Dissemination of blaOXA-370 is mediated by IncX plasmids and the Tn6435 transposon.

In Enterobacteriaceae, the blaOXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the blaOXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the blaOXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The blaOXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.

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.

Analysis of the virulence of an atypical enteropathogenic Escherichia coli strain in vitro and in vivo and the influence of type three secretion system.

Atypical enteropathogenic Escherichia coli (aEPEC) inject various effectors into intestinal cells through a type three secretion system (T3SS), causing attaching and effacing (A/E) lesions. We investigated the role of T3SS in the ability of the aEPEC 1711-4 strain to interact with enterocytes in vitro (Caco-2 cells) and in vivo (rabbit ileal loops) and to translocate the rat intestinal mucosa in vivo. A T3SS isogenic mutant strain was constructed, which showed marked reduction in the ability to associate and invade but not to persist inside Caco-2 cells. After rabbit infection, only aEPEC 1711-4 was detected inside enterocytes at 8 and 24 hours pointing to a T3SS-dependent invasive potential in vivo. In contrast to aEPEC 1711-4, the T3SS-deficient strain no longer produced A/E lesions or induced macrophage infiltration. We also demonstrated that the ability of aEPEC 1711-4 to translocate through mesenteric lymph nodes to spleen and liver in a rat model depends on a functional T3SS, since a decreased number of T3SS mutant bacteria were recovered from extraintestinal sites. These findings indicate that the full virulence potential of aEPEC 1711-4 depends on a functional T3SS, which contributes to efficient adhesion/invasion in vitro and in vivo and to bacterial translocation to extraintestinal sites.

Fimbrial Adhesins Produced by Atypical Enteropathogenic Escherichia coli Strains

Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrheaworldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. Inthis study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Braziland Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic andcommensal E. coli. Here, the most prevalent fimbrial genes found, in descending order, were hcpA (98.6%), ecpA(86%), fimA (76%), elfA (72%), and csgA (19.7%). Phenotypic expression of pili in aEPEC strains was assessedby several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli lamininbindingfimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains,respectively. The E. coli common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering toHeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence forsome aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strainsinvolving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins andindicates that these strains bear several pilus operons that could potentially be expressed in different nichesfavoring colonization and survival in and outside the host.

Fimbrial adhesins produced by atypical enteropathogenic Escherichia coli strains.

Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrhea worldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. In this study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71 aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Brazil and Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic and commensal E. coli. Here, the most prevalent fimbrial genes found, in descending order, were hcpA (98.6%), ecpA (86%), fimA (76%), elfA (72%), and csgA (19.7%). Phenotypic expression of pili in aEPEC strains was assessed by several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli laminin-binding fimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in 59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains, respectively. The E. coli common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering to HeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence for some aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strains involving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins and indicates that these strains bear several pilus operons that could potentially be expressed in different niches favoring colonization and survival in and outside the host.

Distinct Interaction of Two Atypical Enteropathogenic Escherichia coli Strains with Enterocytes In Vitro.

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.

Characterization of Escherichia coli strains isolated from patients with diarrhea in Sao Paulo, Brazil: identification of intermediate virulence factor profiles by multiplex PCR.

Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (EAEC) strains, followed by atypical EPEC (ATEC) strains. Classical enteropathogenic E. coli (EPEC) strains were not detected.

The flagella of an atypical enteropathogenic Escherichia coli strain are required for efficient interaction with and stimulation of interleukin-8 production by enterocytes in vitro.

The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.