Caracterização fenotípica e molecular da resistência à oxacilina em Staphylococcus spp. isolados de mastite subclínica bovina

Abstract

Among the major pathogens of bovine mastitis are bacteria of the genus Staphylococcus. In recent decades, resistance to oxacillin (methicillin) in this genus has been a matter of concern for the possibility of reducing the effectiveness of mastitis treatments, and transferring of resistance determinants. The oxacillin resistance is mediated by PBP 2a protein, encoded by the mecA gene, which confers resistance to all β-lactam antibiotics, including cephalosporins and carbapenems. The mecA gene is part of a genomic island called staphylococcal cassette chromosome mec (SCCmec), which may also include other resistance genes. Oxacillin resistance was studied in 170 Staphylococcus isolated from subclinical mastitis, 79 S. aureus and 91 coagulase-negative staphylococci (CNS). The bacterial strains were isolated from six Brazilian states; four from Minas Gerais, 27 from Paraná, three from Pernambuco, eleven from Rio Grande do Sul, 56 from Santa Catarina and 69 from São Paulo. The susceptibility profile to ten antimicrobial agents used in the veterinary practice was determined by E-TEST® method. Phenotypic characterization of oxacillin resistance was performed by disk diffusion test with oxacillin and cefoxitin, agar dilution test with oxacillin and E-TEST® with oxacillin. All strains were screened by PCR to detect mecA gene using two pairs of primers amplifying different regions of the gene. The strains with oxacillin resistance phenotype were identified by sequencing a 536 bp fragment of 16S rRNA gene. This group was also evaluated for the presence of the gene mecC and blaZ, which encodes for the enzyme β-lactamase. The PCR products were sequenced to confirm the results. Molecular analysis of mecA gene was carried out by the typing of staphylococcal cassette chromosome mec (SCCmec) and the mecA-positive strains by macrorestriction of chromosomal DNA followed by pulsed field gel electrophoresis (PFGE). With the exception of penicillin and oxacillin, more than 86% of the strains presented susceptibility to cephalothin, gentamicin, clindamycin, erythromycin, enrofloxacin, sulfonamide, trimethoprim-sulfamethoxazole and tetracycline. All S. aureus strains were sensitive to oxacillin, while 29.7% of the CNS strains were resistant. The CNS were more resistant than S. aureus to cephalothin and erythromycin (p ≤ 0.01) and gentamicin, clindamycin, and tetracycline (p ≤ 0.05). The agar diffusion test with cefoxitin disc showed higher sensitivity and specificity for the presence of the mecA gene. Considering the total strains studied, 31 CNS were phenotypically resistant to oxacillin in at least one of the tests, and only in ten CNS was detected the mecA gene. mecA-positives bacteria were identified as Staphylococcus epidermidis and classified into three pulsotypes (A, B and C) and four subtypes (A1, B1, B2 and B3). Among strains with oxacillin resistance phenotype, 16 were positive for blaZ gene, seven mecA-negatives and nine mecA-positive strains. Two of the oxacillin resistant strains amplified mecA-like gene of S. sciuri and none amplified mecC. Three SCCmec types were found, types I, IV and V. The results suggest that S. epidermidis can be a reservoir of oxacillin resistance to other species of staphylococci, both of human and animals. Studies that generate information about the molecular and phenotypic profile of antimicrobial resistance in staphylococcal species should be performed for controlling the spread of resistance and selection of appropriate therapeutic measures.