Genomic diversity and fitness of E. coli strains recovered from the intestinal and urinary tracts of women with recurrent urinary tract infection

Abstract

Urinary tract infections (UTIs) are common in women, and recurrence is a major clinical problem. Most UTIs are caused by uropathogenic Escherichia coli (UPEC). UPEC are generally thought to migrate from the gut to the bladder to cause UTI. UPEC form specialized intracellular bacterial communities in the bladder urothelium as part of a pathogenic mechanism to establish a foothold during acute stages of infection. Evolutionarily, such a specific adaptation to the bladder environment would be predicted to result in decreased fitness in other habitats, such as the gut. To examine this prediction, we characterized 45 E. coli strains isolated from the feces and urine of four otherwise healthy women with recurrent UTI. Multilocus sequence typing and whole genome sequencing revealed that two patients maintained a clonal population in both these body habitats throughout their recurrent UTIs, whereas the other two exhibited a wholesale shift in the dominant UPEC strain colonizing both sites. In vivo competition studies in mouse models, using isolates taken from one of the patients with a wholesale population shift, revealed that the strain that dominated her last UTI episode had increased fitness in both the gut and the bladder relative to the strain that dominated in preceding episodes. Increased fitness correlated with differences in the strains' gene repertoires and carbohydrate and amino acid utilization profiles. Thus, UPEC appear capable of persisting in both the gut and urinary tract without a fitness trade-off, emphasizing the need to widen our consideration of potential reservoirs for strains causing recurrent UTI.

Fig. 1. MLST analysis of patient isolates

An eBURST diagram constructed based on publicly available E. coli MLST sequence types. MLST types from patient isolates from the present study are represented by colored circles; the color denotes the patient while the size (diameter) of the circle indicates the proportion of strains that share that same MLST type or an MLST type that differs by only one allele.

Fig. 2. Heatmap of SNP differences between E. coli strains

SNP rates (SNPs/aligned bp) between different sequenced strains were interpreted as a distance matrix. Hierarchical clustering was done on this symmetric matrix of SNP rates. Color in the central heatmap represents SNP rate as shown in the legend at the top left. A tree based on Euclidean distance is shown to left of the central heatmap. The colors of the strains indicate their patient of origin. UTI89_finished refers to the finished UTI89 genome (40), while UTI89_illumina is the re-sequenced and re-assembled genome from a 36 nt read dataset generated for this study. The comparison between the UTI89_finished and UTI89_illumina identified the noise range of the sequencing and assembly pipeline we used.

Fig. 3. OGU-based clustering of sequenced E. coli genomes

The presence or absence of OGUs in the newly sequenced UPEC genomes characterized in the present study and 54 other publicly available E. coli strains were used to construct an OGU matrix. Hierarchical clustering was performed on the matrix based on Euclidean distance. There are two clades suggested by the clustering analysis. Phylogenetic group membership of the strains is indicated with bars at the right of the figure. All isolates from Patient 13 are colored red while all isolates from Patient 72 are colored green. The two strains used for in vivo competition experiments and phenotype microarray analyses are highlighted.

Fig. 4. Growth phenotypes of isolates from Patient 72

(A) Unsupervised hierarchical clustering of growth phenotypes as defined by Biolog phenotype microarrays. Squares represent fecal samples, circles denote urine samples, and the numbers inside squares and circles indicate the episode number from which the strain was derived. (B) Comparison of the growth phenotypes of strains Ec72_E1U1 and Ec72_E3U1 from UTI episodes 1 and 3, on carbohydrate, nucleoside and amino acid substrates. Carbohydrate substrates that result in greater than 10-fold growth of the episode 3 strain are shown. See Fig. S10 for other carbohydrates examined. Utilization of the bracketed carbohydrates requires genes involved in galactose metabolism. The color key denotes growth relative to the reference UPEC strain, UTI89.

Fig. 5. In vivo fitness of urine isolates from Patient 72’s UTI episodes 1 and 3

(A) Urine isolates obtained from UTI episodes 1 (isolate Ec72_E1U1) and 3 (Ec72_E3U1) were introduced separately or together into the bladders of female C3H/HeN mice. Data points represent CFUs/bladder in individual mice, and horizontal bars represent the median of CFUs/bladder of that strain in the experiments. Data on the y-axis are presented in log scale: therefore all 0s were plotted as 1 for visualization (*, p<0.05; two-tailed Mann-Whitney test). (B) The plasmid pACYC184 (Chlor^R) was lost during a two-week colonization of the intestines of gnotobiotic mice or conferred a fitness disadvantage. CFU on LB agar plates without antibiotics represent all Ec72_E1U1 in the fecal samples. CFU on LB agar plates with kanamycin or chloramphenicol represent the Ec72_E1U1 carrying the corresponding antibiotic-resistance plasmid from the same fecal sample (data shown as mean +/− SEM). (C) FitSeq determination of the relative fitness of strains from Patient 72’s UTI episodes 1 and 3 in the gut of gnotobiotic mice (data shown as mean ± SEM).