Optimizing detection methods for MRSA isolated from mastitis cases and assessing virulence genes

Abstract

The primary objective of this study was to evaluate the efficiency and accuracy of bacterial identification methods, BD Phoenix™100 and Matrix Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS), for the identification of Methicillin-resistant Staphylococcus aureus (MRSA) and to select the most reliable method. This study was supported by the inclusion of polymerase chain reaction (PCR) techniques to improve the reliability of MRSA detection. For this purpose, 350 milk samples were collected from different farms and cultured for isolation of Staphylococcus spp., using the salt enrichment culture method. 232 strains were isolated which were further identified with the BD Phoenix™100 system; however, only 204 of these were identified as Staphylococcus spp., 28 of these were unidentified by MALDI-TOF MS. The Maldi Biotype software accurately identified 119 of these isolates at the species level and 85 at the genus level. BD Phoenix™100 demonstrated remarkable accuracy, identifying 100 % of the isolates as Staphylococcus aureus (39/39). In contrast, the MALDI-TOF MS method identified 94.8 % of the isolates as Staphylococcus aureus (37/39). Both identification systems confirmed a total of 37 strains of Staphylococcus aureus. We confirmed Staphylococcus aureus by PCR using the spa 83.7 % (31/37) and nuc 86.4 % (32/37) genes. Using the PCR method, we successfully detected the mecA 0.9 % (2/204), and the blaZ 17.6 % (36/204) gene for beta-lactam antibiotic resistance (beta-lactamase-penicillinase) but did not find mecC and PVL genes from any of the isolates. The antibiotic susceptibility test results were determined for all isolates using the Kirby-Bauer disc diffusion method. This demonstrated that they exhibited the highest resistance to ampicillin 53.9 % and penicillin 52.4 %, followed by tetracycline 21.07 %, clindamycin 18.6 % and oxacillin 18.1 %. Additionally, a total of 18.6 % (38/204) isolates exhibited resistance to antibiotics belonging to more than three groups of antibiotics and were classified as multidrug-resistant (MDR). The results obtained from MALDI-TOF-MS showed a concordance rate of 41.7 % with the findings from the BD Phoenix™100, as confirmed by statistical analysis. This level of agreement indicates a significant divergence between the two methods. This discrepancy highlights the need for additional studies to thoroughly assess the comparative effectiveness and reliability of MALDI-TOF-MS and BD Phoenix™100 in identifying microbial species. Such evaluations could help determine the strengths and limitations of each method, ultimately guiding the laboratory and improving diagnostic accuracy in clinical settings.