ABSTRACT
This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.
Subject(s)
Animals , Cattle , Carrier State/veterinary , Escherichia coli Infections/veterinary , Shiga-Toxigenic Escherichia coli/genetics , Shiga-Toxigenic Escherichia coli/pathogenicity , Virulence Factors/analysis , Abattoirs , Argentina , Bacterial Toxins/analysis , Bacterial Toxins/genetics , Cell Survival , Chlorocebus aethiops , Carrier State/microbiology , Escherichia coli Infections/microbiology , Microbial Sensitivity Tests , Molecular Typing , Polymerase Chain Reaction , Rectum/microbiology , Serotyping , Shiga-Toxigenic Escherichia coli/classification , Shiga-Toxigenic Escherichia coli/isolation & purification , Vero Cells , Virulence Factors/geneticsABSTRACT
This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.
Subject(s)
Carrier State/veterinary , Escherichia coli Infections/veterinary , Shiga-Toxigenic Escherichia coli/genetics , Shiga-Toxigenic Escherichia coli/pathogenicity , Virulence Factors/analysis , Abattoirs , Animals , Argentina , Bacterial Toxins/analysis , Bacterial Toxins/genetics , Carrier State/microbiology , Cattle , Cell Survival , Chlorocebus aethiops , Escherichia coli Infections/microbiology , Microbial Sensitivity Tests , Molecular Typing , Polymerase Chain Reaction , Rectum/microbiology , Serotyping , Shiga-Toxigenic Escherichia coli/classification , Shiga-Toxigenic Escherichia coli/isolation & purification , Vero Cells , Virulence Factors/geneticsABSTRACT
This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.
ABSTRACT
This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.
ABSTRACT
Enteroaggregative Escherichia coli (EAEC) has been implicated in sporadic diarrhea in children and adults and has been identified as the cause of several outbreaks worldwide. The HEp-2 test remains the gold standard for identification of this pathotype. A 60-65 MDa plasmid encodes the aggregative adherence fimbriae (AAF/I and AAF/II), a transcriptional activator (aggR gene), the enteroaggregative heat-stable enterotoxin EAST1 (astA gene) and a cytotoxin (Pet). The standard assay for EAEC is performed only in research laboratories, because it is expensive, labor intensive and time-consuming. The Polymerase Chain Reaction (PCR) offers the possibility of rapid diagnosis. In the current study, a multiplex PCR assay which checks aggR and astA genes was designed. Eigthy-eight E. coli strains, isolated from children with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by aggR-astA PCR. A strong correlation between the presence of the specific marker aggR and the reference test was found. The astA gene had a similar distribution between aggregative and localized strains, indicating that this gene could not be considered as a marker of EAEC. We conclude that aggR may be used to identify EAEC, using the PCR method as a screening test.
Subject(s)
Escherichia coli/genetics , Escherichia coli/isolation & purification , Polymerase Chain Reaction/methods , Bacterial Adhesion/physiology , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/cytology , Escherichia coli Proteins/genetics , Humans , Infant , Serotyping , Trans-Activators/geneticsABSTRACT
Enteroaggregative Escherichia coli (EAEC) has been implicated in sporadic diarrhea in children and adults and has been identified as the cause of several outbreaks worldwide. The HEp-2 test remains the gold standard for identification of this pathotype. A 60-65 MDa plasmid encodes the aggregative adherence fimbriae (AAF/I and AAF/II), a transcriptional activator (aggR gene), the enteroaggregative heat-stable enterotoxin EAST1 (astA gene) and a cytotoxin (Pet). The standard assay for EAEC is performed only in research laboratories, because it is expensive, labor intensive and time-consuming. The Polymerase Chain Reaction (PCR) offers the possibility of rapid diagnosis. In the current study, a multiplex PCR assay which checks aggR and astA genes was designed. Eigthy-eight E. Coli strains, isolated from children with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by aggR-astA PCR. A strong correlation between the presence of the specific marker aggR and the reference test was found. The astA gene had a similar distribution between aggregative and localized strains, indicating that this gene could not be considered as a marker of EAEC. We conclude that aggR may be used to identify EAEC, using the PCR method as a screening test.
Subject(s)
Humans , Infant , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Bacterial Adhesion/physiology , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli/isolation & purification , Polymerase Chain Reaction/methods , Electrophoresis, Agar Gel , Escherichia coli Proteins , Serotyping , Trans-Activators/geneticsABSTRACT
Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4% sensitivity and 78.26% specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.
Subject(s)
Bacterial Adhesion , Diarrhea, Infantile/microbiology , Escherichia coli Infections/microbiology , Escherichia coli/isolation & purification , Argentina/epidemiology , Bacterial Toxins/genetics , Enterotoxins/genetics , Epithelial Cells/microbiology , Escherichia coli/pathogenicity , Fimbriae Proteins/genetics , Fimbriae, Bacterial/genetics , Humans , Infant , Mass Screening , Phenotype , Plasmids , Polymerase Chain Reaction , Sensitivity and Specificity , Tumor Cells, Cultured/microbiology , VirulenceABSTRACT
Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4 sensitivity and 78.26 specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.(AU)
Subject(s)
Humans , Infant , Bacterial Adhesion , Diarrhea, Infantile/microbiology , Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Argentina/epidemiology , Bacterial Toxins/genetics , Enterotoxins/genetics , Epithelial Cells/microbiology , Escherichia coli/pathogenicity , Fimbriae Proteins/genetics , Fimbriae, Bacterial/genetics , Mass Screening , Phenotype , Plasmids , Polymerase Chain Reaction , Sensitivity and Specificity , Tumor Cells, Cultured/microbiology , VirulenceABSTRACT
Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4 sensitivity and 78.26 specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.
Subject(s)
Humans , Infant , Bacterial Adhesion , Diarrhea, Infantile , Escherichia coli , Escherichia coli Infections/microbiology , Argentina , Bacterial Toxins , Epithelial Cells/microbiology , Enterotoxins , Escherichia coli , Fimbriae, Bacterial , Mass Screening , Phenotype , Plasmids , Polymerase Chain Reaction , Fimbriae Proteins/genetics , Sensitivity and Specificity , Tumor Cells, Cultured , VirulenceABSTRACT
The present study evaluates the growth promoting effect of insulin on the proliferative activity of cells from rat mesenteric arteries in culture in order to test the hypothesis that insulin may play a pathogenetic role in the hypertrophy of resistance vessels. The proliferative effect of insulin was studied in cultured vascular smooth muscle cells (SMC) obtained from two functionally different rat vessels: mesenteric arteries and aorta. Growth characteristics (cell number and growth rate) of mesenteric and aortic cells were determined after a quiescent period and followed-up for 24, 72, and 120 h after the addition of insulin. At all studied time intervals, aortic SMC exhibited a significatively higher cell number and specific growth rate than did mesenteric SMC. Aortic SMC also displayed a greater proliferation than did mesenteric SMC in the presence of 10% fetal calf serum (FCS). At a physiological concentration of 100 microU/mL, the proliferative effect of insulin, after a quiescent period, was seen only in aortic SMC, at 72 and 120 h. Higher insulin concentration (500 microU/mL) increased significantly the cell number in SMC of both arteries. The proliferative effect was significant at all studied periods for aortic SMC; however, in mesenteric SMC, insulin increased the cell number only at 72 and 120 h. The proliferative effect of insulin was observed on SMC obtained from functionally different arteries such as aorta and mesenteric, being greater in the former. The different behavior of these SMC in the presence not only of insulin, but also of 10% FCS, provides further evidence for the existence of intervascular heterogeneity. The mild stimulatory effect of insulin in vitro may contribute in this way to the vascular hypertrophy of pathological entities exhibiting hyperinsulinemia.