Mônica A. M. Vieira

Phenotypic and Genotypic Characteristics of Escherichia coli Strains of Non–Enteropathogenic E. coli (EPEC) Serogroups that Carry eae and Lack the EPEC Adherence Factor and Shiga Toxin DNA Probe Sequences

This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying EAE and lacking the enteropathogenic E. coli adherence factor and Shiga toxin probe sequences. In hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences. Of the other 15 virulence DNA sequences tested, HLY was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying EAE only (EAE profile) were the most frequent (35.6%). Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (approximately 75.0%) showed variations of the localized adherence pattern. Actin accumulation was detected in 75.9% of the nondetaching strains. Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%). Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E. coli in 61.0% and 32.0% of the strains, respectively. Thirty-five different serotypes were found. Only strains with the EAE profile were associated with diarrhea (P=.039).

Emerging enteropathogenic Escherichia coli strains

Escherichia coli strains of nonenteropathogenic serogroups carrying eae but lacking the enteropathogenic E. coli adherence factor plasmid and Shiga toxin DNA probe sequences were isolated from patients (children, adults, and AIDS patients) with and without diarrhea in Brazil. Although diverse in phenotype and genotype, some strains are potentially diarrheagenic.

Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil

The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx1 corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx2 was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx1 plus stx2 sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx2 ehxA iha saa and stx1 stx2 ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx1 stx2 ehxA iha, stx2, and stx1 iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx2 alone or associated with stx1 were found more frequently in cattle, those harboring sequences stx1c and stx2d alone or associated with stx1c predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.

Molecular typing and virulence of enteroaggregative Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro city, Brazil

Enteroaggregative Escherichia coli (EAEC) strains have been implicated as emerging aetiological agents of diarrhoea worldwide. In the present study, 43 EAEC strains were serotyped and characterized according to random amplification of polymorphic DNA profiles, PFGE, multilocus enzyme electrophoresis (MLEE) and the presence of putative virulence genes (hly, aero, kps, fim, aggA, aafA, aggR, astA, she, aap, shf and pet). The EAEC strains consisted of a diversity of serotypes including eight O-non-typable and 35 O-typable strains arranged into 21 O : H combinations. Amplification of specific genes revealed that all strains carried at least two of the virulence sequences investigated. fim, aggR and aap were the most frequent genes in both groups studied. hly, aero and aggA sequences were more prevalent in the diarrhoeal group. kps occurred exclusively in strains isolated from symptomatic children and showed strong association with diarrhoeal disease. The molecular approaches used to investigate the relatedness among EAEC strains revealed a high degree of polymorphism, suggesting that these micro-organisms have a non-clonal origin. A closer relationship was observed among EAEC strains sharing O : H types. No significant clustering could be identified related to the virulence traits investigated; however, the she locus showed clonal distribution by MLEE typing. These results are in accordance with previous findings in revealing the conservation of particular EAEC factors, despite the high degree of diversity related to both genotypic and phenotypic markers.

Virulence properties of Escherichia coli isolated from ostriches with respiratory disease.

Eight Escherichia coli isolates from ostriches with respiratory disease were investigated for the presence of genes encoding the following adhesins: type 1 pili (fim), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afaI), temperature regulated adhesin, curli (crl, csgA) and temperature-sensitive hemagglutinin (tsh). Genes for heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf), alpha-haemolysin (hly) and aerobactin (aer) production were also investigated. Other characteristics investigated were the presence of hemagglutination activity, growth on an iron-deficient medium, aerobactin production, serum resistance, adherence to chicken tracheal cells, pathogenicity for day-old chicks, and serogroup. Serogrouping showed that four isolates belonged to serogroup O2, two to serogroup O78, one to serogroup O9, and one to serogroup O21. The virulence genes found were: fim in all eight isolates, csgA in seven, aer in six, and pap, crl and tsh in one isolate each. All isolates analyzed were positive for mannose-resistant hemagglutination, adhered in vitro to ciliated tracheal epithelium, grew on iron-deficient medium, and showed serum resistance. Pathogenicity tests on day-old chickens revealed one highly pathogenic isolate, three of low pathogenicity and four isolates with intermediate pathogenicity.

HeLa-cell adherence patterns and actin aggregation of enteropathogenic Escherichia coli (EPEC) and Shiga-toxin-producing E. coli (STEC) strains carrying different eae and tir alleles.

A collection of 69 eae-positive strains expressing 29 different intimin types and eight tir alleles was characterized with respect to their adherence patterns to HeLa cells, ability to promote actin accumulation in vitro, the presence of bfpA alleles in positive strains, and bundle-forming pilus (BFP) expression. All of the nine typical enteropathogenic Escherichia coli (tEPEC) studied harbored the enteropathogenic E. coli adherence factor (EAF) plasmid, as shown by PCR and/or EAF probe results. In addition, they were positive for bfpA, as shown by PCR, and BFP expression, as confirmed by immunofluorescence (IFL) and/or immunoblotting (IBL) assays. Localized adherence (LA) was exclusively displayed by those nine tEPEC, while localized-adherence-like (LAL) was the most frequent pattern among atypical EPEC (aEPEC) and Shiga-toxinproducing E. coli (STEC). All LA and LAL strains were able to cause attaching and effacing (AE) lesions, as established by means of the FAS test. There was a significant association between the presence of tir allele alpha1 and bfpA-positive strains, and consequently, with the LA pattern. However, intimin type or bfpA was not associated with the adherence pattern displayed in HeLa cells. Among the eight bfpA alleles detected, a new type (beta10; accession number FN391178) was identified in a strain of serotype O157:H45, and a truncated variant (beta3.2-t; accession number FN 391181) in four strains belonging to different pathotypes.

TccP2-mediated subversion of actin dynamics by EPEC 2 – a distinct evolutionary lineage of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott–Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157 : H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspFU (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H−), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2 %) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck−/− cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin-polymerization signalling cascades.

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

Aims:  To determine the prevalence of Shiga toxin‐producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil.

Prevalence and Characteristics of the O122 Pathogenicity Island in Typical and Atypical Enteropathogenic Escherichia coli Strains

ABSTRACT The presence of the pathogenicity island (PAI) O122 genes, efa1 (lifA), sen, pagC, nleB, and nleE, in typical and atypical enteropathogenic Escherichia coli (EPEC) strains was investigated. The simultaneous occurrence of all genes was statistically associated with diarrhea due to atypical EPEC. Detection of the complete PAI O122 could aid in the identification of potential pathogenic strains of atypical EPEC.

Water Buffaloes (Bubalus bubalis) Identified as an Important Reservoir of Shiga Toxin-Producing Escherichia coli in Brazil

ABSTRACT The presence of Shiga toxin-producing Escherichia coli (STEC) in water buffaloes is reported for the first time in South America. The prevalence of STEC ranged from 0 to 64% depending on the farm. STEC isolates exhibiting the genetic profiles stx1stx2ehxA iha saa and stx2ehxA iha saa predominated. Of the 20 distinct serotypes identified, more than 50% corresponded to serotypes associated with human diseases.