Escherichia coli O157:H7 from Argentinean pampean region: pathogenicity islands and effectors genes.

GONZÁLEZ, J., CADONA, J., SANSO, A.M., BUSTAMANTE, A.V.

Mar del Plata

Congreso; . X Congreso Argentino de Microbiología General SAMIGE; 2014

SAMIGE

Verotoxigenic Escherichia coli (VTEC) are important foodborne pathogens associated with sporadic cases and outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). Cattle have been recognized as the main reservoir of VTEC worldwide and O157:H7, as the dominant serotype associated with severe diseases in different parts of the world. Single nucleotide polymorphisms (SNP) typing was previously used to differentiate VTEC O157:H7 into 9 distinct clades, being clade 8 the most virulent one. On the other hand, important virulence properties are carried by pathogenicity islands (PAIs) such as the ?locus of enterocyte effacement? (LEE), which governs the capability of VTEC to colonize the intestinal mucosa of the host. Other PAIs, termed O islands, contain non-LEE (nle) effector genes that encode translocated substrates of type III secretion system. Some of these effectors contribute to the colonization and persistence of VTEC in cattle and interfere with the human inflammatory response. The aim of this study was to evaluate the presence of clade 8 strains in our area and to analyze the distribution of nle genes encoded in different PAIs. A total of 33 VTEC O157:H7 strains isolated, mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle cases and outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). Cattle have been recognized as the main reservoir of VTEC worldwide and O157:H7, as the dominant serotype associated with severe diseases in different parts of the world. Single nucleotide polymorphisms (SNP) typing was previously used to differentiate VTEC O157:H7 into 9 distinct clades, being clade 8 the most virulent one. On the other hand, important virulence properties are carried by pathogenicity islands (PAIs) such as the ?locus of enterocyte effacement? (LEE), which governs the capability of VTEC to colonize the intestinal mucosa of the host. Other PAIs, termed O islands, contain non-LEE (nle) effector genes that encode translocated substrates of type III secretion system. Some of these effectors contribute to the colonization and persistence of VTEC in cattle and interfere with the human inflammatory response. The aim of this study was to evaluate the presence of clade 8 strains in our area and to analyze the distribution of nle genes encoded in different PAIs. A total of 33 VTEC O157:H7 strains isolated, mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle Escherichia coli (VTEC) are important foodborne pathogens associated with sporadic cases and outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). Cattle have been recognized as the main reservoir of VTEC worldwide and O157:H7, as the dominant serotype associated with severe diseases in different parts of the world. Single nucleotide polymorphisms (SNP) typing was previously used to differentiate VTEC O157:H7 into 9 distinct clades, being clade 8 the most virulent one. On the other hand, important virulence properties are carried by pathogenicity islands (PAIs) such as the ?locus of enterocyte effacement? (LEE), which governs the capability of VTEC to colonize the intestinal mucosa of the host. Other PAIs, termed O islands, contain non-LEE (nle) effector genes that encode translocated substrates of type III secretion system. Some of these effectors contribute to the colonization and persistence of VTEC in cattle and interfere with the human inflammatory response. The aim of this study was to evaluate the presence of clade 8 strains in our area and to analyze the distribution of nle genes encoded in different PAIs. A total of 33 VTEC O157:H7 strains isolated, mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle nle genes encoded in different PAIs. A total of 33 VTEC O157:H7 strains isolated, mainly in Argentinean pampean region, from cattle, humans and food, were analyzed. They had been previously characterized as positive for virulence genes eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle eae and vtx2 . The identification of the strains belonging to clade 8 was performed by detecting a SNP located in the rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nle rhsA gene by PCR-RFLP. The restriction digestion of DNA was carried out with HaeII and Sau961 enzimes. In relation to the nlenle genes, nleA, nleH1-2, nleF, nleG, nleG2-1 and nleG9, encoded in genomic island O-I 71, nleB, nleEnleA, nleH1-2, nleF, nleG, nleG2-1 and nleG9, encoded in genomic island O-I 71, nleB, nleE and ent/esp L2, O-I 36, nleB2, nleC, nleH1-1 and nleD, O-I 122, nleG2-3, nleG5-2 and nleG6-2 O-I 57, were amplified. PCR products were visualized in agarose gels stained with SYBR Safe. All isolates belonged to clade 8. Regarding the presence of nle genes, isolates were grouped into six profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were amplified. PCR products were visualized in agarose gels stained with SYBR Safe. All isolates belonged to clade 8. Regarding the presence of nle genes, isolates were grouped into six profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. ent/esp L2, O-I 36, nleB2, nleC, nleH1-1 and nleD, O-I 122, nleG2-3, nleG5-2 and nleG6-2 O-I 57, were amplified. PCR products were visualized in agarose gels stained with SYBR Safe. All isolates belonged to clade 8. Regarding the presence of nle genes, isolates were grouped into six profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. nle genes, isolates were grouped into six profiles. Twenty isolates (61%) were positive for all nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. nle genes analyzed, while the remaining isolates, except two, showed incomplete O-I 71, particularly lacked the nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. nleF gene. No source-specific profiles were observed. According to these studies, O157:H7 strains circulating in the pampean region are a homogeneous group in relation to clades assignment. The belonging of the isolates studied to hypervirulent clade 8, and the high prevalence of nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. nle genes, genetic determinants that enable the pathogen to persist in the host and cause disease, would allow to assign most O157:H7 strains of this region a high risk to public health. X