Desenvolvimento e avaliação de método molecular baseado em PCR multiplex para detecção de genes de resistência a antimicrobianos relevantes para o controle da mastite bovina

Abstract

Bovine mastitis is a disease characterized by the inflammation of the mammary gland and is considered the main disease of dairy herds in the world, causing the greatest economic damage to the dairy sector. Although unavoidable, the use of antibiotics for the control of this disease can lead to the emergence of resistant strains and leave residues in the milk, so these drugs must be used rationally. In order to develop improved, rapid and specific studies for epidemiological studies, as well as for the diagnosis of bovine mastitis, molecular methods have been increasingly studied in order to reduce the time required for the emission of results. Considering the scarcity of data on antibiotic resistance in mastitis pathogens in Brazil and that antibiotic resistance is a genetic phenomenon, the objective of the present work was to develop and validate a multiplex PCR-based method to detect the main genes involved in resistance of pathogens Staphylococcus aureus and Streptococcus agalactiae to the main antibiotics marketed in Brazil for the treatment of bovine mastitis: tetracycline (tetO, tetK, tetM genes), erythromycin (ermB gene), gentamicin (aacA-aphD gene) and penicillin (blaZ gene). A multiplex PCR-based method was developed for amplification of all of the above-mentioned markers as well as the mecA gene, which encodes methicillin/oxacillin resistance and is a marker of multidrug resistance to antibiotics. Preliminary assessments of the detection limit of the assay have demonstrated the need for at least 0,4 ng/μl of bacterial DNA in the reaction so that all markers are amplified simultaneously. The coincidence forces (Kappa index) of the amplification results using the oligonucleotide sets proposed in the present work, by monoplex and multiplex PCR, of the tetO, ermB, mecA, blaZ, tetK and tetM genes ranged from 84% to 100%, while that data relating to the aacA-aphD gene demonstrated a light coincidence force (11%). The method developed in the present work could be used to detect molecular markers of resistance to antibiotics relevant for epidemiological studies and for the control of bovine mastitis in Brazil. Further studies will be carried out aiming at the improvement, adaptation and validation of the assay for the detection of these markers in bovine milk samples.