Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist

a, Mice were pretreated with streptomycin and subsequently colonized via oral gavage (PO) with UTI89, a prototypical human UPEC cystitis isolate. b–e, Colonization of UTI89 in C3H/HeN (b, c) or C57BL/6 (d, e) mice from Envigo was assessed by quantifying CFU in faecal samples collected over the course of 21 days from mice who did not receive streptomycin (white circles) or mice pretreated with the antibiotic (black circles). CFU analysis of levels of colonization in the caecum and colon were defined by analysing tissue homogenates prepared 21 days after colonization. Symbols represent geometric mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney U test. n = 15 mice, 3 biological replicates (b–e).

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a, C3H/HeN mice from Envigo were pretreated with streptomycin and concurrently colonized with 1 × 10⁸ CFU of wild-type UTI89 and UTI89ΔfimH. The wild-type strain outcompetes the strain lacking the FimH adhesin. b, The ability of FimH^LD to bind to Caco-2 cells was assessed by a FimH ELISA. Pre-incubation of FimH^LD with d-mannose (1 mM) or M4284 (1 mM) results in significant reductions in FimH binding to Caco-2 cells while 10% cyclodextrin (M4284 vehicle) had no significant effect. All data shown are normalized to wells that were not exposed to the purified adhesin. Data in a are mean ± s.e.m, and bars in b represent the median. *P < 0.05, **P < 0.01, ***P < 0.001 by Wilcoxon signed-rank test (a). n = 14 mice, 3 biological replicates (a); n = 4 wells examining FimH binding to Caco-2 cells per protein concentration, 4 technical replicates (b).

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C3H/HeN mice received a transurethral inoculation of wild-type UTI89 and UTI89Δucl, concurrently (a, b), or individually (c–e). a, UTI89Δucl and wild-type strains persist at similar levels in the urine over 28 days in competitive infections. b, The two strains are also present at equal levels in the bladder and kidney at the time of euthanization (28 days after infection). c, Single infection with the wild-type strain (black circles) or the F17-like mutant strain (white circles) produces similar levels of bacteruria over 28 days. d, Single strain infection also produces similar levels of viable cells in homogenates of whole bladder or kidneys collected at the time of euthanization (28 days after infection). There was no statistically significant difference in the number of mice that resolved bacteriuria while maintaining bladder-associated CFUs after transurethral infection with either wild-type UTI89 or UTI89Δucl (highlighted in red in d), suggesting that both strains are capable of forming similar numbers of quiescent intracellular reservoirs. e, Mice infected transurethrally with wild-type or Δucl strains of UTI89 exhibit a similar number of IBCs at 6 h in the bladder, indicating that loss of the ucl operon does not alter the ability of UTI89 to form IBCs. Error bars represent mean ± s.e.m. (a, b), geometric mean (c, d) or median (e). No significant difference was detected between any samples by Wilcoxon signed-rank test (a, b) or Mann–Whitney U test (c–e). n = 10 mice, 2 biological replicates (a, b, e); n = 16 mice, 3 biological replicates (c, d).

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The phylogenetic relationships between F17 homologues were estimated using the sequence of the usher genes. Branch colours indicate host strain and pilus identity, and coloured symbols indicate the annotated pathotype of the E. coli strain for each sequence as determined by publically available annotations. Stars indicate extraintestinal pathogenic E. coli (ExPEC) strains, and circles indicate intestinal pathogenic E. coli strains. Carriage of F17-like pili is enriched in UPEC strains, whereas F17 and ECs1278 pili are more common in intestinal pathogens such as ETEC and EHEC, respectively. The strain names for each sequence and ENA accessions are given. Numbers beneath the branches indicate the percentage of support from 1,000 bootstrap replicates (numbers greater than 80% are shown).

The phylogeny of a set of clinical UPEC strains (n = 43 with taxon labels highlighted in green, orange or grey) was contextualized with reference E. coli strains (n = 46, unhighlighted taxon labels) by comparing the concatenated single-copy, core genes of the strains using the RAxML algorithm and the GTRCAT model⁴⁶. Highlighted taxon labels indicate UPEC isolates collected at enrolment (green) and during recurrent UTI (orange). In all cases, patients cleared each infection before recurrence, no patient exhibited signs of asymptomatic bacteriuria. The study design also allowed for the collection, from cohort participants, of E. coli isolates present in the urine in the days leading up to their clinical visit and recurrent UTI diagnosis (highlighted in grey)¹⁷. Branch lines indicate phylogenetic background for strains from clade B2 (red branch lines) and non-B2 clades (blue branch lines). Carriage of F17-like pili (black stars) was limited to the B2 clade and enriched within recurrent UTI UPEC isolates. Bootstrap supports are indicated at internal nodes. Bootstrap values greater than 95 have been removed. The clade to which each strain belongs is indicated in brackets to the right.

a, Experimental design. b, Animals treated as in a show a continued decrease in UTI89 levels in their faeces (samples were processed after 3, 4 and 5 doses of M4284), and at the time of euthanization in the caecum and colon, compared to control mice treated with vehicle alone (control, 10% cyclodextrin). c, d, The effects of mannoside treatment persist 5 days after M4284 exposure. Data are geometric mean ± s.d. *P < 0.05, **P < 0.01 by Mann–Whitney U test. n = 9 mice (control); n = 10 mice (M4284), 2 biological replicates (b); n = 16 mice, 3 biological replicates (d).

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C3H/HeN mice (Envigo) were given an experimental UTI via transurethral inoculation of either 10⁶ or 10⁸ CFU of UTI89. The doses were chosen to represent the reduction observed in intestinal UTI89 titres before and after treatment with the M4284 mannoside. Mice were euthanized 24 h after inoculation, and UTI89 titres in urine, bladder and kidneys were defined by quantifying CFU. Mice receiving the 10⁶ dose of UTI89 had significantly fewer bacteria in all three biospecimen types, indicating an important relationship between the number of bacteria introduced into the urinary tract and the severity of UTI outcome. Bars represent geometric means. **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann–Whitney U test. n = 10 mice, 2 biological replicates.

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a, C3H/HeN mice were treated with M4284 (100 mg kg⁻¹, three doses over 24 h), vehicle alone (10% cyclodextrin, three doses over 24 h), or ciprofloxacin (15 mg kg⁻¹, two doses over 24 h). Untreated mice served as reference controls. Heat maps show the effect of each of the treatments on animals from CRL and Envigo. Each row represents a species-level bacterial taxon, while each column represents a mouse sampled 24 h after the termination of the indicated treatment. Coloured boxes next to the taxon names indicate species whose relative abundance was significantly changed by ciprofloxacin treatment (P < 0.05; Wilcoxon signed-rank test with false discovery rate (FDR) correction). Individual comparisons between untreated and other treatment types did not disclose changes that were statistically significant by Wilcoxon signed-rank test with FDR correction. b, Corresponding faecal samples collected 24 h after treatments (as shown in Extended Data Fig. 8a) were homogenized, diluted serially, and plated on MacConkey medium. The abundance of bacteria capable of growing on the selective medium was similar between faecal samples taken from untreated mice and those collected 24 h after treatment with cyclodextrin and M4284. No colonies were detected from faecal samples collected 24 h after ciprofloxacin treatment. c, Comparison of the representation of bacterial taxa in the faecal microbiota of untreated mice obtained from different vendors or representing different genetic backgrounds. Each row in the heat map represents a species-level taxon, while each column represents a mouse of the indicated genetic background from the indicated vendor. Coloured boxes indicate species whose relative abundances were significantly different (P < 0.05) between all three groups of animals (Kruskal–Wallis test with FDR correction). Rows of each heat map were hierarchically clustered according to pair-wise distances using Pearson correlation. n = 5 mice per treatment type, 1 biological replicate (a); n = 5 mice, 1 biological replicate (b); n = 5 mice per vendor/mouse strain, 1 biological replicate (c). Bars denote median. **P < 0.001, Mann–Whitney U test (b).

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a, C3H/HeN mice from Envigo were pretreated with streptomycin and 24 h later colonized with UTI89 by oral gavage. Three days after inoculation, animals were treated with three doses of M4284 (100 mg kg⁻¹, three doses over 24 h) or vehicle alone (10% cyclodextrin, 3 doses over 24 h). Faecal samples were collected 24 h after the last dose of M4284 or vehicle. b, Heat map showing the effect of each treatment type. Each row represents a bacterial species-level taxon, while each column represents a mouse 24 h after the indicated treatment. Rows of the heat map were hierarchically clustered according to pair-wise distances using Pearson correlation. No treatments produced changes that were statistically significant, as judged by Wilcoxon signed-rank test with FDR correction. n = 4 mice per treatment type, 1 biological replicate.

a, The percentage reduction in CFU for the indicated UPEC strains from M4284-treated versus untreated control C3H/HeN mice obtained from Envigo (based on data in Fig. 4c–f). b, CFU data obtained from C3H/HeN mice from Envigo and CRL and C57BL/6 mice from Envigo (based on data in Fig. 4f–h). P values calculated using Kruskal–Wallis test. n = 14 mice, 3 biological replicates (UTI89); n = 10 mice, 2 biological replicates (CFT073, EC958 and 41.4p) (a); n = 14 mice, 3 biological replicates (C3H/HeN from Envigo); n = 10 mice, 2 biological replicates (C3H/HeN from CRL); and n = 9 mice, 2 biological replicates (C57BL/6 from Envigo) (b).

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