ABSTRACT
UNLABELLED: Escherichia coli is part of the normal microflora of the intestines of mammals. However, among the enteric pathogens, it is one of the leading causes of intestinal diseases, especially Shiga toxigenic E. coli, which can cause diarrhoea, haemorrhagic colitis and complications like haemolytic uraemic syndrome and thrombotic thrombocytopaenic purpura. Escherichia coli is considered a serious public health problem. Water and fish samples were subjected to biochemical tests to confirm the presence of E. coli and by PCR to verify the presence of pathogenic strains (O157, enteropathogenic and shiga toxigenic) in water and fish (skin, gastrointestinal tract and muscles) from pay-to-fish ponds located in the Córrego Rico watershed in the northeastern region of the state of São Paulo, Brazil. Of the 115 E. coli isolates from fish or water, five (4·34%) contained eae and stx2 genes, one had only the eae gene and two had the stx1 gene. An isolate containing the stx2 gene was also found in the water sample. In addition, eight isolates (6·95%) from the fish gastrointestinal tract contained rfbEO157:H7 (O157 gene), and three (2·61%) contained stx2 and eae genes, demonstrating the potential risk to the environment and public health. The results provide useful basic information for the proper management of these environments and animals in order to prevent faecal pollution, reducing health risks to the Brazilian population. SIGNIFICANCE AND IMPACT OF THE STUDY: Pay-to-fish ponds are a common commercial activity in Brazil. Samples of water and Oreochromis niloticus were examined by PCR to detect the presence of pathogenic strains of Escherichia coli (O157, enteropathogenic and shiga toxigenic). Several pathogenic strains were detected in this study, providing useful epidemiological information for the proper management of these environments and animals in order to prevent faecal pollution, reducing health risks to the Brazilian population.
Subject(s)
Cichlids/microbiology , Enteropathogenic Escherichia coli/isolation & purification , Ponds/microbiology , Shiga-Toxigenic Escherichia coli/isolation & purification , Adhesins, Bacterial/genetics , Animals , Brazil , Diarrhea , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Infections/diagnosis , Escherichia coli Proteins/genetics , Feces , Polymerase Chain Reaction , Shiga Toxin/genetics , Shiga-Toxigenic Escherichia coli/pathogenicity , Water MicrobiologyABSTRACT
Analyses of 16S rDNA genes were used to identify the microbiota isolated from the mucus of the zoanthid Palythoa caribaeorum at Porto de Galinhas on the coast of Pernambuco State, Brazil. This study is important as the first report of this association, because of the potential biotechnological applications of the bacterium Alcanivorax dieselolei, and as evidence for the presence of a hydrocarbon degrading bacterium in a reef ecosystem such as Porto de Galinhas.
Subject(s)
Alcanivoraceae/genetics , Anthozoa/microbiology , Mucus/microbiology , Alcanivoraceae/physiology , Animals , DNA, Bacterial/genetics , Polymerase Chain Reaction , RNA, Ribosomal, 16S/geneticsABSTRACT
Analyses of 16S rDNA genes were used to identify the microbiota isolated from the mucus of the zoanthid Palythoa caribaeorum at Porto de Galinhas on the coast of Pernambuco State, Brazil. This study is important as the first report of this association, because of the potential biotechnological applications of the bacterium Alcanivorax dieselolei, and as evidence for the presence of a hydrocarbon degrading bacterium in a reef ecosystem such as Porto de Galinhas.(AU)
Análises dos genes 16S rDNA foram empregadas para identificar a microbiota isolada do muco do zoantídeo Palythoa caribaeorum de Porto de Galinhas, litoral do estado de Pernambuco, Brasil. Este estudo é importante pelo ineditismo dessa associação, pelas relevantes aplicações biotecnológicas da bactéria Alcanivorax dieselolei e pela indicação da presença de uma bactéria degradadora de hidrocarbonetos em um ecossistema recifal como o de Porto de Galinhas.(AU)
Subject(s)
Animals , Alcanivoraceae/genetics , Anthozoa/microbiology , Mucus/microbiology , Alcanivoraceae/physiology , DNA, Bacterial/genetics , Polymerase Chain Reaction , RNA, Ribosomal, 16S/geneticsABSTRACT
Analyses of 16S rDNA genes were used to identify the microbiota isolated from the mucus of the zoanthid Palythoa caribaeorum at Porto de Galinhas on the coast of Pernambuco State, Brazil. This study is important as the first report of this association, because of the potential biotechnological applications of the bacterium Alcanivorax dieselolei, and as evidence for the presence of a hydrocarbon degrading bacterium in a reef ecosystem such as Porto de Galinhas.
Análises dos genes 16S rDNA foram empregadas para identificar a microbiota isolada do muco do zoantídeo Palythoa caribaeorum de Porto de Galinhas, litoral do estado de Pernambuco, Brasil. Este estudo é importante pelo ineditismo dessa associação, pelas relevantes aplicações biotecnológicas da bactéria Alcanivorax dieselolei e pela indicação da presença de uma bactéria degradadora de hidrocarbonetos em um ecossistema recifal como o de Porto de Galinhas.
Subject(s)
Animals , Alcanivoraceae/genetics , Anthozoa/microbiology , Mucus/microbiology , Alcanivoraceae/physiology , DNA, Bacterial/genetics , /genetics , Polymerase Chain ReactionABSTRACT
Livestock manure may contain pathogenic microorganisms which pose a risk to the health of animal or humans if the manure is not adequately treated or disposed of. To determine the fate of Shiga toxigenic Escherichia coli (STEC) non O157 in composted manure from naturally colonized sheep, fresh manure was obtained from animals carrying bacterial cells with stx1/ stx2 genes. Two composting systems were used, aerated and non-aerated, and the experiments were done in Dracena city, São Paulo State. Every week, for seven weeks, one manure sample from six different points in both systems was collected and cultured to determine the presence of E. coli, the presence of the virulence genes in the cells, and also the susceptibility to 10 antimicrobial drugs. The temperature was verified at each sampling. STEC non-O157 survived for 49 days in both composting systems. E. coli non-STEC showing a high degree of antibiotic resistance was recovered all long the composting period. No relationship was established between the presence of virulence genes and antibiotic resistance. The presence of virulence genes and multiple antibiotic resistances in E. coli implicates a potential risk for these genes spread in the human food chain, which is a reason for concern.(AU)
Esterco de animais de criação pode conter microrganismos patogênicos, o que representa um risco para a saúde animal e a humana se o esterco não for adequadamente tratado ou descartado. Determinou-se o tempo necessário para a eliminação de Escherichia coli Shiga toxigenica (STEC) não O157 em esterco ovino composto, obtido de fezes frescas de ovelhas naturalmente colonizadas com cepas STEC não O157 que apresentavam os genes stx1/ stx2. Foram utilizados dois sistemas de compostagem, aerado e não aerado, em experimentos realizados na cidade de Dracena, estado de São Paulo. Todas as semanas, durante sete semanas, uma amostra de compostagem proveniente de seis pontos diferentes na leira, nos dois sistemas, foi coletada e semeada para a determinação da presença de E. coli, da presença de genes de virulência nas células, bem como da sensibilidade dessas células a 10 drogas antimicrobianas. Em cada amostragem, a temperatura da leira foi analisada. Células de STEC não O157 sobreviveram por 49 dias nos dois sistemas de compostagem. E. coli não STEC com um alto grau de resistência a antibióticos foi recuperada ao longo de todo o período de compostagem. Não foi possível estabelecer relação entre a presença de genes de virulência e a resistência a antibióticos. A presença de genes de virulência e a resistência a múltiplos antibióticos em E. coli representam um risco potencial para o espalhamento desses genes na cadeia alimentar humana, o que é motivo de grande preocupação.(AU)
Subject(s)
Animals , Bacterial Shedding/physiology , Shiga-Toxigenic Escherichia coli , Sheep , Manure/analysis , Composting/analysis , NoxaeABSTRACT
Livestock manure may contain pathogenic microorganisms which pose a risk to the health of animal or humans if the manure is not adequately treated or disposed of. To determine the fate of Shiga toxigenic Escherichia coli (STEC) non O157 in composted manure from naturally colonized sheep, fresh manure was obtained from animals carrying bacterial cells with stx1/ stx2 genes. Two composting systems were used, aerated and non-aerated, and the experiments were done in Dracena city, São Paulo State. Every week, for seven weeks, one manure sample from six different points in both systems was collected and cultured to determine the presence of E. coli, the presence of the virulence genes in the cells, and also the susceptibility to 10 antimicrobial drugs. The temperature was verified at each sampling. STEC non-O157 survived for 49 days in both composting systems. E. coli non-STEC showing a high degree of antibiotic resistance was recovered all long the composting period. No relationship was established between the presence of virulence genes and antibiotic resistance. The presence of virulence genes and multiple antibiotic resistances in E. coli implicates a potential risk for these genes spread in the human food chain, which is a reason for concern...
Esterco de animais de criação pode conter microrganismos patogênicos, o que representa um risco para a saúde animal e a humana se o esterco não for adequadamente tratado ou descartado. Determinou-se o tempo necessário para a eliminação de Escherichia coli Shiga toxigenica (STEC) não O157 em esterco ovino composto, obtido de fezes frescas de ovelhas naturalmente colonizadas com cepas STEC não O157 que apresentavam os genes stx1/ stx2. Foram utilizados dois sistemas de compostagem, aerado e não aerado, em experimentos realizados na cidade de Dracena, estado de São Paulo. Todas as semanas, durante sete semanas, uma amostra de compostagem proveniente de seis pontos diferentes na leira, nos dois sistemas, foi coletada e semeada para a determinação da presença de E. coli, da presença de genes de virulência nas células, bem como da sensibilidade dessas células a 10 drogas antimicrobianas. Em cada amostragem, a temperatura da leira foi analisada. Células de STEC não O157 sobreviveram por 49 dias nos dois sistemas de compostagem. E. coli não STEC com um alto grau de resistência a antibióticos foi recuperada ao longo de todo o período de compostagem. Não foi possível estabelecer relação entre a presença de genes de virulência e a resistência a antibióticos. A presença de genes de virulência e a resistência a múltiplos antibióticos em E. coli representam um risco potencial para o espalhamento desses genes na cadeia alimentar humana, o que é motivo de grande preocupação...
Subject(s)
Animals , Bacterial Shedding/physiology , Shiga-Toxigenic Escherichia coli , Manure/analysis , Composting/analysis , Noxae , SheepABSTRACT
The polyphagous pests belonging to the genus Spodoptera are considered to be among the most important causes of damage and are widely distributed throughout the Americas'. Due to the extensive use of genetically modified plants containing Bacillus thuringiensis genes that code for insecticidal proteins, resistant insects may arise. To prevent the development of resistance, pyramided plants, which express multiple insecticidal proteins that act through distinct mode of actions, can be used. This study analyzed the mechanisms of action for the proteins Cry1Ia10 and Vip3Aa on neonatal Spodoptera frugiperda, Spodoptera albula, Spodoptera eridania and Spodoptera cosmioides larvae. The interactions of these toxins with receptors on the intestinal epithelial membrane were also analyzed by binding biotinylated toxins to brush border membrane vesicles (BBMVs) from the intestines of these insects. A putative receptor of approximately 65 kDa was found by ligand blotting in all of these species. In vitro competition assays using biotinylated proteins have indicated that Vip3Aa and Cry1Ia10 do not compete for the same receptor for S. frugiperda, S. albula and S. cosmioides and that Vip3Aa was more efficient than Cry1Ia10 when tested individually, by bioassays. A synergistic effect of the toxins in S. frugiperda, S. albula and S. cosmioides was observed when they were combined. However, in S. eridania, Cry1Ia10 and Vip3Aa might compete for the same receptor and through bioassays Cry1Ia10 was more efficient than Vip3Aa and showed an antagonistic effect when the proteins were combined. These results suggest that using these genes to develop pyramided plants may not prove effective in preventing the development of resistance in S. eridiana.
Subject(s)
Bacterial Proteins/toxicity , Endotoxins/toxicity , Hemolysin Proteins/toxicity , Host-Pathogen Interactions , Insecticides/toxicity , Pest Control, Biological , Spodoptera/drug effects , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Cloning, Organism , Endotoxins/metabolism , Hemolysin Proteins/metabolism , Insect Proteins/metabolism , Insecticides/metabolism , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Larva , Life Cycle Stages , Receptors, Cell Surface/metabolism , Spodoptera/metabolismABSTRACT
A Escherichia coli O157: H7 é uma importante cepa associada a surtos graves de enfermidade em seres humanos, a maioria deles derivada do consumo de carne crua ou mal cozida. É provável que o gado atue como um importante reservatório, sugerindo-se a possibilidade de que a gestão da dieta no confinamento possa influenciar o aparecimento de cepas Shigatoxigênicas. Este estudo teve como objetivo verificar a qualidade microbiológica das carcaças e a ocorrência de E. coli O157: H7, por meio dos resultados obtidos por métodos indicadores (contagem total de microrganismos viáveis, contagem de Coliformes e de E. coli) e por um método automatizado de PCR para detecção de E. coli O157: H7. Foram colhidas amostras de retalhos de carne (carne industrial) e de carcaças de bovinos terminados em pastagem ou em confinamento, permitindo o fornecimento de subsídios necessários para a Análise de Perigos e Pontos Críticos de Controle (HACCP). Desses mesmos animais foram colhidas, também, amostras de swab retal para a detecção experimental de E. coli O157: H7 nas fezes. Um total de 100 swabs retais, 100 amostras de carcaças quentes, além de outras 323 amostras de aparas de carne (retalhos da desossa), foram analisados. Com exceção de uma amostra de retalhos de carne (0,31%), todas as demais, de fezes e de carcaças, foram negativas para a presença de E. coli O157: H7. Não houve diferenças significativas entre os tipos de terminação utilizada para o gado. Os resultados dos métodos indicadores foram considerados aceitáveis em 91%, 85% e 93% das amostras testadas, respectivamente, para a CTV, contagem de Coliformes e de E. coli de carcaças, dando suporte e em acordo com a baixa ocorrência da cepa O157: H7(AU)
Escherichia coli O157:H7, an important bacillus strain associated with serious gastroenteritis in humans, is more frequently derived from the consumption of raw or poorly cooked beef. Cattle are important reservoirs suggesting the possibility that feedlot diet management influences the emergence of Shiga-toxigenic strains. This study evaluates the microbiological quality of carcasses and the occurrence of E. coli O157:H7 using the results from general indicator methods (total viable count, coliform rate and E. coli counts) and by an automated PCR method for the detection of E. coli O157:H7. Samples were collected from (industrially processed) meat trimmings and from carcasses of cattle finished on pasture or in feedlots so that sufficient data for the Hazard Analysis and Critical Control Points (HACCP) could be obtained. Samples of rectal swab for experimental detection of E. coli O157:H7 were also collected. One hundred rectal swabs, 100 samples retrieved from warm carcasses and 323 samples of meat trimmings were analyzed. With the exception of one sample of meat trim (0.31%), all the other samples from excreta and carcasses were negative for the O157:H7 E. coli strain. There were no significant differences between the methods used for cattle finishing. Indicator methods results were considered acceptable in 91%, 85% and 93% of tested samples of carcasses respectively for TVC, coliform and E. coli counts. These results agree with statistical data showing the low occurrence of O157:H7 strain(AU)
Subject(s)
Animals , Cattle , Escherichia coli O157/classification , Microbiology/classification , Microbiology/trends , Polymerase Chain Reaction , Polymerase Chain Reaction/veterinaryABSTRACT
A Escherichia coli O157: H7 é uma importante cepa associada a surtos graves de enfermidade em seres humanos, a maioria deles derivada do consumo de carne crua ou mal cozida. É provável que o gado atue como um importante reservatório, sugerindo-se a possibilidade de que a gestão da dieta no confinamento possa influenciar o aparecimento de cepas Shigatoxigênicas. Este estudo teve como objetivo verificar a qualidade microbiológica das carcaças e a ocorrência de E. coli O157: H7, por meio dos resultados obtidos por métodos indicadores (contagem total de microrganismos viáveis, contagem de Coliformes e de E. coli) e por um método automatizado de PCR para detecção de E. coli O157: H7. Foram colhidas amostras de retalhos de carne (carne industrial) e de carcaças de bovinos terminados em pastagem ou em confinamento, permitindo o fornecimento de subsídios necessários para a Análise de Perigos e Pontos Críticos de Controle (HACCP). Desses mesmos animais foram colhidas, também, amostras de swab retal para a detecção experimental de E. coli O157: H7 nas fezes. Um total de 100 swabs retais, 100 amostras de carcaças quentes, além de outras 323 amostras de aparas de carne (retalhos da desossa), foram analisados. Com exceção de uma amostra de retalhos de carne (0,31%), todas as demais, de fezes e de carcaças, foram negativas para a presença de E. coli O157: H7. Não houve diferenças significativas entre os tipos de terminação utilizada para o gado. Os resultados dos métodos indicadores foram considerados aceitáveis em 91%, 85% e 93% das amostras testadas, respectivamente, para a CTV, contagem de Coliformes e de E. coli de carcaças, dando suporte e em acordo com a baixa ocorrência da cepa O157: H7
Escherichia coli O157:H7, an important bacillus strain associated with serious gastroenteritis in humans, is more frequently derived from the consumption of raw or poorly cooked beef. Cattle are important reservoirs suggesting the possibility that feedlot diet management influences the emergence of Shiga-toxigenic strains. This study evaluates the microbiological quality of carcasses and the occurrence of E. coli O157:H7 using the results from general indicator methods (total viable count, coliform rate and E. coli counts) and by an automated PCR method for the detection of E. coli O157:H7. Samples were collected from (industrially processed) meat trimmings and from carcasses of cattle finished on pasture or in feedlots so that sufficient data for the Hazard Analysis and Critical Control Points (HACCP) could be obtained. Samples of rectal swab for experimental detection of E. coli O157:H7 were also collected. One hundred rectal swabs, 100 samples retrieved from warm carcasses and 323 samples of meat trimmings were analyzed. With the exception of one sample of meat trim (0.31%), all the other samples from excreta and carcasses were negative for the O157:H7 E. coli strain. There were no significant differences between the methods used for cattle finishing. Indicator methods results were considered acceptable in 91%, 85% and 93% of tested samples of carcasses respectively for TVC, coliform and E. coli counts. These results agree with statistical data showing the low occurrence of O157:H7 strain
Subject(s)
Animals , Cattle , /classification , Microbiology/classification , Microbiology/trends , Polymerase Chain Reaction , Polymerase Chain Reaction/veterinaryABSTRACT
The entomophatogenic bacterium Bacillus thuringiensis produces crystal proteins, named Cry proteins which are encoded by the cry genes. This bacterium is used on biological control of important economical pests, as well as in the control of disease´s vectors, such as Aedes aegypti, a mosquito that transmits the dengue viruses. Isolates of this bacterium can be characterized by the content of cry genes and this prediction helps target different insect orders. In this research, we isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela (SP, Brazil), a place where simulids are already biologically controlled by B. thuringiensis, to find bacterial isolates that were capable of controlling A. aegypti. The 16S ribosomal subunit genes of the selected isolates were sequenced, and the isolates were molecularly characterized based on their Dipteran-specific cry gene contents. Eight of the 76 isolates (10.52%) contained the cry4Aa, cry4Ba or cry10Aa genes, these isolates were carried out against A. aegypti larvae on bioassay. The presence or absence of specific cry genes was associated with the observed average larval mortalities. From the 76 isolates, seven (9.2%) were potentially able to control A. aegypti larvae. Therefore these are promising isolates for the biological control of A. aegypti larvae.
Subject(s)
Aedes/drug effects , Bacillus thuringiensis/chemistry , Bacterial Proteins/pharmacology , Endotoxins/pharmacology , Hemolysin Proteins/pharmacology , Soil Microbiology , Animals , Bacillus thuringiensis/isolation & purification , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Biological Assay , Endotoxins/genetics , Endotoxins/isolation & purification , Hemolysin Proteins/genetics , Hemolysin Proteins/isolation & purification , Larva/drug effects , Pest Control, Biological/methodsABSTRACT
The efficiency of microbiological culture and polymerase chain reaction (PCR) for detection of Salmonella Typhimurium is compared in fecal samples of Holstein calves experimentally infected with 10(9) CFU of Salmonella Typhimurium. Seventy-two fecal samples were analyzed by microbiological culture and PCR associated with selenite cystine (SC) and Muller-Kauffmann tethrationate (TMK) selective enrichment broths. Regardless of the selective enrichment broth, the microbiological culture was significantly better than PCR for detection of positive samples of Salmonella Typhimurium. The selective enrichment broths SC and TMK had no effect on the efficiency of the microbiological culture. The SC broth was the best option as selective enrichment associated to PCR.
Subject(s)
Animals , Male , Cattle , Bacteria/isolation & purification , Cattle , Polymerase Chain Reaction/veterinary , Diagnostic Techniques and Procedures/veterinaryABSTRACT
The efficiency of microbiological culture and polymerase chain reaction (PCR) for detection of Salmonella Typhimurium is compared in fecal samples of Holstein calves experimentally infected with 10(9) CFU of Salmonella Typhimurium. Seventy-two fecal samples were analyzed by microbiological culture and PCR associated with selenite cystine (SC) and Muller-Kauffmann tethrationate (TMK) selective enrichment broths. Regardless of the selective enrichment broth, the microbiological culture was significantly better than PCR for detection of positive samples of Salmonella Typhimurium. The selective enrichment broths SC and TMK had no effect on the efficiency of the microbiological culture. The SC broth was the best option as selective enrichment associated to PCR.(AU)
Subject(s)
Animals , Male , Cattle , Cattle , Polymerase Chain Reaction/veterinary , Bacteria/isolation & purification , Diagnostic Techniques and Procedures/veterinaryABSTRACT
O objetivo deste trabalho foi analisar a sensibilidade antimicrobiana in vitro de cepas de Staphylococcus aureusisoladas de tetos de vacas e mãos de retireiros, além de verificar o polimorfismo entre elas pela técnica de PCR-RAPD. Os testes foram realizados pela técnica de difusão em discos e, após a extração do material genético foram desenvolvidas as técnicas de PCR e RAPD, usando para isso 40 iniciadores diferentes. A análise do polimorfismo foi realizada empregando-se o programa de taxonomia numérica NTSYS. As sensibilidades dos antimicrobianos nas cepas obtidas de tetos de vacas foram 4% para a penicilina, 88% para a tetraciclina, 92% para a gentamicina, 96% para a vancomicina e 100% ao cloranfenicol. Para as cepas provenientes das mãos de retireiros, os resultados de sensibilidade foram zero para a penicilina, 70% para a tetraciclina e 90% para a vancomicina e 100% para os antimicrobianos gentamicina e cloranfenicol. A realização do E-teste indicou uma concentração inibitória mínima (CIM) maior que 256 mg/mL para as cepas resistentes ao antimicrobiano vancomicina. Os estudos permitiram detectar a resistência dos S. aureus mediante o uso dos antimicrobianos testados e determinar a diversidade genética entre as cepas de estafilococos devido à presença de muitas bandas polimórficas encontradas em todos os iniciadores.
The aim of this study was to analyze in vitro antimicrobial susceptibility of Staphylococcus aureus strains isolated from teats of cow udders and milking workers' hands as well as to verify polymorphism among them by using RAPD-PCR technique. Tests were conducted by disk diffusion technique and after the collection of the genetic material PCR and RAPD techniques were performed with the use of 40 different initiators. The analysis of polymorphism was conducted by using the NTSYS program of numerical taxonomy. The susceptibility of antimicrobials in the strains collected from teats of cow udders was 4% to penicillin, 88% to tetracycline, 92% to gentamicine, 96% to vancomycin and 100% to chloranfenicol. As for the strains collected from milking workers' hands, susceptibility results were 0% to penicillin, 70% to tetracycline and 90% to vancomycin and 100% to gentamicine and chloranfenicol antimicrobials. E-test showed minimum inhibitory concentration (MIC) greater than 256 ?g/mL to strains resistant to the antimicrobial vancomycin. The studies made it possible to detect S. aureus resistance upon the use of the tested antimicrobials and to determine the genetic diversity found among strains of staphylococcus due to the presence of many polymorphic bands found in all initiators.
Subject(s)
Animals , Female , Cattle , Staphylococcus aureus/genetics , Drug Resistance, Bacterial , Polymorphism, Genetic , Microbial Sensitivity Tests/veterinary , Random Amplified Polymorphic DNA Technique/veterinaryABSTRACT
The efficiency of the Polymerase Chain Reaction (PCR) combined with selective enrichment broth was compared with the standard microbiological techniques for detection of Salmonella Dublin in fecal samples of 10 to 15-days-old Holstein calves, experimentally infected with 10(8) CFU of Salmonella Dublin. Seventy-six fecal samples were analyzed using PCR associated with selenite cystine (SC) and Muller-Kauffmann tetrathionate (TMK) broths and standard microbiological techniques. Regardless of the selective enrichment broth used, the standard microbiological techniques were significantly better than PCR in detection of positive samples of Salmonella Dublin. However, the simultaneous use of both techniques provided detection of a larger number of positive samples. The SC broth was the best option as selective enrichment in both techniques.(AU)
Subject(s)
Animals , Cattle , Salmonella/isolation & purification , Salmonella Infections, Animal/diagnosis , Salmonella Infections, Animal/microbiology , Feces , Polymerase Chain Reaction , Bacteriological TechniquesABSTRACT
The efficiency of the Polymerase Chain Reaction (PCR) combined with selective enrichment broth was compared with the standard microbiological techniques for detection of Salmonella Dublin in fecal samples of 10 to 15-days-old Holstein calves, experimentally infected with 10(8) CFU of Salmonella Dublin. Seventy-six fecal samples were analyzed using PCR associated with selenite cystine (SC) and Muller-Kauffmann tetrathionate (TMK) broths and standard microbiological techniques. Regardless of the selective enrichment broth used, the standard microbiological techniques were significantly better than PCR in detection of positive samples of Salmonella Dublin. However, the simultaneous use of both techniques provided detection of a larger number of positive samples. The SC broth was the best option as selective enrichment in both techniques.
Subject(s)
Animals , Cattle , Salmonella/isolation & purification , Salmonella Infections, Animal/diagnosis , Salmonella Infections, Animal/microbiology , Bacteriological Techniques , Feces , Polymerase Chain ReactionABSTRACT
ABSTRACT The aim of this study was to analyze in vitro antimicrobial susceptibility of Staphylococcus aureus strains isolated from teats of cow udders and milking workers hands as well as to verify polymorphism among them by using RAPD-PCR technique. Tests were conducted by disk diffusion technique and after the collection of the genetic material PCR and RAPD techniques were performed with the use of 40 different initiators. The analysis of polymorphism was conducted by using the NTSYS program of numerical taxonomy. The susceptibility of antimicrobials in the strains collected from teats of cow udders was 4% to penicillin, 88% to tetracycline, 92% to gentamicine, 96% to vancomycin and 100% to chloranfenicol. As for the strains collected from milking workers hands, susceptibility results were 0% to penicillin, 70% to tetracycline and 90% to vancomycin and 100% to gentamicine and chloranfenicol antimicrobials. E-test showed minimum inhibitory concentration (MIC) greater than 256 ?g/mL to strains resistant to the antimicrobial vancomycin. The studies made it possible to detect S. aureus resistance upon the use of the tested antimicrobials and to determine the genetic diversity found among strains of staphylococcus due to the presence of many polymorphic bands found in all initiators.
RESUMO O objetivo deste trabalho foi analisar a sensibilidade antimicrobiana in vitro de cepas de Staphylococcus aureusisoladas de tetos de vacas e mãos de retireiros, além de verificar o polimorfismo entre elas pela técnica de PCR-RAPD. Os testes foram realizados pela técnica de difusão em discos e, após a extração do material genético foram desenvolvidas as técnicas de PCR e RAPD, usando para isso 40 iniciadores diferentes. A análise do polimorfismo foi realizada empregando-se o programa de taxonomia numérica NTSYS. As sensibilidades dos antimicrobianos nas cepas obtidas de tetos de vacas foram 4% para a penicilina, 88% para a tetraciclina, 92% para a gentamicina, 96% para a vancomicina e 100% ao cloranfenicol. Para as cepas provenientes das mãos de retireiros, os resultados de sensibilidade foram zero para a penicilina, 70% para a tetraciclina e 90% para a vancomicina e 100% para os antimicrobianos gentamicina e cloranfenicol. A realização do E-teste indicou uma concentração inibitória mínima (CIM) maior que 256 mg/mL para as cepas resistentes ao antimicrobiano vancomicina. Os estudos permitiram detectar a resistência dos S. aureus mediante o uso dos antimicrobianos testados e determinar a diversidade genética entre as cepas de estafilococos devido à presença de muitas bandas polimórficas encontradas em todos os iniciadores.
ABSTRACT
Salmonella enterica serovar Gallinarum (SG) is an intracellular pathogen of chickens. To survive, to invade and to multiply in the intestinal tract and intracellularly it depends on its ability to produce energy in anaerobic conditions. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. In this study mortality rates of chickens challenged with mutants of Salmonella Gallinarum, which were defective in utilising anaerobic electron acceptors, were assessed in comparison to group of bird challenged with wild strain. The greatest degree of attenuation was observed with mutations affecting nitrate reductase (napA, narG) with additional attenuations induced by a mutation affecting fumarate reductase (frdA) and a double mutant (dmsA torC) affecting DMSO and TMAO reductase.
Subject(s)
Animals , Bacteria, Anaerobic/genetics , Enzyme Activation , Genes, Bacterial , Mutation , Poultry , Salmonella Infections , Salmonella enterica/genetics , Salmonella enterica/isolation & purification , Clinical Enzyme Tests , Methods , Methods , VirulenceABSTRACT
Salmonella enterica serovar Gallinarum (SG) is an intracellular pathogen of chickens. To survive, to invade and to multiply in the intestinal tract and intracellularly it depends on its ability to produce energy in anaerobic conditions. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. In this study mortality rates of chickens challenged with mutants of Salmonella Gallinarum, which were defective in utilising anaerobic electron acceptors, were assessed in comparison to group of bird challenged with wild strain. The greatest degree of attenuation was observed with mutations affecting nitrate reductase (napA, narG) with additional attenuations induced by a mutation affecting fumarate reductase (frdA) and a double mutant (dmsA torC) affecting DMSO and TMAO reductase.
ABSTRACT
Salmonella enterica serovar Gallinarum (SG) is an intracellular pathogen of chickens. To survive, to invade and to multiply in the intestinal tract and intracellularly it depends on its ability to produce energy in anaerobic conditions. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. In this study mortality rates of chickens challenged with mutants of Salmonella Gallinarum, which were defective in utilising anaerobic electron acceptors, were assessed in comparison to group of bird challenged with wild strain. The greatest degree of attenuation was observed with mutations affecting nitrate reductase (napA, narG) with additional attenuations induced by a mutation affecting fumarate reductase (frdA) and a double mutant (dmsA torC) affecting DMSO and TMAO reductase.
ABSTRACT
ABSTRACT The aim of this work was to genetically characterize 1,073 isolates of B. thuringiensis, from three Brazilian collections UNESP/Jaboticabal, ESALQ/Piracicaba and EMBRAPA/Sete Lagoas with the main emphasis on the analysis of the cry1 gene types presented by these isolates. To achieve this purpose, oligonucleotide primers were designed based on 16 conserved and 4 unconserved regions of the corresponding sequences from each one of the 16 subclasses of the cry1 set of genes and used in PCR amplification assays. These sequences were amplified and the presence of amplicons for each subclass was evaluated in terms of percentage of gene type per bacterial collection. As a result, 55.7% of the isolates reacted to the primer Gral cry1, and the subclasses cry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1Ae, cry1Af, cry1Ag, cry1Bf, cry1Ca and cry1Fa were detected in high percentages among the isolates ranging from 43.4 to 54.9%. A subclass distribution was observed among the set of isolates from these collections, with the greater percentage of isolates harboring the cry1Ab (42.12%) and the lowest percentage for thecry1Db subclass (0.6%). The genetic variability of the analyzed bacterial collections seems to indicate the ESALQ/Piracicaba and the UNESP/Jaboticabal subsets as sources of promising isolates for the control of Lepidoptera pest insects. For the EMBRAPA/Sete Lagoas subset of isolates, in which the evaluated subclasses frequencies were considered low (below 20%), the cry1B was the most frequently observed gene type present in 38.5% of the isolates.
RESUMO O presente trabalho teve como objetivo caracterizar geneticamente 1.073 isolados de Bacillus thuringiensis, de três coleções brasileiras, provenientes da UNESP, Jaboticabal, da ESALQ/ Piracicaba e da EMBRAPA. Sete Lagoas, analisando os tipos de genes cry1 apresentados pelos isolados. Para isso, foram elaborados oligonucleotídeos iniciadores a partir de 16 regiões conservadas e 4 regiões não conservadas das seqüências de cada uma das 16 subclasses do gene cry1. Essas seqüências foram amplificadas por PCR e a presença de amplicons para cada subclasse foi calculada em porcentagem por gene e por coleção. Nessa análise, 55,7% dos isolados apresentaram amplificação para o gene cry1, e as subclassescry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1Ae, cry1Af, cry1Ag, e cry1Bf, cry1Ca e cry1Fa estão presentes em alta proporção de isolados, variando de 43,4% a 54,9%. Verificou-se que existe uma distribuição das subclasses dentro do banco de isolados de B. thuringiensis em estudo, com maior porcentagem de isolados portadores dos genes cry1Ab (42,12%) e com menor porcentagem de representantes da subclasse cry1Db (0,6%). A variabilidade gênica, nas coleções analisadas, destaca as coleções de Jaboticabal e Piracicaba como fontes de isolados promissores para uso em programas de Controle Biológico de pragas da ordem Lepidoptera. A coleção de Sete Lagoas, na qual as freqüências das subclasses estudadas foram relativamente baixas (abaixo de 20%), destaca somente o gene cry1Ab, presente em 38,5% dos isolados desta coleção.