Caracterização e epidemiologia molecular de Staphylococcus aureus resistentes aos Beta-lactâmicos isolados de leite de vacas com mastite subclínica

Abstract

Mastitis is a problem both economically and healthily in dairy herds. The most commonly associated pathogen is Staphylococcus aureus bacteria that can also contaminate milk and dairy products causing food poisoning in consumers. Prevention consists of adequate hygiene during milk collection and treatment of sick animals to prevent spread of the disease. This treatment is based on research for the disease-causing agent and the most efficient antibiotic. Among the most effective antibiotics is the β-lactam class, particularly methicillin, which is a penicillin indicated against already resistant penicillin-producing bacteria. Carbapenems, however, are potent β-lactams with broad spectrum of action and are not allowed in dairy animals. Constant and/or abusive use of these drugs may select micro-organisms that have acquired resistance by bacterial mutation, transformation, transduction or conjugation. In this sense, the objective of this study is the screening of blaZ, mecA genes and their variant mecALGA251 that are associated with penicillin resistance, and also the blaKPC and blaOXA23 genes that encode carbapenemic resistance. This research was carried out with samples of bovine milk from teat affected by subclinical mastitis from 15 farms in northern Minas Gerais. Isolation and cultivation of micro-organisms from breast milk were performed, so identification by differential tests and confirmation by MALDI-TOF mass spectrometry. Staphylococcus aureus isolates were then submitted to disc diffusion antibiogram using the antimicrobials amoxacillin, oxacillin, ampicillin/sulbactan, cefoxitin, imipenem and meropenem. Then, DNA was extracted from the resistant bacterial isolates and the blaZ, mecA, mecALGA251, blaKPC and blaOXA23 genes were screened by Single PCR, being sequenced the product of one of the bands of each amplified gene. The RAPD-PCR test was also performed to verify genetic variability among isolates. As a result, 214 microorganisms were found, among them 79 Staphylococcus aureus. Of these, 30 showed resistance to, at least, one of the penicillins tested and 9 showed resistance to meropenem. There was a significant association (p <0.05 by the chi-square test) between antimicrobial resistance and origin farm of isolates. Of the 30 penicillin-resistant isolates, 6 carried blaZ gene, 4 carried mecA gene, and no one carried mecALGA251 gene. No one of the isolates tested carried blaKPC or blaOXA23 gene. A dendrogram was elaborated indicating the genetic proximity between the isolates that, notably, corresponds to the proximity between farms.