The Population Genomics of Increased Virulence and Antibiotic Resistance in Human Commensal Escherichia coli over 30 Years in France

Abstract

Escherichia coli is a commensal species of the lower intestine but is also a major pathogen causing intestinal and extraintestinal infections that is increasingly prevalent and resistant to antibiotics. Most studies on genomic evolution of E. coli used isolates from infections. Here, instead, we whole-genome sequenced a collection of 403 commensal E. coli isolates from fecal samples of healthy adult volunteers in France (1980 to 2010). These isolates were distributed mainly in phylogroups A and B2 (30% each) and belonged to 152 sequence types (STs), the five most frequent being ST10 (phylogroup A; 16.3%), ST73 and ST95 (phylogroup B2; 6.3 and 5.0%, respectively), ST69 (phylogroup D; 4.2%), and ST59 (phylogroup F; 3.9%), and 224 O:H serotypes. ST and serotype diversity increased over time. The O1, O2, O6, and O25 groups used in bioconjugate O-antigen vaccine against extraintestinal infections were found in 23% of the strains of our collection. The increase in frequency of virulence-associated genes and antibiotic resistance was driven by two evolutionary mechanisms. Evolution of virulence gene frequency was driven by both clonal expansion of STs with more virulence genes ("ST-driven") and increases in gene frequency within STs independent of changes in ST frequencies ("gene-driven"). In contrast, the evolution of resistance was dominated by increases in frequency within STs ("gene-driven"). This study provides a unique picture of the phylogenomic evolution of E. coli in its human commensal habitat over 30 years and will have implications for the development of preventive strategies. IMPORTANCE Escherichia coli is an opportunistic pathogen with the greatest burden of antibiotic resistance, one of the main causes of bacterial infections and an increasing concern in an aging population. Deciphering the evolutionary dynamics of virulence and antibiotic resistance in commensal E. coli is important to understand adaptation and anticipate future changes. The gut of vertebrates is the primary habitat of E. coli and probably where selection for virulence and resistance takes place. Unfortunately, most whole-genome-sequenced strains are isolated from pathogenic conditions. Here, we whole-genome sequenced 403 E. coli commensals isolated from healthy French subjects over a 30-year period. Virulence genes increased in frequency by both clonal expansion of clones carrying them and increases in frequency within clones, whereas resistance genes increased by within-clone increased frequency. Prospective studies of E. coli commensals should be performed worldwide to have a broader picture of evolution and adaptation of this species.

Keywords: Escherichia coli; antibiotic resistance; commensal; evolution; genomics; virulence.

FIG 1

Frequency distribution of phylogroups, STs and O-groups between 1980 and 2010. (A) Frequency distribution of phylogroups. The sample size is indicated in brackets. The decline of phylogroup A and the increase of phylogroup B2 were significant (0.05 level). (B) Frequency distribution of STs. The proportion of each ST has been plotted by year ordered by the overall frequency (most common at the bottom). Only the names of STs with an overall frequency >0.03 and ST131 are shown. The only variation that was significant at the 0.05 level was the decline of ST10. (C) Frequency of the O-groups O1, O2, O6, and O25. The sample size is indicated in brackets. No significant variation with time was detected (0.05 level).

FIG 2

Genomic content of three of the most frequent STs. For ST10 (A), ST69 (B), and ST95 (C), in addition to the diversity of O:H serotypes and fimH alleles, we examined the presence of 5 antibiotic resistance genes [bla_TEM-1B, tet(B), tet(A), aph(3″)-Ib, and aph(6)-Id] and of 34 virulence genes (pap [adhesin], chu, iro, iuc [iron capture systems], and kps [protectin] operons). The timetrees were built with BEAST v1.10.4 (65). ST10 exhibits the largest serotype diversity (38 serotypes), among the other STs examined here, ST69 (4 serotypes) and ST95 (6 serotypes). Nodes with a support value (Bayesian posterior probability) of >0.75 are indicated by black circles.

FIG 3

Evolution of virulence and antibiotic resistance genes between 1980 and 2010. (A) Mean virulence score computed for all the 403 commensal E. coli isolates. The virulence score of a strain is defined as the number of the VFs of each category tested that were present in that strain. (B) Frequency of antibiotic resistance through time for all 403 isolates. Both gene acquisition and point mutations are included, but we omitted the macrolide category because more than 99% of the strains were resistant to macrolide antibiotics. For readability, in each panel, the year 2001 includes strains sampled in 2000, 2001, and 2002.

FIG 4

Temporal change of virulence gene frequency (A) between 1980 to 2001 and (B) between 2001 to 2010. The overall frequency change (Δf) for each gene (increases depicted by circles and decreases by triangles) is decomposed in two additive components: change driven by the variation in frequency of STs carrying the focal gene (ST-driven change) and change driven by the variation in frequency of the focal gene (gene-driven change). For readability, only genes for which between-ST change or within-ST change was greater than 0.02 in absolute value are shown (see Tables S13 and S14 for the complete list). The gray ellipses represent the confidence levels computed for NVNR genes. Genes highlighted in bold are those for which the temporal change is significantly different from that of NVNR genes at the 0.05 level (out of the 95% confidence level). (C) Summary of the two additive components of temporal change of virulence genes between 1980 and 2010. We computed the mean change per product (e.g., mean change among iucA, iucB, iucC, iucD, and iutA genes for aerobactin) and reported the mean change among products of a category (e.g., among products classified in the iron acquisition category). CL: confidence level.

FIG 5

Temporal change of antibiotic resistance frequency (A) between 1980 to 2001 and (B) between 2001 to 2010. The overall frequency change (Δf) for each gene (increases depicted by circles and decreases by triangles) is decomposed in two additive components, change driven by the variation in frequency of STs carrying the focal gene (ST-driven change) and change driven by the variation in frequency of the focal gene (gene-driven change). For readability, only genes for which between-ST change or within-ST change was greater than 0.02 are shown here (see Table S15 to S16 for the complete list). The gray ellipses represent the confidence levels computed for NVNR genes. Genes highlighted in bold are those for which the temporal change is significantly different from that of NVNR genes at the 0.05 level (95% confidence level). Note that the scale of the y axis is much smaller for resistance than for virulence genes (Fig. 4), as ST-driven changes are minor compared to gene-driven changes for antibiotic resistance genes. (C) Summary of the two additive components of the temporal change of antibiotic resistance genes between 1980 and 2010. We computed the mean change among genes belonging to a given category [e.g., among tet(A), tet(B), tet(D), and tet(M) for tetracycline]. CL: confidence level.