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 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 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.

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.

PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic) and ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences (rep-PCR) found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis) and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.