Beatriz E. C. Guth

High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil

In order to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains, 197 fecal samples of healthy cattle from 10 dairy farms, four beef farms and one slaughterhouse at Rio de Janeiro State, Brazil, were examined for Shiga toxin (Stx) gene sequences by polymerase chain reaction (PCR). For presumptive isolation of O157:H7 E. coli, the Cefixime-potassium tellurite-sorbitol MacConkey Agar (CT-SMAC) was used. A high occurrence (71%) of Stx was detected, and was more frequently found among dairy cattle (82% vs. 53% in beef cattle), in which no differences were observed regarding the age of the animals. Dot blot hybridization with stx1 and stx2 probes revealed that the predominant STEC type was one that had the genes for both stx1 and stx2 in dairy cattle and one that had only the stx1 gene for beef cattle. Three (1.5%) O157:H7 E. coli strains were isolated from one beef and two dairy animals by the use of CT-SMAC. To our knowledge, this is the first report of O157:H7 isolation in Brazil. A PCR-based STEC detection protocol led to the isolation of STEC in 12 of 16 randomly selected PCR-positive stool samples. A total of 15 STEC strains belonging to 11 serotypes were isolated, and most of them (60%) had both stx1 and stx2 gene sequences. Cytotoxicity assays with HeLa and Vero cells revealed that all strains except two of serotype O157:H7 expressed Stx. The data point to the high prevalence of STEC in our environment and suggest the need for good control strategies for the prevention of contamination of animal products.

Emerging enteropathogenic Escherichia coli strains

Escherichia coli strains of nonenteropathogenic serogroups carrying eae but lacking the enteropathogenic E. coli adherence factor plasmid and Shiga toxin DNA probe sequences were isolated from patients (children, adults, and AIDS patients) with and without diarrhea in Brazil. Although diverse in phenotype and genotype, some strains are potentially diarrheagenic.

Virulence Properties and Characteristics of Shiga Toxin-Producing Escherichia coli in São Paulo, Brazil, from 1976 through 1999

ABSTRACT Twenty-nine Shiga toxin-producing Escherichia coli (STEC) strains were identified in a collection of 2,607 isolates from patients with diarrhea in São Paulo, Brazil, from 1976 to 1999. The STEC strains belonged mainly to serotypes O111:HNM (HNM, nonmotile) (13 of 29 [44.8%]), O111:H8 (7 of 29 [24%]), and O26:H11 (4 of 29 [13.8%]); stx1eae (26 of 29 [89.6%]), in combination with either enterohemorrhagic E. coli hlyA (11 of 26 [42%]) or astA (24 of 26 [92.3%]), prevailed. The O111 STEC strains were distinguished by their inability to decarboxylate lysine. The predominance of STEC O111 and O26 since the late 1970s and the identification of STEC serotypes O55:H19, O93:H19, and O118:H16 in association with human infections in Brazil are described for the first time.

First Shiga toxin-producing Escherichia coli isolate from a patient with hemolytic uremic syndrome, Brazil.

To the Editor: Infection by Shiga toxin (Stx)-producing Escherichia coli (STEC), particularly strains of serotype O157:H7, can cause sporadic cases and outbreaks of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) (1). Some other serotypes (e.g., O26:H11, O111:H8, O111:NM, and O113:H21) share a similar pathogenic potential. STEC are distributed worldwide, but most of the HC and HUS cases were reported from industrialized nations of the Northern and Southern hemisphere (2). In South America, HUS is a major cause of acute renal failure in infants in Argentina (3) and Chile (4). However, in Brazil human STEC infections have been restricted to sporadic cases of nonbloody diarrhea (5,6). Although a high frequency of STEC strains was recently found in foods and animal reservoirs (7,8), only some of the serotypes identified in animals (8) were recognized as causes of human illness (e.g., O157:H7, O22:H16, O82:H8, and NT:H21). Moreover, there is currently no nationwide surveillance system for HUS in Brazil, and STEC-associated HUS has not been previously reported in our country. We describe the case of an 8-month-old boy from a northeastern state in Brazil, who was admitted to the emergency room of Hospital Sao Paulo, Sao Paulo, on March 17, 2001; the boy had anemia, oliguria, and edema of lower extremities. He had an acute diarrheal prodromal illness 3 weeks before hospital admission. On the same day as admission, respiratory failure developed, and the child was transferred to the pediatric intensive-care unit of the hospital. The boy had hemolytic anemia (hemoglobin level 11.9 g/dL at admission, and 9.1 g/dL several days later), renal failure (blood urea nitrogen 43.8 mg/dL and serum creatinine 1.5 mg/dL), and thrombocytopenia (platelet count of 70,000/mm3), leading to a diagnosis of HUS. The patient received treatment with fresh frozen plasma and needed renal support (peritoneal dialysis) for 7 days. Once renal function was reestablished, the patient’s outcome was good. Feces were collected as soon as HUS was suspected and plated onto MacConkey Sorbitol Agar (Difco, Becton Dickinson Microbiology Systems, Sparks, MD). Only sorbitol-positive colonies grew and were biochemically identified as E. coli by standard procedures. The E. coli isolates expressed Stx1, as identified by cytotoxicity and neutralization assays on Vero cells (5). Presence of stx1 and intimin (eae) gene sequences was confirmed by polymerase chain reaction (9,10). The E. coli strain belonged to serotype O26:H11 and produced enterohemorragic E. coli hemolysin (enterohemolysin). This report is the first on the isolation of an STEC strain in a HUS patient in Brazil. The serotype O26:H11 has been described as agent of HC and HUS in other countries and was the second most frequent serotype found in STEC strains isolated from diarrheal cases in our settings (6). Moreover, expression of Stx1 and enterohemolysin and the presence of eae are virulent characteristics usually found in the human STEC strains isolated so far in Brazil. These findings show the importance of looking for non-O157 STEC strains besides O157:H7 in patients with HC and HUS in Brazil. Surveillance for HUS, either nationally or in sentinel population-based studies, should be performed in Brazil, and studies on the occurrence of HUS and its association with STEC infections are under investigation in our laboratory.

Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil

The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx1 corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx2 was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx1 plus stx2 sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx2 ehxA iha saa and stx1 stx2 ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx1 stx2 ehxA iha, stx2, and stx1 iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx2 alone or associated with stx1 were found more frequently in cattle, those harboring sequences stx1c and stx2d alone or associated with stx1c predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.

Serotypes and Shiga toxin genotypes among Escherichia coli isolated from animals and food in Argentina and Brazil

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from animals and food in Argentina (n=44) and Brazil (n=20) were examined and compared in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. The clonal relatedness of STEC O157 isolates (n=22) was established by phage typing (PT) and pulsed-field gel electrophoresis (PFGE). All O157 strains studied carried eae and enterohemorrhagic E. coli (EHEC)-hly sequences. In Argentina, these strains occurred both in cattle and meat, and 50% of them carried stx2/stx2vh-a genes, whereas in Brazil the O157 strains were isolated from animals, and most harbored the stx2vh-a sequence. At least 13 different O:H serotypes were identified among the non-O157 strains studied, with serotype O113:H21 being found in both countries. All but one non-O157 strains did not carry eae gene, but EHEC-hlyA gene was found in 85.7% of them, and the stx2 genotype was also more prevalent in Argentina than in Brazil (P<0.01), where stx1 alone or in association was most common (68.8%). One STEC strain isolated from a calf in Brazil harbored the new variant referred to as stx2-NV206. PFGE analysis showed that STEC O157 strains were grouped in four clusters. One Brazilian strain was considered possibly related (> or =80%) to Argentinean strains of cluster I. Differences in the pathogenic potential, especially in regard to serotypes and stx genotypes, were observed among the STEC strains recovered from animals and food in both countries.

Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA

Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content. All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp. The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established. Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition. The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Determination of Adhesin Gene Sequences in, and Biofilm Formation by, O157 and Non-O157 Shiga Toxin-Producing Escherichia coli Strains Isolated from Different Sources

ABSTRACT Biofilm formation by Shiga toxin-producing Escherichia coli (STEC) has been associated with the expression of different adhesins (type 1 fimbria, curli, Ag43, Cah, and EhaA). In this study, biofilm formation and the presence of adhesin-related gene sequences were determined by PCR in 18 O157 strains and 33 non-O157 strains isolated from different sources (human, animal, food, and water). The expression of different adhesins was also assessed by reverse transcription-PCR (RT-PCR), Congo red agar plates, and mannose-sensitive hemagglutination (MSHA) assay. Biofilm formation occurred in 5/18 (28%) O157 STEC strains and 17/33 (51%) non-O157 STEC strains from different serotypes and sources, when the assays were performed at 28°C for 48 h. Among the non-O157 biofilm-producing isolates, 12/17 (71%) expressed type 1 fimbriae and 11/17 (65%) expressed curli and produced cellulose, while 8/17 (47%) were considered to be Ag43+ by RT-PCR. Among O157 strains, a close correlation was observed between biofilm formation and expression of curli and cellulose. In non-O157 strains, it seems that, in addition to the presence of curli, the ability to form biofilm is associated with the presence of other factors such as type 1 fimbriae and autotransporter proteins, which may contribute to the persistence of these organisms in the environment.

High Occurrence of Shiga-Like Toxin-Producing Strains among Diarrheagenic Escherichia coli Isolated from Raw Beef Products in Rio de Janeiro City, Brazil

Raw beef samples (n = 105) were examined for diarrheagenic Escherichia coli (DEC) using standard methods. The isolates obtained (n = 1,066) were screened for Shiga-like toxins (SLT-I and SLT-II), cytolethal distending toxin (CLDT), enterotoxins (LT-I and STa), and classical enteropathogenic (EPEC) and enteroinvasive (EIEC) serogroups. Seventy-three (6.8%) DEC isolates representing 42 strains isolated from 34 (32.4%) beef samples were detected. SLT-producing E. coli (SLTEC) was the most frequent DEC category found and corresponded to 21 (50%) of the 42 DEC strains. Several serotypes were detected among the SLTEC and some of them have been found previously in animal and human isolates, but E. coli O157:H7 was not isolated. Other virulence markers found in DEC strains included enterotoxin production (38.1%), CLDT (7.1%), and EPEC serogroups (4.3%). This is the first report of CLDT-producing E. coli (CLDTEC) isolated from food samples in Brazil. Production of both SLT-I and LT-I was found in one E. coli isolate, and 3 beef samples harbored both SLTEC and ETEC strains. Although a high frequency of DEC groups was found in commercial beef samples in Rio de Janeiro City, Brazil, the significance of these strains as agents of human diarrhea remains to be established.

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

The heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strains causes diarrhoea by altering the fluid secretion in intestinal epithelial cells. Here, the effectiveness of a flagellin fusion protein of Salmonella containing a 19-amino-acid sequence derived from the ST sequence (FLA--ST) in generating antibodies capable of neutralizing the toxic activity of ST was evaluated. This fusion protein, and an alternative construction where two cysteine residues in the ST sequence were substituted by alanines (ST(mt)), were delivered to the immune system by three distinct strategies: (i) orally, using an attenuated Salmonella strain expressing FLA--ST; (ii) intraperitoneally, by injection of purified FLA--ST; (iii) orally, using attenuated Salmonella carrying a eukaryotic expression plasmid (pCDNA3) with the gene encoding FLA-ST. The results showed that the flagellin system can be used as a carrier to generate ST-neutralizing antibodies. However, it should be mentioned that humoral immune response against ST was only obtained when the mutated ST sequence was employed. FLA-ST was found to be non-immunogenic when delivered via the oral route with attenuated Salmonella strains. However, a flagellin antibody response was obtained by immunizing mice with Salmonella carrying pCDNA3/FLA-ST(mt). Oral immunization with Salmonella carrying the eukaryotic expression plasmid (pCDNA3/FLA--ST(mt)) seems to be a promising method to elicit an appropriate response against fusions to flagellin.