Interactions of Shiga toxin-producing Escherichia coli with leafy green vegetables.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens responsible for a wide spectrum of diseases including diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS). A considerable number of outbreaks and sporadic cases of HUS have been associated with ingestion of fresh ready-to-eat products. Maintenance and persistence of STEC in the environment and foods can be related to its ability to form biofilm. A non-O157 STEC strain isolated from bovine feces was distinguished by its great ability to form biofilm in abiotic surfaces. In the present study, we aimed to investigate the ability of this strain to adhere to rocket leaves (Eruca sativa). Adherence assays were carried out for 3 h at 28 °C and analyzed by scanning electron microscopy. The non-O157 STEC strain adhered to leaf surface and inside the stomata forming several bacterial aggregates. The number of adherent bacteria per square millimeter of leaf was eightfold higher compared with an O157 STEC strain. Deletion of the STEC autotransporter protein contributing to biofilm (Sab) reduced the adherence ability of the non-O157 strain in almost 50%, and deletion of antigen 43 (Ag43) almost abolished this interaction. Very few bacteria were seen on the leaf surface, and these differences were statistically significant, suggesting the role of both proteins and especially Ag43 in the interaction of the non-O157 STEC strain with leaves. The risk posed by non-O157 STEC adherence to leaves on fresh produce contamination should not be neglected, and measures that effectively control adherence should be included in strategies to control non-O157 STEC.

Interactions of Shiga toxin-producing Escherichia coli with leafy green vegetables

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens responsible for a wide spectrum of diseases including diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS). A considerable number of outbreaks and sporadic cases of HUS have been associated with ingestion of fresh ready-to-eat products. Maintenance and persistence of STEC in the environment and foods can be related to its ability to form biofilm. A non-O157 STEC strain isolated from bovine feces was distinguished by its great ability to form biofilm in abiotic surfaces. In the present study, we aimed to investigate the ability of this strain to adhere to rocket leaves (Eruca sativa). Adherence assays were carried out for 3 h at 28 °C and analyzed by scanning electron microscopy. The non-O157 STEC strain adhered to leaf surface and inside the stomata forming several bacterial aggregates. The number of adherent bacteria per square millimeter of leaf was eightfold higher compared with an O157 STEC strain. Deletion of the STEC autotransporter protein contributing to biofilm (Sab) reduced the adherence ability of the non-O157 strain in almost 50%, and deletion of antigen 43 (Ag43) almost abolished this interaction. Very few bacteria were seen on the leaf surface, and these differences were statistically significant, suggesting the role of both proteins and especially Ag43 in the interaction of the non-O157 STEC strain with leaves. The risk posed by non-O157 STEC adherence to leaves on fresh produce contamination should not be neglected, and measures that effectively control adherence should be included in strategies to control non-O157 STEC.

Interactions of Shiga toxin-producing Escherichia coli with leafy green vegetables

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens responsible for a wide spectrum of diseases including diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS). A considerable number of outbreaks and sporadic cases of HUS have been associated with ingestion of fresh ready-to-eat products. Maintenance and persistence of STEC in the environment and foods can be related to its ability to form biofilm. A non-O157 STEC strain isolated from bovine feces was distinguished by its great ability to form biofilm in abiotic surfaces. In the present study, we aimed to investigate the ability of this strain to adhere to rocket leaves (Eruca sativa). Adherence assays were carried out for 3 h at 28 °C and analyzed by scanning electron microscopy. The non-O157 STEC strain adhered to leaf surface and inside the stomata forming several bacterial aggregates. The number of adherent bacteria per square millimeter of leaf was eightfold higher compared with an O157 STEC strain. Deletion of the STEC autotransporter protein contributing to biofilm (Sab) reduced the adherence ability of the non-O157 strain in almost 50%, and deletion of antigen 43 (Ag43) almost abolished this interaction. Very few bacteria were seen on the leaf surface, and these differences were statistically significant, suggesting the role of both proteins and especially Ag43 in the interaction of the non-O157 STEC strain with leaves. The risk posed by non-O157 STEC adherence to leaves on fresh produce contamination should not be neglected, and measures that effectively control adherence should be included in strategies to control non-O157 STEC.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype.

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like?+?strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like?+?strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.

Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we have not identified any plasmid genes specifically present in all LA/AA-like+ strains and absent in the LA+ strains, these results suggest the presence of an unknown mechanism to promote the AA-like pattern production and biofilm formation by the LA/AA-like+ strains. Because their ability to produce A/E lesions and biofilm concomitantly could exacerbate the clinical condition of the patient and lead to persistent diarrhea, the mechanism underlying the enhanced biofilm formation by the LA/AA-like+ O119:H6 strains and their spread and involvement in severe diarrheal diseases should be more intensively investigated.

Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli.

The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliCa nd fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The a EPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process.