Molecular detection of methicillin-resistant staphylococcus aureus from bovine mastitic milk and milker hand swabs

Introduction

On one hand, Staphylococcus aureus is the most frequently isolated pathogen from bovine mastitis worldwide, responsible for chronic intramammary infections that reduce milk yield and quality [1] . On the other hand, the same organism is a leading cause of hospital-acquired and community-acquired infections in humans, where methicillin-resistant strains (MRSA) complicate treatment and raise mortality [2] . The overlap between these two epidemiological realities becomes a tangible public health concern whenever dairy workers handle infected udders without gloves and then touch their own skin, food, or family members. MRSA is defined by the acquisition of the mecA gene, which encodes an altered penicillinbinding protein (PBP2a) with low affinity for all β-lactam antibiotics, rendering the organism resistant to the entire class from penicillin through carbapenems [3] . The mecA gene is carried on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec), which can transfer horizontally between strains and even between Staphylococcus species, facilitating the spread of resistance across ecological niches [4] . In Nigeria, where an estimated 70% of dairy production occurs in smallholder pastoral and peri-urban systems, milking is done exclusively by hand, often without teat disinfection or personal protective equipment [5] . This practice creates repeated, high-contact opportunities for bacterial exchange between the cow's teat canal and the milker's skin. Previous Nigerian research has documented S. aureus prevalence in mastitic milk ranging from 30% to 55%, but molecular data on mecA carriage are limited to a few reports from northern states [6,7] . Lagos State, with its dense peri-urban dairy belt supplying raw milk to millions of consumers, has not been surveyed for livestock-associated MRSA (LA-MRSA) using PCR-based methods. Bridging this gap matters for two reasons. First, identifying the mecA gene by PCR provides definitive confirmation of methicillin resistance that phenotypic disc diffusion alone can miss in borderline cases [8] . Second, paired sampling of milk and milker hands from the same farm can reveal whether identical resistant clones are circulating between animals and humans, evidence that strengthens the case for One Health interventions. This research set out to determine the prevalence of MRSA in bovine mastitic milk and milker hand swabs across peri-urban dairy farms in Lagos State, confirm resistance genotypically through mecA PCR, characterise the antimicrobial susceptibility profile of confirmed MRSA isolates, and assess the concordance of paired animal-human isolates as a measure of zoonotic transmission risk.

Material and Methods

Reagents and Chemicals

Mannitol salt agar (MSA), brain heart infusion broth (BHI), Mueller-Hinton agar (MHA), rabbit plasma for the coagulase test, and DNase agar were obtained from Oxoid (Basingstoke, UK). Antibiotic discs (penicillin 10 U, oxacillin 1 µg, cefoxitin 30 µg, tetracycline 30 µg, erythromycin 15 µg, ciprofloxacin 5 µg, gentamicin 10 µg, trimethoprimsulfamethoxazole 25 µg, clindamycin 2 µg, chloramphenicol 30 µg, vancomycin 30 µg, linezolid 30 µg, rifampicin 5 µg) were sourced from HiMedia (Mumbai, India). DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Germany). PCR reagents included DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, USA), mecAspecific primers (forward: 5′-GTAGAAATGACTGAACGTCCGATAA-3′; reverse: 5′-CCAATTCCACATTGTTTCGGTCTAA-3′; amplicon 310 bp) [8] , and S. aureus ATCC 43300 as a mecA-positive control.

Material

Methods

Milk and swab samples were streaked onto MSA plates and incubated aerobically at 37°C for 24-48 hours. Yellow colonies (mannitol fermenters) were subcultured on blood agar and subjected to Gram staining (Gram-positive cocci in clusters), catalase test (positive), tube coagulase test (positive), and DNase test (positive) for definitive S. aureus identification [1] . Confirmed isolates underwent phenotypic MRSA screening by cefoxitin disc diffusion on MHA following CLSI 2023 guidelines: zone diameter ≤21 mm was classified as methicillin-resistant [8] . For molecular confirmation, genomic DNA was extracted from overnight BHI cultures using the QIAamp kit. mecA gene amplification used the primers above under these conditions: initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 60 s; final extension at 72°C for 7 min. Amplicons were resolved on 1.5% agarose gel stained with ethidium bromide and visualised under UV transillumination. Antimicrobial susceptibility of all mecApositive MRSA isolates was determined by the Kirby-Bauer disc diffusion method on MHA, interpreting zone diameters per CLSI breakpoints. Data were analysed in SPSS 28.0. Prevalence was expressed as percentages with 95% CI. Chisquare or Fisher's exact test compared categorical variables. Concordance of paired milk-hand mecA status was assessed by Cohen's kappa.

Quality Control

Quality assurance measures were embedded at every stage. Culture media were batch-tested with S. aureus ATCC 25923 (methicillin-susceptible) and ATCC 43300 (methicillinresistant) reference strains before use. Each PCR run included a positive control (ATCC 43300 DNA), a negative extraction control (reagent blank processed alongside samples), and a no-template control (nuclease-free water). Gel electrophoresis included a 100 bp DNA ladder (Thermo Fisher) on every gel. Antibiotic susceptibility testing was validated weekly against ATCC 25923 reference ranges. Any isolate with discordant phenotypic and genotypic results (cefoxitin-resistant but mecA-negative, or vice versa) was re-tested in duplicate before final classification.

Ethical Considerations

Informed consent was obtained from each milker before hand swab collection. Participants were assured of confidentiality and received individual MRSA screening results with a brief educational pamphlet on hand hygiene. Milkers identified as MRSA carriers were referred to the nearest primary health centre for clinical evaluation. Animal sampling followed routine veterinary practice and caused no pain beyond standard CMT and milk collection. Farm owners received aggregated mastitis prevalence results for their herds with recommendations for teat dipping and dry cow therapy.

Results

Comprehensive Interpretation

The resistance profile (Figure 3) paints a concerning picture: MRSA from both milk and hands showed near-universal penicillin and oxacillin resistance, high tetracycline and erythromycin resistance, and multi-drug resistance patterns (resistance to ≥3 antibiotic classes) in 84.6% of milk isolates and 77.8% of hand isolates. Critically, all isolates retained susceptibility to vancomycin and linezolid the last-resort agents for systemic MRSA infection in humans. The similarity of resistance profiles between milk and hand isolates from the same farm supports active bidirectional transmission. Among 18 farm-level paired sets where both milk and hand MRSA were detected, 11 (61.1%) shared identical antibiograms, consistent with a common clonal origin.

Discussion

The overall mecA-confirmed MRSA prevalence of 20.8% in this Lagos peri-urban dairy survey exceeds the 12-15% range reported from northern Nigerian states [6, 7] and the 9.4% recorded in a Kenyan dairy chain investigation [9] . Higher prevalence in Lagos may reflect the density of peri-urban farming, where farms share grazing corridors and livestock markets that facilitate strain circulation [10] . The tropical humid climate (≥70% RH during the rainy season) may also prolong bacterial survival on skin and milking utensils, sustaining transmission chains that would fade faster in drier environments.

The concordance between phenotypic cefoxitin screening and mecA PCR (κ=0.80) was reassuring and aligns with published validation data from European and North American laboratories [8,11] . The seven phenotype-positive but mecAnegative isolates likely harbour mecC (a mecA homologue found in LA-MRSA CC130 lineages) or express borderline resistance through β-lactamase hyperproduction [12] . Screening for mecC was beyond the scope of this research but should be included in future surveys. The multi-drug resistance profile with over 80% of MRSA isolates resistant to three or more antibiotic classes limits treatment options for both bovine mastitis and potential human infections. That vancomycin and linezolid remain effective is encouraging but should not breed complacency; vancomycin-intermediate S. aureus (VISA) has been reported from neighbouring West African countries [13] . Implementing basic milking hygiene pre-milking teat disinfection, disposable gloves, post-milking teat dip costs less than USD 0.05 per milking session and could interrupt the transmission circuit at its most vulnerable point [14] .

Conclusion

This research set out to determine the prevalence of MRSA in bovine mastitic milk and milker hand swabs from peri-urban dairy farms in Lagos State, confirm methicillin resistance at the molecular level through mecA gene PCR, profile the antimicrobial resistance patterns of confirmed MRSA isolates, and assess the evidence for zoonotic transmission between cattle and their handlers.

The key findings can be stated directly.

Acknowledgement

Acknowledgements Contributions Not Qualifying for AuthorshipThe authors thank field veterinarians Dr. Funke Adeyemi and Dr. Emeka Obi for coordinating farm visits and sample collection across the four local government areas.

References

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