Evidence-based identification and characterization of methicillin-resistant Staphylococcusaureus isolated from subclinical mastitis in dairy buffaloes of Pakistan

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA), affecting livestock and human beings, has become a global public health hazard with economic consequences.

Aims: The current study was designed to investigate the prevailing MRSA-associated subclinical mastitis and associated risk factors in dairy buffaloes. The study also highlighted the genetic variations and in silico-based proteomic differences among MRSA isolates.

Methods: Out of 516 milk samples, 45.93% (237/516) were found positive for subclinical mastitis, while the prevalence of S. aureus was recorded 56.12%. The methicillin resistance in S. aureus isolates was evaluated by oxacillin disc diffusion test and molecular identification of the mecA gene.

Results: The results revealed a phenotypic and molecular prevalence of MRSA at 45.11% and 18.79%, respectively. The risk factor analysis revealed that among various assumed risk factors, parity, milking hygiene, milker care during milking, milk yield, housing system, and floor type were significantly associated with subclinical mastitis in buffaloes. The sequencing and phylogenetic analysis showed no significant genetic variations among study isolates and depicted a high similarity with isolates from Africa, USA, India, Italy, Turkey, and Iran. The in-silico protein analysis showed that all sequences had the same protein motifs resembling penicillin protein 2a except Buff-13, whose protein structure resembles alpha-catenin-like protein hmp-1.

Conclusion: The current study was the first report of the genotypic characterization and in silico protein analysis of MRSA from dairy buffaloes in Pakistan. The result highlighted the importance of antimicrobial resistance (AMR) and development of control strategies against MRSA infections.

Keywords: Antimicrobial resistance; Buffaloes; Mastitis; Methicillin-resistant Staphylococcus aureus; Phylogenetic analysis.

Fig. 1

GIS map of the study districts

Fig. 2

Risk factors associated with the occurrence of mastitis in dairy buffaloes

Fig. 3

Multiple clustal W alignment of the local isolates with reported isolates

Fig. 4

Phylogenetic tree showing the relationship of local isolates of MRSA with previous reported isolates

Fig. 5

Nucleic acid alignment of penicillin-binding protein 2a gene

Fig. 6

Conserved motifs of penicillin-binding protein 2a gene

Fig. 7

Structure of penicillin-binding protein 2a gene

Fig. 8

Protein sequence alignment of penicillin-binding protein 2a

Fig. 9

Conserved motifs of protein sequence (penicillin-binding protein 2a sequences)

Fig. 10

(a) Protein structure of reference protein, (b) Protein structure of Buff-12, Buff-4, Buff-3, and Buff-5, and (c) Protein structure of Buff-13

Fig. 11

Protein-protein interaction of the reference protein, Buff-12, Buff-4, Buff-3, and Buff-5 proteins

Fig. 12

Other protein interactions with the reference protein, Buff-12, Buff-4, Buff-3, and Buff-5 proteins