Phenotypic and genotypic characteristics of shiga toxin-producing Escherichia coli strains isolated from children in São Paulo, Brazil

The biochemical and serological characteristics, virulence properties, and genetic relatedness of Shiga toxin-producing Escherichia coli (STEC) strains isolated in São Paulo, from April 1989 through March 1990, were determined. This is also the first report on clinic findings of human STEC infections in Brazil. The only three STEC strains identified in that period were lysine decarboxylase negative, belonged to serotype O111ac: non-motile, were Stx1 producers, carried the eae and astA genes, and 2 of them also presented the EHEC-hly sequence. The children carrying STEC were all boys, with less than two years old, and had no previous history of hospitalization. None of them presented blood in stools. Vomiting, cough and coryza were the most common clinical manifestations observed. Although the STEC strains were isolated during summer months, and presented similar phenotypic and genotypic characteristics, carbohydrate fermentation patterns and PFGE analysis suggested that these diarrheal episodes were not caused by a single clone.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29% of the strains. Enterohaemolysin production was detected in 29% of the strains. Most of the bacterial strains (95%) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50% of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

Detection of virulence factors and antimicrobial resistance patterns in shiga toxin-producing Escherichia coli isolates from sheep

Abstract: In order to detect virulence factors in Shiga toxin-producing Escherichia coli (STEC) isolates and investigate the antimicrobial resistance profile, rectal swabs were collected from healthy sheep of the races Santa Inês and Dorper. Of the 115 E. coli isolates obtained, 78.3% (90/115) were characterized as STEC, of which 52.2% (47/90) carried stx1 gene, 33.3% (30/90) stx2 and 14.5% (13/90) both genes. In search of virulence factors, 47.7% and 32.2% of the isolates carried the genes saa and cnf1. According to the analysis of the antimicrobial resistance profile, 83.3% (75/90) were resistant to at least one of the antibiotics tested. In phylogenetic classification grouped 24.4% (22/90) in group D (pathogenic), 32.2% (29/90) in group B1 (commensal) and 43.3% (39/90) in group A (commensal). The presence of several virulence factors as well as the high number of multiresistant isolates found in this study support the statement that sheep are potential carriers of pathogens threatening public health.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC) on dairy farms

Milkborne transmission of Shiga toxin- producing Escherichia coli (STEC) has raised considerable concern due to recent outbreaks worldwide and poses a threat to public health. The aim of this study was to develop a sensitive and specific multiplex PCR assay to detect the presence of STEC in bovine raw milk. To identify E. coli (ATCC 25922) contamination, the gene uspA was used, and PCR sensitivity and specificity were accessed by testing diluted samples ranging from 2 to 2.0 × 10(6) CFU/mL. To detect STEC, the stx1 and stx2 genes were selected as targets. After reaction standardization, the multiplex assay was tested in raw milk collected from 101 cows on dairy farms. PCR assay for E. coli detection had a specificity of 100% and sensitivity of 79% (P<0.0001), with a lower detection limit of 2 CFU/mL. Multiplex PCR assay had 100% sensitivity for E. coli positive raw milk samples, and 31.1% were contaminated with STEC, 28.3% of stx2, and 1.9% of stx1. The multiplex PCR assay described in the present study can be employed to identify and screen E. coli harboring stx1 and stx2 genes in raw milk on dairy farms and in industries.

Survival of Shiga toxin-producing Escherichia coli O157:H7 in Minas frescal cheese

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains (isolated by cattle’s faeces and a reference strain, EDL933), were inoculated into pasteurized milk (102 and 103 cells.mL–1) to prepare the Minas frescal cheese. As control was used uninfected milk. Physicochemical and microbiological analyses were performed to milk and elaborated cheese. The O157:H7 strains were quantified in the stages of cheese processing and during 0, 2, 4, 5, 7, 10 and 15 storage days at 8 °C onto Sorbitol MacConkey Agar supplemented with potassium tellurite and cefixime (CT-SMAC). O157:H7 was not present in the pasteurised milk prior to the artificial inoculation. At the end of the processing the cheese had 10 to 100 times more STEC O157:H7 than the initial inoculum. During the storage, the Minas frescal cheese exhibited the largest population increase on the 4th and 5th day when inoculated with 102 and 103 cells.mL–1, respectively. Additionally, viable cells were found up to the 10th and 15th day, according to the amount of initial inoculum. This number of cells is able to cause infection in humans, and therefore, Minas frescal cheese, even when stored under refrigeration, is a potential vehicle of disease caused by STEC O157:H7.

ISOLATION AND MOLECULAR IDENTIFICATION OF POTENTIALLY PATHOGENIC Escherichia coli AND Campylobacter jejuni IN FERAL PIGEONS FROM AN URBAN AREA IN THE CITY OF LIMA, PERU

SUMMARY Feral pigeons (Columbia livia) live in close contact with humans and other animals. They can transmit potentially pathogenic and zoonotic agents. The objective of this study was to isolate and detect strains of diarrheagenic Escherichia coli and Campylobacter jejuniof urban feral pigeons from an area of Lima, Peru. Fresh dropping samples from urban parks were collected for microbiological isolation of E. coli strains in selective agar, and Campylobacterby filtration method. Molecular identification of diarrheagenic pathotypes of E.coliand Campylobacter jejuni was performed by PCR. Twenty-two parks were sampled and 16 colonies of Campylobacter spp. were isolated. The 100% of isolates were identified as Campylobacter jejuni. Furthermore, 102 colonies of E. coli were isolated and the 5.88% resulted as Enteropathogenic (EPEC) type and 0.98% as Shiga toxin-producing E. coli (STEC). The urban feral pigeons of Lima in Peru can act as a reservoir or carriers of zoonotic potentially pathogenic enteric agents.

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hibridization whit the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O?H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.

Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10% of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products - for example, adhesins and toxins - and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

Videira 'Syrah' sobre diferentes porta-enxertos em ciclo de inverno no sul de Minas Gerais

O objetivo deste trabalho foi avaliar a influência de porta-enxertos sobre as características agronômicas, ecofisiológicas e qualitativas da videira 'Syrah' manejada por meio da técnica da dupla poda. As videiras foram enxertadas nos porta-enxertos 'SO4', '110 Richter' e '1103 Paulsen', e sustentadas em sistema espaldeira sem irrigação. Foram avaliadas as características ecofisiológicas, de produção e de composição físico-química das bagas maduras em três safras (2007, 2008 e 2010). Os porta-enxertos não exerceram influência sobre o potencial hídrico de base, que apresentou valores próximos a -0,2 MPa, o que indica que não houve restrição hídrica no solo ao final da maturação (junho). Também não houve diferença significativa quanto à produção. O porta-enxerto '1103 Paulsen' conferiu menor vigor, menor taxa fotossintética e melhores resultados de maturação, para as safras com menores precipitações. Os porta-enxertos '110 Richter' e 'SO4' apresentaram maior vigor nas condições meteorológicas de 2010 e as maiores taxas fotossintéticas para o mesmo período. As condições meteorológicas tiveram efeito significativo na maturação tecnológica e fenólica, com os melhores resultados para os anos mais secos. O porta-enxerto '1103 Paulsen' fornece melhor equilíbrio entre vigor e produção, o que aumenta a qualidade da uva.

Influência da altitude na qualidade das uvas 'Chardonnay' e 'Pinot Noir' em Minas Gerais

No Sul e Sudeste brasileiros, o excesso de chuvas durante o período de maturação afeta negativamente a qualidade dos vinhos tintos. Por outro lado, estas regiões possuem potencial para a elaboração de espumantes, uma vez que, para a elaboração desta bebida, a uva é colhida antes de completar o amadurecimento. No Estado de Minas Gerais, as condições de verão chuvoso estão presentes em todas as regiões de potencial vitícola, e a variação de altitude entre elas pode exercer influência na composição das uvas. Desta forma, este estudo buscou avaliar o potencial de maturação de uvas 'Chardonnay' e 'Pinot Noir' destinadas à elaboração de espumantes em dois locais de Minas Gerais: Cordislândia (873m) e Caldas (1.150m). As plantas foram enxertadas sobre 1.103 Paulsen e conduzidas em espaldeira. Foram avaliados os teores de sólidos solúveis totais, acidez total, ácidos málico e tartárico, e pH do mosto, tamanho e massa das bagas, compostos fenólicos nas cascas e sementes, antocianinas na casca e açúcares solúveis nas bagas, em duas safras consecutivas. As bagas apresentaram maior tamanho e massa quando cultivadas em Caldas. As uvas colhidas em Cordislândia apresentaram maior grau de maturação, sendo observados maior pH, maiores teores de glicose e frutose, e quantidade inferior de acidez e fenólicos totais nas sementes. Os maiores teores de ácido málico presentes nas uvas provenientes de Caldas sugerem que esta região pode ser mais indicada à produção de uvas para elaboração de vinhos espumantes.

Composição de bagas de 'Niágara Rosada' e 'Folha-de-Figo' relacionadas ao sistema de condução

RESUMO Este trabalho teve como objetivo avaliar a influência do sistema de condução na qualidade de bagas de uvas 'Niágara Rosada' e 'Folha-de-Figo' cultivadas em Caldas-MG. Foram avaliados a produção por planta, o diâmetro, a massa, o teor de potássio e a temperatura das bagas, pH, teor de sólidos solúveis, açúcares redutores, acidez total e ácidos orgânicos do mosto, compostos fenólicos nas cascas e sementes, e antocianinas. As plantas foram conduzidas em espaldeira, lira, latada ou cordão simples, e os frutos, avaliados nas safras de 2006 e 2007. As videiras de 'Niágara Rosada' cultivadas em latada e 'Folha-de-Figo' em lira apresentaram produção significativamente superior aos demais sistemas de condução. Em todos os casos, as bagas apresentaram temperatura inferior à ambiente, indicando sombreamento completo ou parcial dos cachos. No sistema latada, houve menor acúmulo de sólidos solúveis e açúcares redutores nas duas cultivares. Estes resultados preliminares indicam que a composição das bagas foi pouco influenciada pelo sistema de condução.

Composição físico-química de uvas para vinho fino em ciclos de verão e inverno

Este trabalho teve como objetivo avaliar o potencial de maturação das cultivares Pinot Noir, Tempranillo, Merlot, Cabernet Sauvignon, Syrah, Chardonnay e Sauvignon Blanc submetidas ao regime de dupla poda, em Cordislândia, região cafeeira do sul de Minas Gerais. As plantas foram submetidas a dois ciclos de produção, um de primavera-verão, compreendido entre agosto e janeiro, e outro ciclo de outono-inverno, entre janeiro e julho. Como parâmetros de qualidade, foram avaliados os diâmetros transversal e longitudinal da baga, acidez, ácidos tartárico e málico, pH, sólidos solúveis, antocianinas, fenólicos totais e os teores de glicose, frutose e sacarose. Todas as variedades apresentaram maiores teores de pH, sólidos solúveis, açúcares, antocianinas e fenólicos totais, e redução nos diâmetros transversal e longitudinal na safra de inverno. A cultivar Syrah destacou-se das demais no conteúdo de antocianinas e fenólicos totais tanto no verão quanto no inverno, entretanto apresentou o menor conteúdo de açúcares. A alteração do ciclo de produção da videira através da técnica da dupla poda para colheita, no período de inverno, na região cafeeira de Minas Gerais, favorece a maturação dos frutos e melhora consideravelmente a qualidade das uvas para vinificação.

Analysis of Escherichia coli isolated from bovine mastitic milk

Mastitis has been recognized for some time as the most costly disease in dairy herds. From February to November 2004, 670 samples of bovine mastitic milk from which 231 Escherichia coli strains were isolated, were collected from two Brazilian states. The strains were screened for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twenty (8.6%) strains were detected by PCR to harbor the Shiga toxin genes (8 the stx 1 gene, 12 the stx 2 gene and none both of them). Two (0.8%) of the Escherichia coli strains studied were eae positive non Shiga toxin-producing. The strains were also examined for resistance to 12 antimicrobial agents. The predominantly observed resistance was to tetracycline (92.2%), streptomycin (90.4%), nalidixic acid (88.3%), amikacin (86.5%) and cephalothin (84.8%). Multidrug resistance was found among 152 isolates (65.8%).