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.
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-108 bovine strains of S. aureus were selected from our European strain collection which includes 456 strains . The selection was made in a way, that the distribution of CC types among strains was the same as observed for intramammary infections in our European survey study (Boss et al., 2016). Accordingly, 37 strains of CC8, 33 of CC705, 18 of CC97, and 20 strains of other CCs were selected. Within each CC, they were randomly selected. Subsequently, additional 41 CC8 strains were randomly selected for more detailed investigation, as the preliminary results obtained with the different methods frequently showed discrepancies. These 149 strains all originated from different herds, located troughout Europe: Austria, Belgium, France, Germany, Ireland, Italy, Norway, Sweden, and Switzerland. They had all been isolated from aseptically collected milk samples of cows with intramammary infection, and stored in skim milk at -20°C. Previous characterization included PCR for the nuc gene (highly specific for S. aureus (Brakstad et al., 1992; Graber et al., 2007), RS-PCR for genotyping and CC atribution, and spa type as described (Boss et al., 2016; Fournier et al., 2008). Furthermore, as genotypes may have variants (±1 additional band) they had been combined to genotypic clusters (CL) as described (Cosandey et al., 2016; Fournier et al., 2008). -Bacteriological culture and extraction by boiling prep -All strains were tested for methicillin sensitivity by PCR using the commercial SureFast® MRSA 4plex kit (Congen Biotechnologie GmbH, Berlin, Germany). The assay is a real-time PCR for the direct, qualitative detection of S. aureus, of the mecA and mecC genes as well as of the junction between the orfX gene and the staphylococcal cassette chromosome (SCCmec) carrying mecA/mecC -All strains were analyzed for the presence of blaI, blaR1, and blaZ of the bla operon using singleplex melting curve PCR (design of new primers for the PCR) -Phenotypic test to check the susceptibility or resistance to the penicillin antibiotics (MIC,DD, Nitrocefin test) -Extraction and whole genome sequencing by Illumina for a selected group of 45 samples -In silico analysiss of plasmids and in silico analysis of teh bal operon -Statistical analyses to investigate the agreement between the different analysis done (DDC: disk diffusion for penicillin according to CLSI protocol, DDE: disk diffusion test for peniciilin according to the EUCAST protocol, mPCR: melting curve PCR for blaI, blaR and blaz genes)