Penicillin resistance in bovine Staphylococcus aureus: genomic evaluation of the discrepancy between phenotypic and molecular test methods

Staphylococcus aureus is a major pathogen in humans and animals. In cattle, it is one of the most important agents of mastitis causing serious costs in the dairy industry. Early diagnosis and adequate therapy are, therefore, two key factors to deal with the problems caused by this bacterium, whereby benzylpenicillin (penicillin) is usually the first choice to treat these infections. Unfortunately, penicillin resistance testing in bovine S. aureus strains show discrepancy results between the tests used, and consequently, the best method for assessing penicillin resistance is still unknown. The aim of this study was, therefore, to find a method that assesses penicillin resistance in S. aureus and to elucidate the mechanisms leading to the observed discrepancies.A total of 146 methicillin-sensitive S. aureus strains isolated from bovine mastitis were tested for penicillin resistance using a broth microdilution (MIC) and two different disc diffusion (DD) protocols. Furthermore, the strains were analyzed for the presence of the bla operon genes (blaI, blaR1, blaZ) by PCR, while a subset of 45 strains was also subjected to whole genome sequencing. Penicillin resistance in S. aureus is highly dependent on the completeness of the bla operon promotor. When the bla operon was complete based on WGS analysis, all strains showed MIC ≥1 µg/mL, whereas, when the bla operon was mutated (31-nucleotide deletion), they were penicillin sensitive except in those strains where an additional, bla operon-independent resistance mechanism was observed.The WGS of S. aureus analysis fully explained the discrepant results of different resistance testing methods, such as DD and PCR, compared with MIC used as reference method. However, caution is required when interpretating such results