Identification and Discrimination of Staphylococcus aureus Causing Mastitis Using Protein Fingerprinting (MALDI-TOF)

Abstract

Mastitis is one of the most serious diseases facing livestock keepers in livestock farms worldwide. Nevertheless, phenotypic and genotypic techniques represent the most straightforward approaches for the detection of Staphylococcus aureus (S. aureus) mastitis; these techniques are arduous, luxurious, and, until now, haven’t been able to differentiate between methicillin‐sensitive S. aureus (MSSA) and methicillin‐resistant S. aureus (MRSA). From this perspective, our investigation is designed to examine the proteomic characteristics of S. aureus isolates recovered from various farm animal species with a history of mastitis throughout a survey carried out from 2019 to 2020 in the Qassim region, Saudi Arabia. The classical phenotypic techniques used for identification of S. aureus were compared to the proteomic identification by MALDI Biotyper. Also, a distinction between MRSA and MSSA via Principle Component Analysis (PCA) and Single-Peak Intensity yielded by MALDI Biotyper were applied. The vulnerability of S. aureus strains against the most common antibiotics used for the treatment of S. aureus mastitis were also evaluated. To achieve these goals, 400 mastitic milk samples from different animal farms (cows, sheep, goats, and dromedary camels) with a high prevalence of S. aureus mastitis were selected conveniently based on the availability of dairy animals. The isolates were firstly submitted to routine microbiological diagnostics, and then the phenotypic identification of S. aureus was made through a catalase test, MASTASAPH Latex test, STAPH ID 32 (API system), and Vitek 2 Compact system. The proteomic identification of S. aureus was done by MALDI Biotyper (MBT). The Kirby Bauer method was accomplished to detect the degree of resistance of S. aureus strains to various types of antimicrobial agents. The findings of thisstudy revealed that out of 400 milk samples, 120 (30%) Staphylococcus isolates were isolated. Forty-eight of these isolates were recovered from cows (40%), 32 (26.6%) from goats, 28 2 (23.3%) from sheep, and 12 (10%) from camels. The morphology of the cultures, and the results of the MASTASAPH Latex test, revealed that out of 120 Staphylococcus isolates, 54 (46%) were identified as coagulase positive Staphylococci (S. aureus) and 66 isolates were recognized as coagulase negative Staphylococci. All S. aureus strains (100%) were identified rapidly and accurately by MALDI-TOF MS with a score value of ≥2.00 as compared to the Staph ID 32 and Vitek 2 Compact system, in which 48 (89%) and 51 (94.47%) were identified as S. aureus, respectively. After analysis of peak intensities created by MBT, we observed that several peaks were identified in MSSA in the mass of 4590 Da,4826 Da, 4863 Da, and 4938 Da but were absent in MRSA. Likewise, distinct peaks were identified in MRSA in the mass of 2636 Da and 3009 Da but were absent in MSSA. The main spectra profiles (MSP) dendrogram produced by MBT demonstrated that the verified S. aureus isolates were closely related to each other and classified into one group with a distance level of ≤400. The results of the antimicrobial resistance of S. aureus revealed a higher degree of resistance (94.4%) against carbenicillin of the β-lactam group, whereas 38.88% and 33.33% of S. aureus isolates were resistant to erythromycin and kanamycin, respectively. In contrast, 94.44% and 81.48% of S. aureus strains were sensitive to clindamycin and gentamycin, respectively. In conclusion, S. aureus bacteria are among the key triggers for mastitis in Saudi Arabia. MALDI-TOF-MS compared to the classical methods is reported to be not only the most rapid and sensitive instrument to identify S. aureus but also able to discriminate between MSSA and MRSA using single peak and computer analyses.