Molecular biology tools to study microbial diversity and characterization of Staphylococcus aureus in organic and conventional dairy in São Paulo

Abstract

Cheese is an important food of animal origin and can be vehicle to pathogen transmission. Among of the most prevalent microorganisms in cheese is the S. aureus that has the capacity to produce enterotoxin, to form biofilm, and some strains are resistant to antibiotics and sanitizers. The objectives of the study are to observe the microbial diversity, to perform the bacterial isolation,to identify and to characterize molecularly and phenotypically Staphylococcus aureus and to evaluate the genetic transfer of resistance genes in biofilm. For microbial diversity study, we will be performed using metagenomic sequencing of 16S rRNA gene from total DNA samples from six cheese processing plants, three organic and three conventional. From the samples will be carryied out bacterial isolation and identification of S. aureus by phenotypic analysis and the specific nuc gene. In the characterization of antibiotic resistant strains, disc infusion method will be completed testing several antimicrobial agents (oxacillin, cefoxitin, chloramphenicol, clindamycin, erythromycin, penicillin, tobramycin, tetracycline and gentamicin). Using Polymerase Chain Reaction (PCR), genes encoding biofilm formation (icaA, icaD, bap) and enterotoxins (sea-see, seg-sej) production will be identified. For molecular comparison of strains will be performed agr-type, spatyping , typing of SCCmec, and PFGE. Strains with biofilm-forming ability and with the presence of the mecA gene will be selected to the study of genetic transfer of resistance genes into biofilms, which will be perform at Department of Clinical Microbiology at Education and Research Centre Royal College of Surgeons in Ireland under supervision of Dr. Deirdre Fitzgerald Hughes. (AU)