouse strains was categorized separately as “deficient” (blue stippled outline), “variable” (orange outline), or “robust” (green outline). Bladder colonization between C3H/HeN and C57BL/6 mice was significantly different for strains 31.1a and 41.1a by Mann-Whitney U test with **, P<0.01.

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