ANTIBIOTIC RESISTANCE AND PHYLOGENETIC PROFILING OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) FROM BOVINE MILK
Dr. Sujata Bhatkar
Abstract
Antimicrobial resistance (AMR) poses a significant public health threat, necessitating a “One Health” approach considering human, animal, and environmental reservoirs. In the present study, out of 50 bovine milk samples, 24 (24%) were confirmed as S. aureus, with 90% resistant to methicillin. 77.77% isolates with methicillin resistance were found to harbour mecA gene.The representative -positive samples were subjected to mec A gene sequencing and phylogenetic analysis. Sequence of isolates showcasing (96-98%) homology with the sequence obtained from human (Homo sapiens). Antimicrobial resistant bacteria in their environment may be more common and spread due to husbandry procedures used in the dairy farming business, as suggested by the findings of this study. The tendency for higher rates of resistance among strains from bovine milk may be important for future decision-making around milk plants or milk holders .
Introduction
Antimicrobial resistance (AMR) is a significant health issue resulting from the persistent use and abuse of antimicrobials. The majority of infections around the world are caused by Staphylococcus aureus, a common foodborne bacterium that can be found in fresh and ready-to-eat foods. (Diep et al., 2006). Milk, a vital food rich in proteins, vitamins, and minerals, is often linked to the growth of Staphylococcus aureus, a potential cause of food poisoning (Pandey et al., 2014). The use of methicillin in clinical settings and food animal production facilities has led to a significant rise in the isolation of Methicillin-resistant Staphylococcus aureus (MRSA) strains (Oliveira et al., 2002). The study utilizes microbiological and biochemical analysis to confirm and characterize Staphylococcus aureus isolates, DNA sequencing, multiple sequence alignment, and phylogenetic tree construction to reveal genetic variation and evolutionary relationships among them (Janda et al., 2007).In this study, we compare the rates of AMR determined S. aureus isolates from local milk holders in Nagpur region to assess any differences such farming systems may have on the emergence of AMR in this ubiquitous and model organism.
Methodology
50 bovine milk samples from Nagpur region were tested for S. aureus on Baird Parker agar, supplemented with egg yolk and potassium tellurite, and incubated overnight at 37℃ ( Ghabbour et al., 2022). The presumptive positive isolates were confirmed using nuc gene-based polymerase chain reaction (Ghabbour et al.,2022). The final protocol for PCR reaction was finalized as 12.5 µL of 2X PCR mastermix, 1 µL (20 pmol )of each primer, 5 µL(10 ng) of DNA template and final volume of 25 µL reaction was made using nuclease free water. The amplification was carried out in a Thermal cycler (Eppendrof, Germany) with a pre-heated lid with an initial denaturation at 94°C for 5 min. followed by 35 cycles consisting of denaturation at 94°C for 30 sec, annealing at 60°C for 60 min, extension at 72°C for 1 min and a final extension at 72°C for 10 min. Cooling was allowed at 4 oC .The PCR, products were removed from the Thermocycler Pros and were kept at – 20°C until further analysis by agarose gel electrophoresis.
All the confirmed isolates were subjected to in vitro antibiogram using a number of antibiotics according to the guidelines of Clinical and Laboratory Standards（CLSI 2018). Isolates which showed resistance against methicillin were characterized for the presence of mecA gene (McClure et al., 2006). The final protocol for PCR reaction was finalized as 12.5 µL of 2X PCR mastermix , 0.24 µM of forward and reverse primer, 2 µL(10 ng) of DNA template and final volume of 25 µL reaction was made using nuclease free water. The amplification was carried out in a Thermal cycler (Eppendrof, Germany) with a pre-heated lid with an initial denaturation at 94°C for 5 min. followed by 35 cycles consisting of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 30 sec and a final extension at 72°C for 7 min. Cooling was allowed at 4 oC The PCR, products were removed from the Thermocycler Pros and were kept at – 20°C until further analysis by agarose gel electrophoresis as described in 3.4.2. About 5 µL each PCR product was visualized using 1.2 % agarose prepared in TBE.
One representative-positive samples (M5) was subjected to mec A gene sequencing and phylogenetic analysis along with poultry meat and chevon samples tested (P4, G11). Phylogenetic analysis of Staphylococcus aureus gene sequences for respective antibiotic resistance genes through Maximum likelihood methods were carried out using MEGA X software. It reveals different clade showing their evolutionary relationship within different isolates of Staphylococcus aureus and the sequences which lie in the same cluster are closely related (Kumar et al.,2020).
Results
15/50 (30%) bovine milk samples yielded S.aureus on Baired Parker agar and 12/50 ( 24%) samples were confirmed using nuc gene-based PCR for molecular confirmation of S. aureus .(Plate 2) 
The in vitro antibiogram revealed that 9/15 (60 %) S. aureus isolates were resistant to methicillin. (Plate 3)
Furthermore, the study found that 7 out of 9 (77.77%) bovine milk samples were found to possess mecA gene which generated amplicon size of 310 bp. (Plate 4)
Sequence of isolates M5 and G11 showcasing (96-98%) homology with the sequence obtained from human (Homo sapiens) of Australia and USA. (Figure 1)
Fig. 1. Phylogenetic analysis of partial gene (mec A) of samples P4, G11, M5 with Indian and world isolates using MEGA X
Conclusion
Assessing the origin and prevalence of AMR is a priority in today’s climate of escalating AMR concern. As previously mentioned, antibiotics are used extensively in dairy farms, leading to the introduction of AMR genes in milk and dairy products. In this perspective, the growing preference for raw milk, which has already demonstrated to pose a consumer risk due to the presence of foodborne pathogens, is even more concerning.Our data confirms the alarming increase in the prevalence of community-acquired MRSA in bovine milk from local milk-holders of Nagpur region and its emerging multiple antibiotic resistances in foods as serious problem for public health.
Also, highlighted their significant public health hazards linked to the consumption of those foods contaminated with S. aureus, particularly milk, chevon and poultry meat. Investigation of the presence of AMR genes in the milk production environment provided precious data to estimate the public health risk associated with antibiotic usage in dairy industry.
References
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