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Escherichia coli (EC) strains belong to several pathotypes capable of infecting both humans and animals. Some of them have zoonotic potential and can sporadically cause epidemic outbreaks. Our aim was to screen for the distribution of these pathotypes in broilers and their related products. Therefore, E. coli strains were isolated (n = 118) from poultry intestine (n = 57), carcass (n = 57), and wastewater (n = 4) samples from one slaughterhouse with own reared poultry source and the National Reference Laboratory (NRL) poultry E. coli collection (n = 170) from the year 2017 was also studied. All 288 E. coli strains were screened by PCR for pathotype-specific genes stx, eae, st-lt, aggR, ipaH, and for further EPEC-specific virulence genes (bfp, EAF, tir, perA, ler). Altogether 35 atypical enteropathogenic E. coli (aEPEC) strains from the slaughterhouse and 48 aEPEC strains from the NRL collection were found. Regarding the phylogenetic groups of aEPEC, all four main groups were represented but there was a shift toward the B2 group (25%) as compared with the non-EPEC isolates (3%). The aEPEC isolates belonged to serogroups O14, O108, and O45. Multidrug resistance (MDR) was abundant in aEPEC strains (80 out of 83 aEPEC) with a diverse resistance pattern (n = 56). Our results of this study indicate that the high frequency of aEPEC in broilers and on their carcass surface, with frequent MDR to several antibiotic groups, raises the possibility that these strains pose a zoonotic risk to humans.

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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.

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Atypical enteropathogenic Escherichia coli (aEPEC) is an emerging pathogen that has been implicated in outbreaks of diarrhea worldwide. The objective of this study was to investigate the occurrence of aEPEC in retail foods at markets in the People's Republic of China and to characterize the isolates for virulence genes, intimin gene ( eae) subtypes, multilocus sequence types (STs), and antimicrobial susceptibility. From May 2014 to April 2015, 1,200 food samples were collected from retail markets in China, and 41 aEPEC isolates were detected in 2.75% (33 of 1,200) of the food samples. The virulence genes tir, katP, etpD, efa/lifA, ent, nleB, and nleE were commonly detected in these isolates. Nine eae subtypes were detected in the isolates, among which θ (23 isolates) and ß1 (6 isolates) were the most prevalent. The 41 isolates were divided into 27 STs by multilocus sequence typing. ST752 and ST10 were the most prevalent. Antibiotic susceptibility testing revealed high resistance among isolates to streptomycin (87.80%), cephalothin (73.16%), ampicillin (51.22%), tetracycline (63.42%), trimethoprim-sulfamethoxazole (43.90%), and kanamycin (43.90%). Thirty isolates (73.17%) were resistant to at least three antibiotics, and 20 (53.66 %) were resistant to five or more antibiotics. Our results suggest that retail foods in markets are important sources of aEPEC. The presence of virulent and multidrug-resistant aEPEC in retail foods poses a potential threat to consumers. Surveillance of aEPEC contamination and prudent use of antibiotics is strongly recommended in China.

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Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E.coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

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Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

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Using clonal phylogenetic methods, it has been demonstrated that O111:H25 atypical enteropathogenic E. coli (aEPEC) strains belong to distinct clones, suggesting the possibility that their ability to interact with different hosts and abiotic surfaces can vary from one clone to another. Accordingly, the ability of O111:H25 aEPEC strains derived from human, cat and dogs to adhere to epithelial cells has been investigated, along with their ability to interact with macrophages and to form biofilms on polystyrene, a polymer used to make biomedical devices. The results demonstrated that all the strains analyzed were able to adhere to, and to form pedestals on, epithelial cells, mechanisms used by E. coli to become strongly attached to the host. The strains also show a Localized-Adherence-Like (LAL) pattern of adhesion on HEp-2 cells, a behavior associated with acute infantile diarrhea. In addition, the O111:H25 aEPEC strains derived either from human or domestic animals were able to form long filaments, a phenomenon used by some bacteria to avoid phagocytosis. O111:H25 aEPEC strains were also encountered inside vacuoles, a characteristic described for several bacterial strains as a way of protecting themselves against the environment. They were also able to induce TNF-α release via two routes, one dependent on TLR-4 and the other dependent on binding of Type I fimbriae. These O111:H25 strains were also able to form biofilms on polystyrene. In summary the results suggest that, regardless of their source (i.e. linked to human origin or otherwise), O111:H25 aEPEC strains carry the potential to cause human disease.

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Autotransporter proteins (AT) are associated with bacterial virulence attributes. Originally identified in enteroaggregative Escherichia coli (EAEC), Shigella flexneri 2a and uropathogenic E. coli, the serine protease Pic is one of these AT. We have previously detected one atypical enteropathogenic E. coli strain (BA589) carrying the pic gene. In the present study, we characterized the biological activities of Pic produced by BA589 both in vitro and in vivo. Contrarily to other Pic-producers bacteria, pic in BA589 is located on a high molecular weight plasmid. PicBA589 was able to agglutinate rabbit erythrocytes, cleave mucin and degrade complement system molecules. BA589 was able to colonize mice intestines, and an intense mucus production was observed. The BA589Δpic mutant lost the capacity to colonize as well as the above-mentioned in vitro activities. Thus, Pic represents an additional virulence factor in aEPEC strain BA589, associated with adherence, colonization and evasion from the innate immune system.

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Atypical enteropathogenic Escherichia coli (aEPEC) strains produce attaching-effacing (AE) lesions on enterocytes due to the interaction of the adhesin intimin with its translocated receptor. aEPEC strain 1551-2 was previously shown to invade HeLa and T84 cells by means of the uncommon intimin subtype omicron. Other aEPEC strains carrying uncommon intimin subtypes have also been shown to invade differentiated T84 intestinal cells. In this study, seven aEPEC strains carrying the most common EPEC intimin subtypes (alpha, beta, and gamma) were evaluated regarding the ability to invade differentiated intestinal Caco-2 cells. Although all strains adhered to and promoted AE lesions, the numbers of cell-associated bacteria varied significantly between the different strains regardless of the intimin subtype (P < 0.05). Gentamicin protection assay and transmission electron microscopy analyses showed that in comparison with the invasive strain 1551-2, only one strain (aEPEC EC423/03, intimin beta) was invasive (P = 0.05). Although both strains persisted intracellularly until 48 h, the number of viable bacteria of EC423/03 decreased, whereas that of 1551-2 increased significantly up to 24 h and then decreased. In conclusion, invasiveness is a sporadic property among aEPEC strains carrying some common intimin subtypes.

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Typical and atypical Enteropathogenic Escherichia coli (EPEC) promote attaching-effacing lesions in intestinal cells but only typical EPEC carry the EPEC adherence factor plasmid. Atypical EPEC (aEPEC) are emerging agents of acute and persistent diarrhea worldwide. We aimed at comparing the ability of two aEPEC strains, 1711-4 (serotype O51:H40) and 3991-1 (serotype O non-typeable:non-motile) to invade, persist inside Caco-2 and T84 cells, and to induce IL-8 production. Typical EPEC strain E2348/69 was used for comparisons. The strains associated more significantly with T84 than with Caco-2 cells, with 3991-1 being the most adherent (P < 0.001). In contrast, aEPEC 1711-4 was significantly more invasive than the other strains in both cell lines, and was found within vacuoles near the basolateral cell surfaces. Strains persisted within both cell lines for at least 48 hours, but the persistence index was higher for 3991-1 in Caco-2 cells. IL-8 production was significantly higher from Caco-2 cells infected with 1711-4 for at least 48 hours (P < 0.001), and from T84 cells after 24 and 48 h than with the other strains (P = 0.001). We demonstrated that aEPEC are heterogeneous in various aspects of their interaction with enterocytes in vitro.