Genomic characterization of enteroaggregative Escherichia coli from children in Mali

Abstract

Background: Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood.

Methods: We characterized 121 EAEC strains isolated in 2008 as part of a case-control study of moderate to severe acute diarrhea among children 0-59 months of age in Bamako, Mali. We applied multiplex polymerase chain reaction and comparative genome hybridization to identify potential virulence factors among the EAEC strains, coupled with classification and regression tree modeling to reveal combinations of factors most strongly associated with illness.

Results: The gene encoding the autotransporter protease SepA, originally described in Shigella species, was most strongly associated with diarrhea among the EAEC strains tested (odds ratio, 5.6 [95% confidence interval, 1.92-16.17]; P = .0006). In addition, we identified 3 gene combinations correlated with diarrhea: (1) a clonal group positive for sepA and a putative hemolysin; (2) a group harboring the EAST-1 enterotoxin and the flagellar type H33 but no other previously identified EAEC virulence factor; and (3) a group carrying several of the typical EAEC virulence genes.

Conclusion: Our data suggest that only a subset of EAEC strains are pathogenic in Mali and suggest that sepA may serve as a valuable marker for the most virulent isolates.

Figure 1.

Classification and regression tree (CART) classification tree topology reveals combinations of factors most strongly associated with moderate to severe diarrhea. We considered all genotypic and phenotypic assays performed: aatA, aggR, aaiC, aap, ORF3, sat, sepA, pic, sigA, pet, astA, aafC, agg3/4C, aafA, agg3A, aggA, agg4A, air, capU, eilA, ORF61, virulence factor score (VSF), and flagellum type H33. Each branch of the CART tree ends in a terminal “node” (red boxes), and each terminal node is uniquely defined by the presence or absence of a predictive factor such as a gene or VFS. The tree is hierarchical in nature. C701-09, C718-09, C801-09, and C46-10 are also shown on the dendrogram.

Figure 2.

Cladogram of comparative genomic hybridization data of sepA-positive isolates (C34-10, C35-10, C679-09C, C682-09, C693-09, C701-09, C703-09, C716-09, C718-09, C719-09, C729-09, C736-09, C745-09, C764-09, C765-09, C769-09, C771-09, C778-09, C783-09, C796-09, C697-09, C707-09, C710-09, C734-09, and C748-09), sepA-negative isolates (C46-10 and C801-09), and reference isolates (C1096, 042, 55989, JM221, 17-2, 34b, 101-1, and HS). Notably, C46-10 was most closely related to Mexican enteroaggregative Escherichia coli (EAEC) strain JM221 (isolated from an adult traveler to Guadalajara [27]), and strain C801-09 was most closely related to EAEC strain 042, isolated from a child with diarrhea in Lima, Peru [28]. The phylogenetic comparison was performed using the 12 673 variable features of the 36 hybridizations included. The tree is built using a hierarchical clustering with Pearson correlation using both the absolute distance and complete linkage and viewed in FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Isolates represented in black are reference isolates, controls are indicated in blue, and cases in red. The serotypes of the strains are indicated to the right. The gray boxes identify clusters of serotypes within the context of the larger tree, indicating that those serogroups are genomically similar.

Figure 3.

Classification and regression tree analysis described in Figure 1, adding the genes shiA, espy2, rmoA (hemolysin expression-modulation protein). See Figure 1 legend for details of analysis