Species identification of Staphylococcus spp. isolated from blood culture, mecA gene of oxacillin resistance and the genes icaA, to icaD icaC and biofilm production by multiplex PCR

Abstract

In the last decade there has been a clear shift in the etiology of bacterimias. Data from the National Nosocomial infects Study (NNIS) show that the Gram-positive cocci have exceeded the aerobic Gram-negative bacteria as the main etiological agents, where coagulase negative (SCN) became the first SCN isolated. The SCN agents were previously considered non-pathogenic or only part of the normal flora of human skin. But recently they have been implicated in a wide variety of infections in both hosts immunossupressed as in healthy people. Of the Staphylococcus spp. the most common blood cultures are found in S. aureus, S. epidermidis and S. haemolyticus. The main virulence factor of Staphylococcus spp. is associated with biofilm formation may facilitate the accession of these plastic surfaces. The adhesion is mediated by a polysaccharide intracellular adhesin (PIA), encoded by icaADBC operon, composed of four genes icaA, icaD, icaC, icaB. This extracellular matrix may act to protect the microorganisms from antimicrobial agents, which necessitated the removal of foreign body so that healing is achieved. Another relevant factor involved in the pathogenesis and considered important is the high isolation rate of Staphylococcus spp. methicillin resistant, allowing it to large-scale use of toxic or more expensive antibiotics such as Vancomycin. Antibiotics ² - lactam antibiotics produced a bactericidal effect by inhibiting the PBPs in the synthesis of peptidoglycan. When the peptidoglycan can not be formed, there is a disturbance of the control mechanism of hydrolases (autolysins) and the bacteria enters the lytic cycle and this process leads to cell death. The resistance of Staphylococcus spp. ²-lactam antibiotics is mainly due to two distinct mechanisms, but both may interact. The first mechanism is the production of the enzyme ²-lactamase that hydrolyzes the antibiotic, and the second is associated with the change of site of action of ² - lactam antibiotics by producing a new penicillin-binding protein, the PBP2a, encoded by the mecA gene, contained in chromosomal mobile element termed SCCmec (Staphyloccocal Chromossome cassette mec), widely distributed among Staphylococcus. Many laboratories do not identify the species of SCN, only identified in S. aureus and SCN by catalase and coagulase tests, since identification of the SCN consists of a series of biochemical tests, which require long incubation period makes it of little practical application in clinical microbiology laboratories routinely. Even with the automation of detection of microorganisms in the blood, identification and determination of antimicrobial susceptibility are still time consuming, requiring more rapid and efficient methods, especially when dealing with urgent cases of bacteremia. The application of molecular biology techniques, including the polymerase chain reaction (PCR) for identification of bacteria proved to be a promising technique due to its speed, accuracy and sensitivity in obtaining results. The techniques of PCR have undergone improvements and now with the multiplex PCR able to detect multiple pathogens and more than one gene for each pathogen, using multiple sets of primers in a single reaction. This study aims to evaluate the efficiency, accuracy and sensitivity of multiplex PCR for detection of species of Staphylococcus spp. most often found in blood cultures, the presence of mecA gene for resistance to oxacillin and gene icaA, icaD, icaC responsible for the production of biofilm in the studied samples. (AU)