Identificação de Staphylococcus aureus, S. intermedius e S. hyicus através de testes bioquímicos e da amplificação por PCR de sequências dos genes coa E nuc

Abstract

Staphylococcus aureus was considered, for many years, the only species of the Staphylococcus genus with capacity to produce enterotoxins and coagulase. Later, other enterotoxin and coagulase producing species, such as S. hyicus and S. intermedius, had been identified. These three species have extremely similar morphologic characteristics, as well as biochemical reactions, making their differentiation difficult through the conventional methods of microbiological analysis. The development of modern molecular biology techniques, such as in vitro amplification of DNA through the polymerase chain reaction (PCR), allowed several studies in the search of more efficient methods for identification and characterization of microrganisms. Objective of this work was to compare a biochemical method and a molecular method, based in PCR, for the identification of S. aureus, S. intermedius and S. hyicus. Sixty five strains of coagulase positive staphylococci, previously submitted to tests of Gram staininy, catalase, thermonuclease and free coagulase production, were differentiated and identified at species level through biochemical analysis, using the tests of pigment production, hemolytic activity in blood agar, production of β-galactosidase, acetoin production, aerobic fermentation of maltose, anaerobic fermentation of manitol, lipolytic activity in polisorbate, growth in BairdParker agar and P agar supplemented with acriflavina, as well as through the PCR amplification of a specific S. aureus coa gene sequence and specific S. intermedius and S. hyicus nuc gene sequences. The combination of biochemical testes, as well as the amplification of coa and nuc gene sequences, allowed the identification and differentiation between the three species of coagulase positive staphylococci. Due to the variability of results of biochemical tests, it was concluded that they can be used for differentiation of the three species when applied and analyzed together. The testsfor acriflavina resistance and β-galactosidase production showed no variability of results, being the best biochemical differentiation tests. The PCR amplification of coa and nuc genes sequences, with primers COAG2, COAG3, NUC1, NUC2, NUC3 and NUC4, allowed S. aureus, S. intermedius and S. hyicus species identification and differentiation. Differences on the discriminatory power were not verified using the combination of biochemical tests or the molecular method that uses the PCR amplification of the coa and nuc genes sequences.