SHORT - COURSE: MOLECULAR BIOLOGY APPLIED TO CLINICAL ANALYZES

 In recent years, genetics, cellular biology, genome sequencing of pathogens, among other resources, have significantly changed the opportunities for epidemiological investigations, pathogenesis studies, diagnosis and control of microbial diseases. The PCR technique has expanded the possibilities of DNA analysis and has made molecular biology find new applications even in areas outside its traditional field, such as medicine, agriculture and biotechnology. PCR presents a wide range of applications in various branches of scientific research. This reaction allows a particular region of the genome of any organism to be multiplied in millions of copies, which facilitates genetic analysis and allows the development of diagnostic techniques, much more sensitive and more specific than those traditionally used. The mini-course will have a brief presentation on molecular techniques and after the practical part with the following steps: Extraction of bacterial DNA, Quantification of DNA by the absorption spectrum of nucleic acids, Polymerase Chain Reaction (PCR) associated with length polymorphism DNA fragments (PCR_RFLP / ARDRA) terminating with Sample Analysis using the technique of Agarose Gel Electrophoresis.Keywords: Genetics. Epidemiological. Polymerase Chain Reaction

MOLECULAR ANALYSIS AND PROFILES OF ANTIBIOTICS RESISTANCE OF SALMONELLA ENTERITIDIS ISOLATES FROM POULTRY CHAIN PRODUCTION OF THE BRAZILIAN SOUTHERN REGION

The agroindustrial sector of meats production and derivatives presents importance for the economic development in the Brazil's Southern States. Salmonella Enteritidis was isolated in all sectors of the productive chain of poultry and PCR-based techniques have demonstrated good applicability for identification and epidemiology studies of food pathogens. The aim of this work was to select molecular techniques with greatest potential to detect polymorphism of Salmonella Enteritidis strains, besides to determine the antibiotic resistance profile, and to find correlation between them. Sixty-eight Salmonella Enteritidis strains isolated from the poultry production chain in the southern States of Brazil were analyzed with the PAST program by UPGMA method and the similarity index Euclidean. The molecular technique RAPD, using the 23L primer, presented highest polymorphism, with 10 polymorphic groups and a Discriminatory Power of 0.96. For the resistance test, 75% of the isolates showed multiresistance, moreover the isolates were 100% resistant to erythromycin and 77% resistant to nalidixic acid. In conclusion, the RAPD molecular technique with the primer 23L have good applicability in epidemiological studies and the high incidence of mutiresistance isolates serves as an alert for the indiscriminate use of antibiotics in productions animals

PREVALENCE OF SALMONELLA ENTERICA HEIDELBERG IN THE PRODUCTIVE CHAIN OF BROILERS

Salmonella enterica subsp. enterica Heidelberg is one of the most invasive serovar  and deserves attention due to its prevalence in the productive chain of borilers. This research analysed  the percentage of S. Heidelberg serotype samples in semi-solid medium (MSRV) and to evaluate the profile of antimicrobial susceptibility.  Isolation of samples from avian environment was performed from MSRV method  recommended by ISO 6579. Aliquots removed from the MSRV migration zone were inoculated in selective medium agar XLD and VB agar (DIFCO®) with incubation at 37ºC/24h. Were perfomed biochemical  and serological analysis of colonies as well molecular analysis based on PCR.  A total of 1506 samples were obtained, of which n=376 samples (24.96%) were positive for S. Heidelberg. From isolates, n=1130 did not migrate in MSRV (75.03%). However, n=62 (16.49%) confirmed as being S. Heidelberg. Isolates with migration in MSRV were  susceptible to all antimicrobials tested, whereas all at the group without migration (flagellar injury?) showed resistance to Ceftiofur and Neomycin.  It is early  to affirm the existence of a  relation  of using antimicrobials in PCB with the flagellar injury of the pathogen. However, monitoring should be performed, due we detect isolates resistant to at least two broad-spectrum antibiotics at the group of isolates with no ability to migrate in MSRV medium.Keywords: Produtive chain. Salmonella. Antimicrobials. Flagella

AVALIAÇÃO DA IDENTIFICAÇÃO DE SALMONELLA ENTERITIDIS DA CADEIA PRODUTIVA DE AVES COM BASE NA ANÁLISE DE POLIMORFISMO DE DNA POR RAPD

Nesta pesquisa o objetivo foi isolar, determinar o perfil de susceptibilidade aantibióticos e caracterizar bioquímica e molecularmente linhagens deSalmonella enterica subesp. enterica Enteritidis de diferentes pontos dacadeia produtiva de frango. Sessenta linhagens de Salmonella Enteritidistiveram seu perfil de susceptibilidade (resistência/sensibilidade) aantimicrobianos determinado por teste antibiograma. Isolados dos quinzesorotipos mais prevalentes no estudo e cinqüenta e dois isolados deSalmonella Enteritidis obtidos em estudo prévio foram analisados quanto aopolimorfismo de DNA através da técnica de RAPD com o primer OPB15. Paraa análise do polimorfismo gerado foi utilizado o software WinBoot, de acordocom os princípios adotados em taxonomia numérica UPGMA (UnweighedPair-Group Methods Analysis), a partir dessa análise foram obtidosdendrogramas, detectando similaridades entre os sorotipos de Salmonella, esuas capacidades na identificação, caracterização e diferenciaçãogenética entre os isolados. A partir do antibiograma, observou-se resistênciaaos antibióticos Eritromicina e Espiramicina em 100% dos isolados. Todos osisolados foram sensíveis a Fosfomicina. Foram encontrados 56 (93,33%)isolados multi-resistentes (resistentes a três ou mais antimicrobianos). Atécnica mostrou-se altamente promissora para estudos de caracterização ediferenciação dos sorotipos de Salmonella e ainda, um alto potencial dediscriminação entre os isolados de S. Enteritidis, evidenciando pelodendrograma de similaridade obtido por UPGMA baseado no coeficiente deJacard.Palavras-chaves: Rastreabilidade. Enterobactéria. Resistência aantimicrobianos

SHORT - COURSE: MOLECULAR BIOLOGY APPLIED TO CLINICAL ANALYZES

 In recent years, genetics, cellular biology, genome sequencing of pathogens, among other resources, have significantly changed the opportunities for epidemiological investigations, pathogenesis studies, diagnosis and control of microbial diseases. The PCR technique has expanded the possibilities of DNA analysis and has made molecular biology find new applications even in areas outside its traditional field, such as medicine, agriculture and biotechnology. PCR presents a wide range of applications in various branches of scientific research. This reaction allows a particular region of the genome of any organism to be multiplied in millions of copies, which facilitates genetic analysis and allows the development of diagnostic techniques, much more sensitive and more specific than those traditionally used. The mini-course will have a brief presentation on molecular techniques and after the practical part with the following steps: Extraction of bacterial DNA, Quantification of DNA by the absorption spectrum of nucleic acids, Polymerase Chain Reaction (PCR) associated with length polymorphism DNA fragments (PCR_RFLP / ARDRA) terminating with Sample Analysis using the technique of Agarose Gel Electrophoresis.Keywords: Genetics. Epidemiological. Polymerase Chain Reaction

MOLECULAR ANALYSIS AND PROFILES OF ANTIBIOTICS RESISTANCE OF SALMONELLA ENTERITIDIS ISOLATES FROM POULTRY CHAIN PRODUCTION OF THE BRAZILIAN SOUTHERN REGION

The agroindustrial sector of meats production and derivatives presents importance for the economic development in the Brazil's Southern States. Salmonella Enteritidis was isolated in all sectors of the productive chain of poultry and PCR-based techniques have demonstrated good applicability for identification and epidemiology studies of food pathogens. The aim of this work was to select molecular techniques with greatest potential to detect polymorphism of Salmonella Enteritidis strains, besides to determine the antibiotic resistance profile, and to find correlation between them. Sixty-eight Salmonella Enteritidis strains isolated from the poultry production chain in the southern States of Brazil were analyzed with the PAST program by UPGMA method and the similarity index Euclidean. The molecular technique RAPD, using the 23L primer, presented highest polymorphism, with 10 polymorphic groups and a Discriminatory Power of 0.96. For the resistance test, 75% of the isolates showed multiresistance, moreover the isolates were 100% resistant to erythromycin and 77% resistant to nalidixic acid. In conclusion, the RAPD molecular technique with the primer 23L have good applicability in epidemiological studies and the high incidence of mutiresistance isolates serves as an alert for the indiscriminate use of antibiotics in productions animals

AVALIAÇÃO DA IDENTIFICAÇÃO DE SALMONELLA ENTERITIDIS DA CADEIA PRODUTIVA DE AVES COM BASE NA ANÁLISE DE POLIMORFISMO DE DNA POR RAPD

Nesta pesquisa o objetivo foi isolar, determinar o perfil de susceptibilidade aantibióticos e caracterizar bioquímica e molecularmente linhagens deSalmonella enterica subesp. enterica Enteritidis de diferentes pontos dacadeia produtiva de frango. Sessenta linhagens de Salmonella Enteritidistiveram seu perfil de susceptibilidade (resistência/sensibilidade) aantimicrobianos determinado por teste antibiograma. Isolados dos quinzesorotipos mais prevalentes no estudo e cinqüenta e dois isolados deSalmonella Enteritidis obtidos em estudo prévio foram analisados quanto aopolimorfismo de DNA através da técnica de RAPD com o primer OPB15. Paraa análise do polimorfismo gerado foi utilizado o software WinBoot, de acordocom os princípios adotados em taxonomia numérica UPGMA (UnweighedPair-Group Methods Analysis), a partir dessa análise foram obtidosdendrogramas, detectando similaridades entre os sorotipos de Salmonella, esuas capacidades na identificação, caracterização e diferenciaçãogenética entre os isolados. A partir do antibiograma, observou-se resistênciaaos antibióticos Eritromicina e Espiramicina em 100% dos isolados. Todos osisolados foram sensíveis a Fosfomicina. Foram encontrados 56 (93,33%)isolados multi-resistentes (resistentes a três ou mais antimicrobianos). Atécnica mostrou-se altamente promissora para estudos de caracterização ediferenciação dos sorotipos de Salmonella e ainda, um alto potencial dediscriminação entre os isolados de S. Enteritidis, evidenciando pelodendrograma de similaridade obtido por UPGMA baseado no coeficiente deJacard.Palavras-chaves: Rastreabilidade. Enterobactéria. Resistência aantimicrobianos

PREVALENCE OF SALMONELLA ENTERICA HEIDELBERG IN THE PRODUCTIVE CHAIN OF BROILERS

Salmonella enterica subsp. enterica Heidelberg is one of the most invasive serovar  and deserves attention due to its prevalence in the productive chain of borilers. This research analysed  the percentage of S. Heidelberg serotype samples in semi-solid medium (MSRV) and to evaluate the profile of antimicrobial susceptibility.  Isolation of samples from avian environment was performed from MSRV method  recommended by ISO 6579. Aliquots removed from the MSRV migration zone were inoculated in selective medium agar XLD and VB agar (DIFCO®) with incubation at 37ºC/24h. Were perfomed biochemical  and serological analysis of colonies as well molecular analysis based on PCR.  A total of 1506 samples were obtained, of which n=376 samples (24.96%) were positive for S. Heidelberg. From isolates, n=1130 did not migrate in MSRV (75.03%). However, n=62 (16.49%) confirmed as being S. Heidelberg. Isolates with migration in MSRV were  susceptible to all antimicrobials tested, whereas all at the group without migration (flagellar injury?) showed resistance to Ceftiofur and Neomycin.  It is early  to affirm the existence of a  relation  of using antimicrobials in PCB with the flagellar injury of the pathogen. However, monitoring should be performed, due we detect isolates resistant to at least two broad-spectrum antibiotics at the group of isolates with no ability to migrate in MSRV medium.Keywords: Produtive chain. Salmonella. Antimicrobials. Flagella