Production of Staphylococcus aureus Isolates

Staphylococcus aureus which is an important pathogen, is known to have several virulence factors. The pathogenicity of S.aureus isolates are related with features like adherence, various toxins, enzymes, structural and extracellular factors. Slime factor and biofilm formation are also the pathogenicity factors of the bacteria. Biofilm formation is usually associated with chronic infections and has become a subject of interest in a wide area of research. Biofilm is an adherent structure formed by bacteria encased within a matrix produced on natural body surfaces or medical devices. As the biofilm producing S.aureus isolates are more resistant to antibiotics and and their biofilms prevent phagocytosis, the treatment of infections caused by biofilm positive S.aureus isolates become difficult. The icaADBC locus and some proteins have provento be responsible for the formation of S.aureus type biofilm. The aim of this study was to investigate the relationship between the genotypic and phenotypic detection methods of biofilm formation with icaA, icaD and bap genes and phenotype expressions which are responsible for the formation of biofilm in S.aureus isolates. One hundred seventy five S.aureus isolates from various clinical specimens were included in the study. For the phenotypic detection of biofilm producing isolates Congo red agar (CRA) medium and microplate method were used. The biofilm-producing strain Staphylococcus epidermidis ATCC 35984 and S. epidermidis ATCC 12228 were used as positive and negative controls, respectively. One hundred fifty two S.aureus biofilm producing isolates were detected at least by either Congo Red agar or microplate method and all isolates were screened for icaA, icaD and bap genes by PCR. The production of polysaccharide intracellular adhesins /poly-N-acetyl-beta-1-6-glucosamine (PIA/ PNAG) was also investigated in S.aureus isolates. For the detection of PIA/PNAG chemiluminescence dot-blot method was used. According to the phenotypic evaluations based on colony morphologies in CRA medium, biofilm formation were found as negative in 101 of 175 isolates (57.7%), while biofilm formation were positive in 74 (42.3%) of the isolates. As a result of quantitative evaluation by microplate method based on absorbance measurement, biofilm production was determined as negative in 34 (19.4%) specimens, moderate in 112 (64.0%) specimens and strong in 29 (16.6%) specimens. Microplate and CRA methods were incompatible with each other when compared for their biofilm production determination (p< 0.001). Among the 152 clinical isolates used to determine the presence of icaA and icaD genes responsible for the formation of biofilms, the genes were detected in 136 (89.5%) of the isolates and in the S.epidermidis ATCC 35984 strain used as a positive control. icaA and icaD genes were not detected in sixteen of the 152 (10.5%) clinical isolates and in the S.epidermidis ATCC 12228 strain used as a negative control. A weak-moderate correlation was found between icaA and icaD genes and biofilm production determined in CRA medium.A good correlation was found between icaA and icaD genes and biofilm production detected in microplate method. bap gene was determined in only one of the 152 clinical S.aureus isolates studied and in S.aureus V329 strain used as positive control. For the detection of PIA/PNAG which was synthesized by icaADBC genes, 50 clinical S.aureus isolates were used. PNAG production was determined in 42 isolates with positive icaA and icaD genes by the chemiluminescence dot-blot method, and no PNAG production was determined among eight isolates with negative ica genes. As a result, it was found that the microplate method was more sensitive and reliable for the phenotypic determination of biofilm formation in S.aureus isolates, high level of biofilm formation among clinical S.aureus isolates (about 80%), the role of icaA and icaD genes and products (PIA/PNAG) in biofilm production was determined