Transposon mutagenesis of atypical enteroaggregative Escherichia coli reveals a hemagglutinin-associated protein that mediates cell adhesion and contributes to the Galleria mellonella virulence.

Twenty-two atypical enteroaggregative Escherichia coli isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes afpA2 and afpR were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in E. coli strain 7-233-03_S3_C2. Therefore, the ORF was named orfHA. The regions flanking orfHA were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An orfHA mutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHA strain did not adhere to HeLa cells, whereas Q015BΔ orfHA transformed with a pACYC184 plasmid carrying orfHA restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHA mutant had a marked effect on the ability of strain Q015B to kill the larvae of Galleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the G. mellonella model.

Large plasmids encoding antibiotic resistance and localized-like adherence in atypical enteropathogenic Escherichia coli strains.

BACKGROUND: In previous studies, we have shown that atypical enteropathogenic Escherichia coli (aEPEC) strains are important diarrheal pathogens among Brazilian children. In the characterization of a collection of 126 aEPEC strains, we identified 29 strains expressing the localized-like adherence (LAL) pattern on HEp-2 cells and harboring large plasmids in the range of 60 to 98 MDa. In this study, we examined 18 of these strains for their ability to transfer the LAL phenotype to a E. coli K-12 C600 strain. RESULTS: In conjugation experiments, using eight strains which were resistant to one or more antimicrobials and positive for F-pili genes (traA), we were able to cotransfer antimicrobial resistance markers along with adhesion genes. By transforming E. coli DH5α with plasmid DNA from strains A46 (pIS46), A66 (pIS66) and A102 (pIS102), we were able to demonstrate that genes encoding ampicillin, tetracycline and LAL were encoded on a 98-MDa conjugative plasmid. To identify a gene responsible for LAL, we constructed a transposon mutant library of A102 strain. Among 18 mutants that did not adhere to HeLa cells, four carried insertions within fimbrial genes (fimA and traJ) and agglutinin genes (tia and hek). Using these Tn5 mutants as donors, we were able to obtain kanamycin-resistant E. coli MA3456 transconjugants. Sequence analysis of the plasmid genes revealed a region exhibit to 80 and 73% amino acid similarities to the agglutinins Tia and Hek, respectively. CONCLUSION: In this study, we have identified three large conjugative plasmids, pIS46, pIS66 and pIS102, coding for antimicrobial resistance and localized-like adherence (LAL) to HeLa cells. In addition, we identified a tia/hek homolog encoded on the pIS102 plasmid, which seems to be involved in adhesion of A102 strain.

Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities.

BACKGROUND: Childhood diarrheal diseases remain highly endemic in developing areas of Brazil. The importance of Escherichia coli among children with diarrhea in these areas was unknown. This study determined the prevalence of different E. coli categories in symptomatic and asymptomatic children from low socioeconomic level rural communities in southeastern Brazil. METHODS: A total of 560 stool samples were collected from 141 children with diarrhea (< 10 years) and 419 apparently healthy controls who resided in 23 communities. E. coli isolates (n = 1943) were subjected to two multiplex PCRs developed for the detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and Shiga toxin-producing E. coli (STEC). Strains were also examined for the presence of EPEC, EAEC, and DAEC by assays of adhesion to HEp-2 cells and by hybridization with specific DNA probes. RESULTS: Diarrheagenic E. coli strains were isolated from 253 (45.2%) children, and were associated with diarrhea in children aged < 5 years (p < 0.001). EAEC (20.9%), DAEC (11.6%), EPEC (9.3%) were the most frequent pathotypes, followed by ETEC (2.7%), EIEC (0.5%), and STEC (0.2%). Depending of the assay, EPEC, EAEC, and DAEC (collectively termed enteroadherent E. coli) strains were isolated in 45% to 56% of diarrhea cases, a significantly higher incidence than in controls (P < 0.05). Individually, only DAEC showed significant association with diarrhea (p < 0.05), particularly in children aged 2-5 years. CONCLUSION: This study indicates that enteroadherent E. coli is an important cause of diarrhea in children living in low socioeconomic level communities in southeastern Brazil. Our results reveal that the PCR1 assay is an excellent tool for the identification of EAEC and DAEC.