Virulence exaltation of Clostridium perfringens strains from bovines

Ten out of eighty-nine strains biochemically identified as Clostridium perfringens, isolated from bovine organs, were selected by their different results showed in toxigenicity test on mice. Those and the standard strains, ATCC types A, B, C, and D, had their virulence exalted through serial intramuscular inoculation into guinea pigs. Results showed that, for toxigenic strains (6), one or two passages were enough to cause exaltation, while for the atoxigenic (4), five or six inoculations were needed. Esterase electrophoresis of standard and isolated strains, with and without exaltation, was performed. Electrophoresis analysis permits the following conclusions: strains that do not show any clinical symptoms in mice, when exalted demonstrate decreased esterase activity; on the contrary, it is increased when correlated with animal symptoms.

Esterase electrophoresis of Clostridium perfringens bovine strains

Eighty-nine of 144 isolates of Clostridium perfringens abtained from 187 samples of 71 bovine in several Brazilian states were submitted to esterase electrophoresis for typing. Mobilites electrophoresis, as parameter, were settled down by isolates from ATCC pattern of types A, B, C, and D. Of the 89 isolates, 43 (48.3 per cent) were characterized as electrophoretic type A, 20 (22.5 per cent) as D, 18 (20.2 per cent) as C, and 3 (3.4 per cent) as B. Five (5.6 per cent) isolates did not identity with any type. Similarly esterase electrophoresis enabled the typing of 94.4 per cent of the isolates, demonstrating to be an appropriate method for animal sample analyses.

Toxigenicity characterization of Clostridium perfringens from bovine isolates

Clinical samples from 71 bovine from different Brazilian states were processed for the analysis of anaerobe organisms with emphasis on the isolation and characterization of Clostridium spp. From these eighty-nine Clostridium perfringens strains were recovered: 32 from liver, 19 from intestinal contents, 14 from kidney, 6 from rumen, 5 from nervous system, 4 from bone marrow, 2 from udder tract, blood, spleen and lung, and one from muscle. Four reference Clostridium perfringens types A, B, C, and D were used as controls in this study. All isolates were cultivated in appropriate media, and after centrifugation the supernatant and sediment were separated. From pure supernatant post exopolysaccharide (EPS) extraction, mouse toxigenicity tests were performed, determining protein and protein plus carbohydrate, respectively. ELISA was performed from sediments. The results showed that 51 (57.3 per cent) of the isolates were toxigenic to mice when inoculated by intraperitoneal route; bacteria from different organs had variable patterns of toxigenicity. Toxigenicity of EPS extracts was only expressed when protein concentration was 0.04 mg/mL and between 0.31 and 0.5 mg/mL for carbohydrate. Isolates were characterized as toxigenic when showing optimum protein and carbohydrate concentrations.

Removal of Escherichia coli O157:H7 from surface tissues of beef carcasses inoculated with wet and dry manure.

Beef tissues were contaminated with wet and dry manure. The manure was previously inoculated with Escherichia coli O157:H7 GFP, genetically modified with a plasmid encoding a protein that fluoresces green when exposed to long-wave ultraviolet light. After incubation at 37 degrees C for 5 days, the wet manure was spread on the surface of beef tissues at an average E. coli O157:H7 GFP level of 6.62 log CFU/cm2. Dry manure was obtained by subjecting wet manure to natural drying (simulating dry manure adhering to the hides of cattle) and was also applied to the surfaces of beef tissues. The degree of removal of E. coli O157:H7 GFP by washing was compared to the removal of cells of the same strain that had been inoculated as a suspension. The E. coli O157:H7 mixed into feces of cattle adhered more strongly to meat surfaces than that applied as a suspension, complicating the removal by conventional washing procedures. The fate of the bacterium mixed into wet or dry manure was evaluated. An initial decrease of the inoculated population was observed; this was probably an effect of the changed environment represented by the manure. After adaptation, the inoculated bacteria grew in the wet manure; a maximum population was reached in 5 days at 37 degrees C; levels declined with drying. The use of the GFP marker was of great value, since it allowed enumeration of E. coli O157:H7 in the presence of the natural flora of manure.

Survival of Vibrio cholerae 01 strains in shrimp subjected to freezing and boiling.

This research was undertaken to assess the resistance of Vibrio cholerae 01 strains inoculated into white shrimp, Penaeus schimitti, to heating and freezing treatments. Shrimp samples with and without carapace were obtained from Sao Luis, Brazil. Microbial analysis revealed the presence of marine vibrios including Vibrio alginolyticus, Vibrio parahaemolyticus, and other vibrios and aerobic gram-negative and gram-positive bacteria that grew on selective medium, thiosulfate-citrate-bile salt-sucrose agar. Samples with and without carapaces were heated before inoculating with cells of V. cholerae and then one-half of the samples was stored frozen at -200 degrees C and the other one-half was heated to boiling temperatures. Viable cells of the test organism were recovered from samples without carapaces, stored under frozen conditions, after 36 days. In contrast, no living cells were recovered after 26 days from samples with carapaces. Boiling temperatures were very damaging to V. cholerae 01 in shrimp samples with and without carapaces. Total destruction of the cells occurred within 1 to 2 min of exposure to heating.

Decontaminating beef for Escherichia coli O157:H7.

Beef lean, fat, and connective tissues were inoculated with Escherichia coli O157:H7 before and after a prewashing procedure to compare the efficacy of prewashing and no prewashing on bacterial adherence and, consequently, on the removal of bacteria from the inoculated surfaces. Prewashing consisted of spraying tissues with tap water before inoculation. Final washing with disinfectant solutions compared the efficacy of several chemicals for the removal or destruction of E. coli O157:H7. The results showed that prewashing was very effective in reducing the numbers of bacterial cells on beef tissues, mainly lean tissue, in the control samples which received final washing with water. An opposite effect of prewashing was observed when disinfectant solutions were used for final washing; this may be due to dilution by water carried on the tissues after prewashing. The efficacy of chemicals was dependent on the type of exposed tissue. Hydrogen peroxide (3%) was more efficient in the removal of E. coli O157:H7 from connective tissues, with reductions greater than 4 log CFU/cm2, compared to a normally washed control (P < 0.01). Chlorhexidine (0.1%) was very efficient on fat and lean tissues, causing reductions over 5 log CFU/cm2 on not prewashed fat and lean tissues, compared to the control (P < 0.01). Acetic acid (5%) was the least effective, decreasing the number of CFU by under 1 log/cm2 as compared to the control; and no statistically significant difference was found among tissues, even though the removal of bacteria seemed less in lean tissue compared to fat or connective tissues.

[Effect of pluviometric precipitation on the physical, chemical, and health/hygiene aspects of water from three public water supplies]. / Influência da precipitação pluviométrica nas características físicas, química e higiênico-sanitária da água de trés mananciais de abastecimento público.

Aiming to know the influence of the pluviometric precipitation over the physical chemical and bacteriological characteristics of the water coming from three sources of public supply, and to verify the probable origin of faecal contamination, tests of total coliforms, faecal coliforms, faecal streptococci and mesophilic microorganisms, as well as the determination of pH, temperature and turbidity of 156 water samples were done. The samples were taken from a stream, a drain and an artesian well, located in the city of Jaboticabal, SP, Brazil. The results obtained showed that the occurrence of pluviometric precipitation determined a significant increase of the number of total coliforms in the water samples from the stream. The use of the ratio CF/EF, indicate the probable origin of the faecal contamination only in the samples of the water coming from the stream, which were defined as being of human origin. In relation to the physical-chemical composition it was noticed that the influence of the pluviometric precipitation over the turbidity appeared to be higher and significant in the samples of water of the stream.

[Salmonellas and fecal coliforms in drinking water for animals]. / Salmonelas e coliformes fecais em águas de bebida para animais.

Sixty small farms of Botucatu County, S. Paulo, Brazil, given over to cattle breeding were studied. These farms account for 15% of the total of 402 farms that exist in the county. The sample was drawn by simple probabilistic technique. There were found to be one hundred and thirteen drinking places located on the farms. Samples of water were taken from these drinking places and examined for bacteria of the genus Salmonella, for the determination of Most Probable Number (MNP) of fecal coliform bacteria as well as determination of the water's pH. Water temperature was measured before collection. Samples from 15 drinking places (13.3%) were positive for Salmonella. The drinking places belonged to 12 of the sixty farms studied (20%). The following serotypes were identified: S. dublin, S. newport, S. madelia, S. IV 43:g,z57:-, S. saphra, S. glostrup, S. IV ochsenzool; S. I9,12:i:- and two new serotypes S. IV 41:z52:- and S. IV 50:d:-. Of the 113 samples studied 14 (12.4%) presented MPN/100ml of fecal coliforms above 4,000. There was no relationship between MPN/100ml of fecal coliforms above 4,000 and positivity for Salmonella. Highest positivity both for Salmonella and MPN/100mL of fecal coliforms over 4,000 occurred at temperatures above 18 degrees C. As regards pH, in both situations the highest positivity occurred between 6.0 and 7.0.

[Changes in physical, chemical, and microbiological characteristics of brines applied in the salting of mozzarella cheese during the period of utilization]. / Variação das características físico-químicas e microbiológicas das salmouras empregadas na salga de queijos tipo mussarela durante o período de sua utilização.

Variation of the physical, chemical and microbiological characteristics of brines during their utilization for salting mozzarella cheese. Forty brine samples used for submersion salting of mozzarella cheese in a dairy industry in the State of S. Paulo, Brazil, were analysed for the purpose of discovering the variation in the physical, chemical and microbiological characteristics observed over their period of utilization. The mean values preparation up to the 21st day of utilization of pH, sodium chloride and protein concentration varied from 7.21 to 5.76, from 27.1 to 24.5 and from zero to 0.126 mg/ml, respectively over the period from their. The mean values of the mesophilic microorganism counts and of the total and fecal coliforms MPN varied from 5.8 x 10 CFU/ml to 6.9 x 10(4) CFU/ml, from zero to 1.6 x 10(5)/100 ml and from zero to 1.1 x 10(5)/100 ml, respectively. Moreover, the mean values of mould and yeast and Staphylococcus positive coagulase counts varied from 0.4 x 10 CFU/ml to 2.0 x 10(3) CFU/ml and from zero to 1.3 x 10 CFU/ml, respectively. The results obtained suggest that the hygienic conditions during the preparation and the utilization of the brine were not satisfactory so that they may represent an important source of contamination for the cheeses. The quality of the product may be harmed, as a result of this fact, in such a way as to represent a potential hazard for the health of the consuming population.

Staphylococcus aureus enterotoxigenico em doces cremosos vendidos em padarias e confeitarias do municipio de Sao Paulo, Brasil. / Enterotoxic Staphylococcus aureus in creamy pastries sold in bakery pastry shops in the municipality of Sao Paulo, Brasil

A partir de 100 amostras de doces cremosos coletados em 40 padarias e confeitarias da cidade de Sao Paulo (Brasil), foi realizada a contagem de S. aureus por grama de alimento.As cepas isoladas, apos terem sido identificadas morfologica e bioquimicamente, foram submetidas a provas de fagotipagem e a verificacao da capacidade produtora de enterotoxina. Das 100 amostras de doces examinadas, 38,0% foram positivas para S. aureus e originarias de 21 (52,5%) dos 40 estabelecimentos visitados. Do total de doces analisados, 7% foram positivos para cepas enterotoxigenicas sendo 5% do tipo C, 1% do B e 1% do D das 76 cepas isoladas de S. aureus, 39(51,5%) revelaram-se nao fagotipaveis. Das fagotipaveis houve predominancia das que foram lisadas por fagos do grupo I isoladamente (21,2%) ou em associacao com fagos de outros grupos (35,5%) Cepas nao tipaveis de S. aureus estavam presentes em 76,2% dos estabelecimentos em que houve amostras positivas para esta bacteria

Alguns grupos de microrganismos em manteigas vendidas no municipio de Sao Paulo. / Some groups of microorganisms in butters sold in the municipality of Sao Paulo

Foram colhidas 105 amostras de manteiga de 5 marcas diferentes vendidas em supermercados da cidade de Sao Paulo (Brasil) com a finalidade de verificar as condicoes microbiologicas de manteigas e compara-las com os padroes recomendados. Semanalmente foi colhida uma amostra de cada marca, durante 21 semanas. A partir da parte aquosa de cada amostra, foram realizadas as contagens de bacterias mesofilas e psicrofilas (em agar padrao e agar gelisato), coliformes, proteoliticas e de bolores e leveduras e os resultados comparados com alguns parametros propostos por varios pesquisadores. Os valores obtidos nas contagens dos varios grupos de microrganismos estudados, em muitos casos podem ser considerados altos, os quais podem ser resultado do processamento e/ou conservacao, realizados em condicoes nao satisfatorias

Bacterias indicadoras de poluicao fecal em aguas de irrigacao de hortas que abastecem o municipio de Natal - Estado do Rio Grande do Norte (Brasil). / Bacterial indicators of fecal pollution in irrigation water of vegetable gardens which supply the municipality of Natal State of Rio Grande do Norte (Brazil)

Foram coletadas 50 amostras de agua usadas na irrigacao de hortalicas em 10 hortas, situadas no municipio de Extremoz e de Sao Goncalo do Amarante, RN (Brasil). A maior parte da producao destas hortas e destinada ao abastecimento do municipio de Natal. Todas as amostras de agua analisadas mostraram-se com numeros elevados de bacterias coliformes totais e fecais e de estreptococos fecais. Em todas as hortas, as aguas utilizadas na irrigacao de hortalicas revelaram-se com poluicao fecal e os valores dos NMP/100 ml, tanto de coliformes totais quanto de coliformes fecais ultrapassaram de muito os limites tolerados pela legislacao brasileira vigente