Enteroaggregative Escherichia coli as etiological agent of endemic diarrhea in Spain: A prospective multicenter prevalence study with molecular characterization of isolates

Abstract

Background: Enteroaggregative Escherichia coli (EAEC) is increasingly associated with domestically acquired diarrheal episodes in high-income countries, particularly among children. However, its specific role in endemic diarrhea in this setting remains under-recognized and information on molecular characteristics of such EAEC strains is limited. We aimed to investigate the occurrence of EAEC in patients with non-travel related diarrhea in Spain and molecularly characterize EAEC strains associated with illness acquired in this high-income setting.

Methods: In a prospective multicenter study, stool samples from diarrheal patients with no history of recent travel abroad (n = 1,769) were collected and processed for detection of EAEC and other diarrheagenic E. coli (DEC) pathotypes by PCR. An additional case-control study was conducted among children ≤5 years old. Whole-genome sequences (WGS) of the resulting EAEC isolates were obtained.

Results: Detection of DEC in the study population. DEC was detected in 23.2% of patients aged from 0 to 102 years, with EAEC being one of the most prevalent pathotypes (7.8%) and found in significantly more patients ≤5 years old (9.8% vs. 3.4%, p < 0.001). Although not statistically significant, EAEC was more frequent in cases than in controls. WGS-derived characterization of EAEC isolates. Sequence type (ST) 34, ST200, ST40, and ST10 were the predominant STs. O126:H27, O111:H21, and O92:H33 were the predominant serogenotypes. Evidence of a known variant of aggregative adherence fimbriae (AAF) was found in 89.2% of isolates, with AAF/V being the most frequent. Ten percent of isolates were additionally classified as presumptive extraintestinal pathogenic E. coli (ExPEC), uropathogenic E. coli (UPEC), or both, and belonged to clonal lineages that could be specifically associated with extraintestinal infections.

Conclusion: EAEC was the only bacterial enteric pathogen detected in a significant proportion of cases of endemic diarrhea in Spain, especially in children ≤5 years old. In particular, O126:H27-ST200, O111:H21-ST40, and O92:H33-ST34 were the most important subtypes, with all of them infecting both patients and asymptomatic individuals. Apart from this role as an enteric pathogen, a subset of these domestically acquired EAEC strains revealed an additional urinary/systemic pathogenic potential.

Keywords: children; diarrheagenic Escherichia coli; enteroaggregative Escherichia coli; extraintestinal infection; molecular characterization; non-travel related diarrhea; whole-genome sequencing.

Figure 1

Distribution of different sequence types (ST) (A), serogenotypes (B), and aggregative adherence fimbriae (AAF) variants (C) among enteroaggregative Escherichia coli isolates. Other serogenotypes and STs (with less than five isolates each) are summarized in Supplementary Table 2. When O antigen was not predicted, it was considered not typeable (ONT).

Figure 2

Phylogenomic analysis of the enteroaggregative Escherichia coli genomes. The whole-genome phylogeny was constructed from 690,639 conserved SNP sites per genome that were identified compared against the reference genome of the E. coli strain IAI39 (GenBank accession no. NC_011750.1). The isolates from Spain (this study) are colored in red, those from Egypt, Kenya, or Peru are colored in purple, and those from the United Kingdom are not colored. EAEC and ExPEC reference genomes are colored in gray and yellow, respectively. Isolates obtained from asymptomatic controls are in bold and indicated by a star in the outer ring of labels. The most important serogenotype-ST combinations identified in this study are highlighted in green. The E. coli phylogroups are designated by letters (A, B1, B2, and D) on the interior of the phylogeny based on the inclusion of both EAEC isolates sequenced in this study and reference strains. The tree scale indicates the distance of 0.01 nucleotide changes per site.