Identification of a hybrid atypical enteropathogenic and enteroaggregative Escherichia coli (aEPEC/EAEC) clone of serotype O3:H2 associated with a diarrheal outbreak in Brazil

Abstract

Enteropathogenic (EPEC) and enteroaggregative (EAEC) Escherichia coli are two of the major pathotypes of diarrheagenic E. coli causing disease worldwide. Here, we report a diarrheal outbreak caused by E. coli of serotype O3:H2, harboring virulence markers from EPEC (eae) and/or EAEC (aggR). This is likely the first E. coli diarrheal outbreak caused by a hybrid atypical-EPEC/EAEC clone reported in Brazil.

Escherichia coli isolates associated with diarrheal disease are collectively termed diarrheagenic E. coli (DEC). On the basis of a set of molecular and phenotypic features, DEC can be divided into six main pathotypes: enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) [1–3].

EPEC produces a histopathological lesion in infected cells, termed attaching, and effacement (AE), which is characterized by intimate adherence of bacteria to the host cell, leading to microvillus effacement and accumulation of mainly F-actin but also other cytoskeleton elements, underneath adherent bacteria [4, 5]. All the proteins necessary for the AE lesion formation are encoded by genes located on a chromosomal pathogenicity island termed LEE (locus of enterocyte effacement) region [6]. Among these proteins, it is important to highlight the adhesin intimin encoded by the eae (E. coli attaching and effacing) gene [7].

One of the first virulence properties identified in EPEC isolates was the formation of very compact clusters of bacteria on the surface of infected epithelial cells (HeLa or Hep-2), which is referred as localized adherence (LA) pattern [8]. The adhesin responsible for the establishment of this phenotype is called bundle-forming pilus (BFP) and is encoded by a set of 14 genes (bfp operon) located in the EPEC adherence factor (EAF) plasmid [9, 10]. However, a study with adult volunteers showed that EPEC lacking the EAF plasmid can also cause diarrhea [11], thus leading to the subdivision of this pathotype into two subgroups: typical (tEPEC) and atypical (aEPEC), with the EAF (bfp⁺) plasmid present only in tEPEC [12–14]. Since aEPEC do not produce BFP, these isolates produce adherence patterns other than LA, such as the localized adherence-like (LAL) pattern, which differs from the LA pattern by the formation of less compact bacterial microcolonies on the surface of infected cells, detected only in assays performed with extended periods of bacteria-cell incubation [15]. Moreover, there are a large number of isolates that do not produce a characteristic adherence pattern or are non-adherent [16, 17].

The hallmark of EAEC is the production of a bacterial arrangement that resembles stacked bricks on epithelial cells, that is, known as aggregative adherence (AA) pattern [18], as well as biofilm formation on abiotic surface and intestinal mucosa [19, 20]. The formation of the AA pattern depends on the production of one of the five distinct chaperone-usher aggregative adherence fimbriae (AAF/I—AAF/V) described so far [21–25]. The genes encoding the proteins necessary for the AAFs biogenies are located in a plasmid (pAA), which also carries genes that encode other EAEC-associated virulence factors and the global virulence regulator AggR, which activates the expression of several plasmid and chromosomal virulence factor-encoding genes [26, 27].

In 2011, an outbreak of diarrhea in Germany, caused by a hybrid STEC/EAEC of serotype O104:H4, was responsible for the surprising number of diarrhea cases, namely 3816, of which 845 progressed to hemolytic-uremic syndrome, and a total of 54 deaths were registered [28, 29]. This outbreak drew attention to the emergence of hybrid DEC pathotypes and their high pathogenic potential, and since then, many other hybrid DEC pathotypes have been associated with diarrheal disease worldwide, such as: STEC/ETEC [30, 31] and aEPEC/ETEC [32].

A recently published epidemiological surveillance study carried out in Brazil between 2011 and 2016, involving a total of 5047 patients with acute diarrhea, demonstrated that aEPEC and EAEC appear as the most common DEC pathotypes in the Brazilian scenario [33].

Here, we report an investigation of a community-acquired diarrheal outbreak involving eight patients with acute diarrhea, occurring in São Paulo State, Brazil, during the year of 2010. Briefly, colony-forming bacteria, observed on MacConkey agar plates seeded with the stool samples collected from the diarrheal patients, were biochemically identified as E. coli. The presence of the molecular markers used for DEC pathotype identification (eae, bfpB, stx1, stx2, aggR, elt, est, and ipaH) was detected by PCR, using primers and conditions as previously described [33], and the somatic (O) and flagellar (H) antigens of the DEC isolates were serologically typed by tube agglutination assays employing absorbed somatic (O1-O188) and flagellar (H1-H56) antisera produced at Institute Adolfo Lutz (IAL). Furthermore, the adherence pattern produced in HeLa cells was determined, using 3 and 6 h of co-incubation of bacteria and epithelial cells, as previously described [15].

The presence of virulence factor-encoding genes revealed that one diarrheal patient was infected with aEPEC (eae⁺/bfpB⁻), four with EAEC (aggR⁺), and three with a hybrid aEPEC/EAEC (eae⁺/bfpB⁻/aggR⁺), with all DEC isolates obtained classified in the serotype O3:H2. Other enteropathogens, such as Salmonella spp., Shigella spp., Campylobacter spp., Aeromonas spp., Plesiomonas spp., Yersinia enterocolitica, rotavirus, and norovirus were not detected in the stool samples obtained from the 8 diarrheal patients evaluated in this study. One DEC isolate per patient was then selected and subjected to pulsed-field gel electrophoresis (PFGE) analysis, showing the DEC isolates of serotype O3:H2 with more than 74.1% similarity, regardless of the pathotype. Of importance, the three hybrid aEPEC/EAEC isolates showed more than 94.2% similarity (Fig. 1), thus confirming the first diarrheal outbreak caused by a hybrid aEPEC/EAEC of serotype O3:H2 in Brazil.

Fig. 1.

Dendrogram showing the relationships between EAEC, aEPEC, and hybrid aEPEC/EAEC isolates of serotype O3:H2 obtained during a diarrheal outbreak investigation. The XbaI DNA restriction profiles were analyzed in Bionumerics v7.5 (Applied Maths), and a dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA) applying the Dice coefficient with optimization and tolerance set at 1.0%

EAEC of serotype O3:H2 has been identified in Brazil in diarrheal patients, healthy subjects [33–37], and bovine meat [38], thus pointing out the circulation of this pathogen in our setting, as well as in distinct case–control studies performed worldwide [39–42]. The occurrence of hybrid aEPEC/EAEC isolates appears to be rare in Brazil, but combination of virulence factor-encoding genes from these two DEC pathotypes has already been observed in E. coli isolates obtained from patients with acute diarrhea, from serotypes O142:H34 and O158:HNM [43, 44].

As expected, the four EAEC isolates displayed an AA pattern in HeLa cells in assays performed with 3 and 6 h of incubation. On the other hand, the three hybrid aEPEC/EAEC demonstrated only a trend to form the AA pattern in 3 h, with this phenotype being better observed in the 6-h assay, concomitantly with short chain-like adherence (CLA). In contrast, the aEPEC isolate did not produce a characteristic adherence pattern, adhering only sporadically to HeLa cells, even in assays performed with long incubation periods (Fig. 2).

Fig. 2.

Adherence pattern displayed in HeLa cells, using 3 (A, C, and E) and 6 (B, D, and F) hours of incubation of cells and bacteria, by representatives EAEC (A and B), aEPEC (E and F), and hybrid aEPEC/EAEC (C and D) isolates of serotype O3:H2. (Bar scale = 10 μm)

Here, we report, to the best of our knowledge, the first diarrheal outbreak due to a hybrid aEPEC/EAEC of serotype O3:H2 in Brazil. Future whole genome sequencing studies will be necessary to better understand the genetic background of these hybrid isolates, the horizontal gene transfer events involved in their emergence, and how this particular combination of virulence factors can influence their pathogenic potential.

Funding

This study was supported in part by the São Paulo Research Foundation (FAPESP grant 2017/14821–7).

Declarations

Conflict of interest

The authors declare no competing interests.