Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131

Abstract

f Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum ␤-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the bla CTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111: H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/ CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance. Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea in children and adults, in both developing (1) and industrialized (2, 3) countries. Several outbreaks of gastroenteritis linked to EAEC have been reported (4, 5). EAEC is also the second most common cause of travelers' diarrhea (6). EAEC is defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture (7). Although detection of the AA phenotype in the HEp-2 cell adherence assay is still the gold standard for EAEC identification, the transcriptional regulator AggR, which controls the expression of multiple EAEC virulence genes, is also used as a typical EAEC marker for PCR detection (8).The pathogenesis of EAEC infection involves the adherence of the bacterium to the intestinal mucosa, followed by the secretion of one or more enterotoxins and/or cytotoxins that induce mucosal inflammation (9). EAEC adherence to the mucosa is characterized by the formation of a thick biofilm (10), which may promote persistent infection. The majority of EAEC strains carry a large (ϳ100-kb) plasmid, which encodes most putative EAEC virulence factors, such as AggR, aggregative adherence fimbriae (AAF), the dispersin protein Aap (11), its transporter Aat (12), the plasmid-encoded toxin Pet (13), enteroaggregative heat-stable toxin 1 (EAST-1) (14), and the...

Emergence of enteroaggregativeEscherichia coliwithin the ST131 lineage as a cause of extraintestinal infections

“…Like other DEC pathotypes, EAEC has been shown to encompass diverse genetic lineages, reflecting a high level of phylogenetic heterogeneity ( 16, 34 ). Although until recently no extraintestinal EAEC ST131 had been documented, recent reports have described the occurrence in certain ST131 subclonal lineages of EAEC-specific traits in geographically distinct areas ( 10, 13, 16 ). ESBL-producing ST38 and other ST types of various serotypes have been indicated as emerging hybrid strains of UPEC and EAEC involved primarily in UTIs from Germany, the Netherlands and the United Kingdom ( 35 ).…”

“…In a study of fecal and urine isolates from Danish patients in 1998-2000, we made the novel observation of EAEC ST131 of serotype O25 ( 10 ). Subsequently, another study documented the emergence of a bla CTX-M-14 -containing EAEC ST131 O25:H4 strain in stool samples of diarrheic patients in Japan from 2003 onwards ( 13 ).…”

“…This pathotype gained particular attention following a major foodborne outbreak in Germany in 2011 that was caused by a Shiga toxin (Stx)-producing O104:H4 EAEC strain and resulted in 3,842 confirmed cases and 54 deaths ( 12 ). Additionally, a worryingly high prevalence of multidrug resistance among EAEC strains has been reported, and reports of CTX-M-type ESBL-producing EAEC have appeared recently from around the world ( 13–16 ). EAEC pathogenesis involves adherence to human intestinal mucosa by virtue of aggregative adherence fimbriae (AAF) and subsequent biofilm formation.…”

Prevalence and Molecular Characteristics of Extended-Spectrum β-Lactamase Genes in Escherichia coli Isolated from Diarrheic Patients in China

“…It would appear that the CTX-M resistance mechanism has spread to several types of E. coli . Moreover EAEC O25:H4/CG131 is thought to have evolved to harbor CTX-M plasmids ( Imuta et al, 2016 ).…”

“…Five distinct classes of diarrheagenic E. coli (DEC) are recognized as being associated with diarrheal disease: enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC). PCR was used to distinguish these E. coli pathotypes by amplification of the following gene targets: typical EPEC ( eae and bfp ), atypical EPEC ( eae or bfp ), STEC ( eae and stx1 and/or stx2 ), ETEC ( elt and/or estIa or estIb ), EIEC ( invE and ipaH ), and EAEC ( aggR and/or aatA or aaiC ) ( Imuta et al, 2016 ; Tian et al, 2016 ).…”