Renal damage and death in weaned mice after oral infection with Shiga toxin 2-producing Escherichia coli strains.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infections are considered a public health problem in both developed and developing countries because of their increasing incidence and the severity of clinical presentation. Approximately 10% of infected patients develop complications such as haemolytic uraemic syndrome (HUS) characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. The precise sequence of events leading to HUS is still understood incompletely. Because of the lack of a reproducible small animal model for EHEC infections, in vivo studies examining EHEC-host early interactions are limited and insufficient. The aim of this study was to characterize the weaned BALB/c mouse as a model of E. coli O157:H7 infection. In this paper we report that human Shiga toxin 2 (Stx2)-producing EHEC strains can adhere to the intestinal epithelium of weaned BALB/c mice, and produce local damage which leads to systemic disease and death in a percentage of infected mice. The lethality of the EHEC strain is closely age-dependent, and is related to the bacterial ability to colonize intestine and to produce Stx2. It can be concluded that the weaned BALB/c mouse can be used as a small animal model to study host early responses, and the role of bacterial pathogenic factors in the induction of systemic disease, thus providing a useful tool for the evaluation of therapeutic or vaccine approaches.

Isolation and characterisation of Shiga toxin-producing Escherichia coli O157:H7 from calves in Argentina.

Four hundred and twenty-two calves were examined for intestinal carriage of Shiga toxin-producing Escherichia coli O157:H7 using conventional plating. Two (0.5%) E. coli O157 were recovered. They were compared with 96 Argentine strains of different origin by pulsed-field gel electrophoresis, phage typing and PCR-RFLP of stx2 genes. One strain isolated from a calf, was closely related with 18 strains of clinical origin.

[Isolation and characterization of Shiga-toxin-producing Escherichia coli from frozen hamburgers and soft cheeses]. / Aislamiento y caracterización de Escherichia coli productor de toxina Shiga en hamburguesas supercongeladas y quesos de pasta blanda.

Shiga toxin producing-Escherichia coli (STEC), an important emerging foodborne pathogen, has been associated with bloody and non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. The cattle have been shown to be a major reservoir of STEC and raw foods such as ground beef and milk are the most common vehicles of infection. In the present study, the prevalence of STEC in 95 samples of frozen hamburgers and in 114 samples of soft cheese was established in 8.4% and 0.9%, respectively. The genotypic and phenotypic characteristics of the strains were determined. The virulence genes stx1, stx2, eaeA and EHEC-hlyA were identified by PCR and by colony blot hybridization assays. Serotyping, antimicrobial susceptibility and production of Stx using specific cytotoxicity assays on Vero cells were also determined. All STEC strains were characterized as eaeA-/EHEC-hlyA+. The stx2 genotype was prevalent (77.8%), and four different O:H serotypes were found, comprising: O8:H19 (5 strains), O113:H21 (1), O8:H16 (1), and O39:H49 (1). One STEC strain was nontypable. Although soft cheese complimented the microbiological quality controls for the coliform counts, the detection of STEC in one sample raises doubts concerning the effectiveness of the current quality controls. These data contribute to the implementation of strategies for the prevention and control of HUS.

Detection of Shiga toxin-producing Escherichia coli by PCR in cattle in Argentina. Evaluation of two procedures.

Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.

Aislamiento y caracterización de Escherichia coli productor de toxina Shiga en hamburguesas supercongeladas y quesos de pasta blanda. / [Isolation and characterization of Shiga-toxin-producing Escherichia coli from frozen hamburgers and soft cheeses]

Shiga toxin producing-Escherichia coli (STEC), an important emerging foodborne pathogen, has been associated with bloody and non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. The cattle have been shown to be a major reservoir of STEC and raw foods such as ground beef and milk are the most common vehicles of infection. In the present study, the prevalence of STEC in 95 samples of frozen hamburgers and in 114 samples of soft cheese was established in 8.4

and 0.9

, respectively. The genotypic and phenotypic characteristics of the strains were determined. The virulence genes stx1, stx2, eaeA and EHEC-hlyA were identified by PCR and by colony blot hybridization assays. Serotyping, antimicrobial susceptibility and production of Stx using specific cytotoxicity assays on Vero cells were also determined. All STEC strains were characterized as eaeA-/EHEC-hlyA+. The stx2 genotype was prevalent (77.8

), and four different O:H serotypes were found, comprising: O8:H19 (5 strains), O113:H21 (1), O8:H16 (1), and O39:H49 (1). One STEC strain was nontypable. Although soft cheese complimented the microbiological quality controls for the coliform counts, the detection of STEC in one sample raises doubts concerning the effectiveness of the current quality controls. These data contribute to the implementation of strategies for the prevention and control of HUS.

Isolation and characterization of Escherichia coli O157:H7 from retail meats in Argentina.

Between February and May 2000, 279 meat samples were collected from 136 retail stores in Gualeguaychú City, Argentina. Samples were assayed for Escherichia coli O157:H7 by selective enrichment in modified EC broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto both sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and a chromogenic medium. Eleven E. coli O157:H7 isolates were detected in 6 (3.8%) of 160 ground beef samples, in 4 (4.8%) of 83 fresh sausages, and in 1 (3.3%) of 30 dry sausages. E. coli O157:H7 was not isolated from five hamburger patties or one barbecue-type fresh sausage assayed. The isolates were tested for virulence-related genes. Ten additional Shiga toxin-producing E. coli (STEC) O157:H7 isolates of food origin, recovered from different locations in Argentina, were included for comparison purposes. All 21 isolates harbored both eae and EHEC-hlyA genes, and 12 (57.1%) encoded stx2/stx2vh-a. The isolates were of phage types 87 (seven strains), 14 (four strains), 4 (three strains), and 26 (one strain). Six strains were nontypable by phage typing. Pulsed-field gel electrophoresis (PFGE) revealed 19 XbaI-PFGE profiles. Fifteen (71%) strains were grouped in four clusters, which shared more than 80% of DNA restriction fragments. The enrichment culture method with IMS was a sensitive procedure to detect E. coli O157:H7 strains in retail meats. Some of the isolates from different stores presented a high clonal relatedness, as determined by XhaI-PFGE and phage typing, and harbored the virulence factors associated with human illness.

Intestinal bleeding and occlusion associated with Shiga toxin-producing Escherichia coli O127:H21.

We report a case of a nine-year old boy with vomiting, abdominal pain and fever, who underwent surgery with a diagnosis of appendicitis in Mendoza and from whom a Shiga toxin-producing Escherichia coli (STEC) O127:H21 strain was recovered. Forty-eight hours after surgery he presented bilious vomiting and two episodes of intestinal bleeding. Laboratory findings included: hematocrit, 35%; blood urea nitrogen, 0.22 g/L. The urinary output was normal. The following day physical examination showed an alert mildly hydrated child, without fever but with distended and painful abdomen. The patient was again submitted to surgery with a diagnosis of intestinal occlusion. Bleeding and multiple adhesions in jejunum and ileum were found. The patient still had tense and painful abdomen and presented two bowel movements with blood; hematocrit fell to 29% and blood urea nitrogen rose to 0.32 g/L. STEC O127:H21 eae(-)/Stx2/Stx2vh-b(+)/E-Hly(+) was isolated from a stool sample. He was discharged after 10 days of hospitalization and no long-term complications such as HUS or TTP were observed. This is the first report, to our knowledge, on the isolation of E. coli O127:H21, carrying the virulence factors that characterize STEC strains, associated to an enterohemorrhagic colitis case. This serotype was previously characterized as a non-classic enteropathogenic E. coli (EPEC). STEC infections can mimic infectious or noninfectious pathologies. Therefore an important aspect of clinical management is making the diagnosis using different criteria thereby avoiding misdiagnoses which have occasionally led to invasive diagnostic and therapeutic procedures or the inappropriate use of antibiotics.

Intestinal bleeding and occlusion associated with Shiga toxin-producing Escherichia coli O127:H21.

We report a case of a nine-year old boy with vomiting, abdominal pain and fever, who underwent surgery with a diagnosis of appendicitis in Mendoza and from whom a Shiga toxin-producing Escherichia coli (STEC) O127:H21 strain was recovered. Forty-eight hours after surgery he presented bilious vomiting and two episodes of intestinal bleeding. Laboratory findings included: hematocrit, 35

; blood urea nitrogen, 0.22 g/L. The urinary output was normal. The following day physical examination showed an alert mildly hydrated child, without fever but with distended and painful abdomen. The patient was again submitted to surgery with a diagnosis of intestinal occlusion. Bleeding and multiple adhesions in jejunum and ileum were found. The patient still had tense and painful abdomen and presented two bowel movements with blood; hematocrit fell to 29

and blood urea nitrogen rose to 0.32 g/L. STEC O127:H21 eae(-)/Stx2/Stx2vh-b(+)/E-Hly(+) was isolated from a stool sample. He was discharged after 10 days of hospitalization and no long-term complications such as HUS or TTP were observed. This is the first report, to our knowledge, on the isolation of E. coli O127:H21, carrying the virulence factors that characterize STEC strains, associated to an enterohemorrhagic colitis case. This serotype was previously characterized as a non-classic enteropathogenic E. coli (EPEC). STEC infections can mimic infectious or noninfectious pathologies. Therefore an important aspect of clinical management is making the diagnosis using different criteria thereby avoiding misdiagnoses which have occasionally led to invasive diagnostic and therapeutic procedures or the inappropriate use of antibiotics.

Casos de gastroenteritis asociados a Vibrio cholerae no O1 en Orán, Salta / Non- O group 1 Vibrio cholerae gastroenteritis in Oran, Salta

Entre febrero de 1992 y 1995 se detectaron 41 casos de gastroenteritis asociada a Vibrio cholerae no O1 en Orán, Salta. La frecuencia de aislamiento fue del 0,9 o/o, entre los casos de diarrea. El 51,2 o/o de los casos correspondió a mayores de 15 años y el 60,9, al sexo masculino. Todos los pacientes tuvieron diarrea, en 24 (58,5 o/o) fue líquida y en 6 (14,5 o/o) con aspecto coleriforme. Diez (24,4 o/o) de los pacientes presentaron vómitos y 12 (29,3 o/o) deshidratación leve o moderada. Seis pacientes pediátricos, desnutridos de 2º y 3º grado, que presentaron diarrea de más de una semana de evolución y deshidratación moderada, requirieron hospitalización durante 7 días. Durante el primer brote, en un paciente se aisló simultáneamente V. cholerae no O1 y Shigella flexneri y en el cu

Casos de gastroenteritis asociados a Vibrio cholerae no O1 en Orán, Salta / Non- O group 1 Vibrio cholerae gastroenteritis in Oran, Salta

Entre febrero de 1992 y 1995 se detectaron 41 casos de gastroenteritis asociada a Vibrio cholerae no O1 en Orán, Salta. La frecuencia de aislamiento fue del 0,9 o/o, entre los casos de diarrea. El 51,2 o/o de los casos correspondió a mayores de 15 años y el 60,9, al sexo masculino. Todos los pacientes tuvieron diarrea, en 24 (58,5 o/o) fue líquida y en 6 (14,5 o/o) con aspecto coleriforme. Diez (24,4 o/o) de los pacientes presentaron vómitos y 12 (29,3 o/o) deshidratación leve o moderada. Seis pacientes pediátricos, desnutridos de 2§ y 3§ grado, que presentaron diarrea de más de una semana de evolución y deshidratación moderada, requirieron hospitalización durante 7 días. Durante el primer brote, en un paciente se aisló simultáneamente V. cholerae no O1 y Shigella flexneri y en el cuarto brote en otro se detectó la asociación V. cholerae no O1 y Salmonella subespecie IV 50: b:-. Una mujer de 72 años falleció durante el segundo brote. El cuadro clínico estuvo caracterizado por diarrea líquida, vómitos, fiebre y deshidratación moderada. De su coprocultivo se recuperó V. cholerae O5 negativo para los siguientes factores de virulencia: toxina de cólera (CT), enterotoxina termoestable, hemolisina (El Tor) y hemaglutinina asociadas a células resistentes a D-manosa y L-fucosa. La caracterización bioquímica de los 41 aislamientos correspondió a V. cholerae con serología negativa para los serogrupos O1 y O139. Ningún aislamiento produjo CT. El 19,5 o/o presentó resistencia a la ampiclina y 4,9 o/o, a trimetoprimasulfametoxazol. La vigilancia activa de las diarreas en Orán, ha demostrado que V. cholerae no O1 no es un agente causal importante de las mismas

[Cases of gastroenteritis associated to Vibrio cholerae no 01 in Oran, Salta]. / Casos de gastroenteritis asociados a Vibrio cholerae no O1 en Orán, Salta.

Forty-one sporadic cases of non-O group 1 Vibrio cholerae gastroenteritis were detected in Orán, Salta, between February 1992 and February 1995. The frequency of isolation was 0.9% of the diarrhea cases. Out of 41 patients, 21 (51.2%) were older than 15 years and 25 (60.9%) were male. All the patients had diarrhea, 24 (58.5%) had watery stools and 6 (14.6%) cholera-like diarrhea; 10 (24.4%) presented vomiting and 12 (29%) mild dehydration. Six malnourished children who suffered from diarrhea with moderate dehydration for more than a week, were hospitalized. V. cholerae non O1 and Shigella flexneri were isolated from one patient, during the first outbreak and V. cholerae non O1 and Salmonella IV 50:b:- were recovered simultaneously from another patient during the fourth outbreak. A 72 year old woman died during the second cholera outbreak. The symptoms were: watery diarrhea, vomiting, fever and mild dehydration. A strain of V. cholerae O5, that did not produce cholera toxin, heat-stable enterotoxin, Kanagawa-like hemolysin or verocitotoxin was detected. It was positive for El Tor hemolysin and D-mannose and L-fucose resistant cells-associated hemagglutinins. Among the 41 isolates studied, all were oxidase and indole positive, fermented glucose, saccharose and mannitol. They were all motile, produced lysine and ornithine decarboxylases but not arginine dihydrolase or hydrogen sulfide. They were sensitive to O129 vibriostatic compound. None of them belonged to O1 or O139 serogroup and they did not produce cholera troxin. Among the V. cholerae non O1 strains isolated, 9.5% were resistant to ampicillin and 4.9% to trimethoprim-sulfamethoxazole. Active surveillance had shown that V. cholerae non-O1 is not an important agent of diarrhea in Orán, Salta.