Extensive Within-Host Diversity in Fecally Carried Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolates: Implications for Transmission Analyses

Abstract

Studies of the transmission epidemiology of antimicrobial-resistant Escherichia coli, such as strains harboring extended-spectrum beta-lactamase (ESBL) genes, frequently use selective culture of rectal surveillance swabs to identify isolates for molecular epidemiological investigation. Typically, only single colonies are evaluated, which risks underestimating species diversity and transmission events. We sequenced the genomes of 16 E. coli colonies from each of eight fecal samples (n = 127 genomes; one failure), taken from different individuals in Cambodia, a region of high ESBL-producing E. coli prevalence. Sequence data were used to characterize both the core chromosomal diversity of E. coli isolates and their resistance/virulence gene content as a proxy measure of accessory genome diversity. The 127 E. coli genomes represented 31 distinct sequence types (STs). Seven (88%) of eight subjects carried ESBL-positive isolates, all containing blaCTX-M variants. Diversity was substantial, with a median of four STs/individual (range, 1 to 10) and wide genetic divergence at the nucleotide level within some STs. In 2/8 (25%) individuals, the same blaCTX-M variant occurred in different clones, and/or different blaCTX-M variants occurred in the same clone. Patterns of other resistance genes and common virulence factors, representing differences in the accessory genome, were also diverse within and between clones. The substantial diversity among intestinally carried ESBL-positive E. coli bacteria suggests that fecal surveillance, particularly if based on single-colony subcultures, will likely underestimate transmission events, especially in high-prevalence settings.

FIG 1

Genetic characteristics of Escherichia coli carriage isolates from subject IHD244 (A), subject IHD86 (B), subject IHD232 (C), subject IHD357 (D), and subject IHD45 (E). Black circles at tree nodes indicate bootstrap values of >95%. Colored circles represent genetic similarity of isolates within STs and are sized and labeled according to the number of isolates sharing identity. Branches between circles represent a core, single nucleotide variant. Labels adjacent to circles represent bla_CTX-M variants (colored by variant type). Within-host resistance and virulence gene profiles are represented in the heat map at the bottom of each panel, with colored blocks representing presence and gray blocks representing absence of a gene. Resistance genes are represented as red blocks; virulence factors are colored by functional category (sea green, adhesins; olive, toxins; light blue, nutritional factors; dark blue, immune evasion; lilac, miscellaneous). Isolates from within the cefpodoxime zones are labeled 1 to 8 (e.g., IHD244_1) and those from outside the zones are labeled 9 to 16 (e.g., IHD244_9), where applicable.

FIG 1

Genetic characteristics of Escherichia coli carriage isolates from subject IHD244 (A), subject IHD86 (B), subject IHD232 (C), subject IHD357 (D), and subject IHD45 (E). Black circles at tree nodes indicate bootstrap values of >95%. Colored circles represent genetic similarity of isolates within STs and are sized and labeled according to the number of isolates sharing identity. Branches between circles represent a core, single nucleotide variant. Labels adjacent to circles represent bla_CTX-M variants (colored by variant type). Within-host resistance and virulence gene profiles are represented in the heat map at the bottom of each panel, with colored blocks representing presence and gray blocks representing absence of a gene. Resistance genes are represented as red blocks; virulence factors are colored by functional category (sea green, adhesins; olive, toxins; light blue, nutritional factors; dark blue, immune evasion; lilac, miscellaneous). Isolates from within the cefpodoxime zones are labeled 1 to 8 (e.g., IHD244_1) and those from outside the zones are labeled 9 to 16 (e.g., IHD244_9), where applicable.

FIG 1

Genetic characteristics of Escherichia coli carriage isolates from subject IHD244 (A), subject IHD86 (B), subject IHD232 (C), subject IHD357 (D), and subject IHD45 (E). Black circles at tree nodes indicate bootstrap values of >95%. Colored circles represent genetic similarity of isolates within STs and are sized and labeled according to the number of isolates sharing identity. Branches between circles represent a core, single nucleotide variant. Labels adjacent to circles represent bla_CTX-M variants (colored by variant type). Within-host resistance and virulence gene profiles are represented in the heat map at the bottom of each panel, with colored blocks representing presence and gray blocks representing absence of a gene. Resistance genes are represented as red blocks; virulence factors are colored by functional category (sea green, adhesins; olive, toxins; light blue, nutritional factors; dark blue, immune evasion; lilac, miscellaneous). Isolates from within the cefpodoxime zones are labeled 1 to 8 (e.g., IHD244_1) and those from outside the zones are labeled 9 to 16 (e.g., IHD244_9), where applicable.

FIG 1

Genetic characteristics of Escherichia coli carriage isolates from subject IHD244 (A), subject IHD86 (B), subject IHD232 (C), subject IHD357 (D), and subject IHD45 (E). Black circles at tree nodes indicate bootstrap values of >95%. Colored circles represent genetic similarity of isolates within STs and are sized and labeled according to the number of isolates sharing identity. Branches between circles represent a core, single nucleotide variant. Labels adjacent to circles represent bla_CTX-M variants (colored by variant type). Within-host resistance and virulence gene profiles are represented in the heat map at the bottom of each panel, with colored blocks representing presence and gray blocks representing absence of a gene. Resistance genes are represented as red blocks; virulence factors are colored by functional category (sea green, adhesins; olive, toxins; light blue, nutritional factors; dark blue, immune evasion; lilac, miscellaneous). Isolates from within the cefpodoxime zones are labeled 1 to 8 (e.g., IHD244_1) and those from outside the zones are labeled 9 to 16 (e.g., IHD244_9), where applicable.

FIG 1

Genetic characteristics of Escherichia coli carriage isolates from subject IHD244 (A), subject IHD86 (B), subject IHD232 (C), subject IHD357 (D), and subject IHD45 (E). Black circles at tree nodes indicate bootstrap values of >95%. Colored circles represent genetic similarity of isolates within STs and are sized and labeled according to the number of isolates sharing identity. Branches between circles represent a core, single nucleotide variant. Labels adjacent to circles represent bla_CTX-M variants (colored by variant type). Within-host resistance and virulence gene profiles are represented in the heat map at the bottom of each panel, with colored blocks representing presence and gray blocks representing absence of a gene. Resistance genes are represented as red blocks; virulence factors are colored by functional category (sea green, adhesins; olive, toxins; light blue, nutritional factors; dark blue, immune evasion; lilac, miscellaneous). Isolates from within the cefpodoxime zones are labeled 1 to 8 (e.g., IHD244_1) and those from outside the zones are labeled 9 to 16 (e.g., IHD244_9), where applicable.

FIG 2

Pairwise distance comparisons of differences in the accessory gene component in clones/singletons between and within study subjects. Each dot represents a single pairwise comparison of differences in accessory gene content between each set of clones/singletons, depending on whether these strains were present in the same individual (within) or in different individuals (between). Gray lines represent the median pairwise difference for each subgroup; dotted gray lines represent the upper and lower quartiles for each subgroup.