Molecular Epidemiology and Antimicrobial Resistance of Methicillin Resistant Staphylococcus aureus (MRSA) from Milk and Nasal Samples of Dairy Buffalos in Nueva Ecija, Philippines

Abstract

Methicillin Resistant Staphylococcus aureus (MRSA) is a zoonotic pathogen that carries the mecA gene responsible for the loss of susceptibility to methicillin and other βlactam antibiotics. Only MRSA human rate has been reported in the Philippines and baseline

data

on MRSA prevalence in livestock is lacking. Moreover, dairy buffalos in the Philippines contribute to 35% of local milk production, thus, there is a need to examine if its milk or the cows’ nasal carriage does not pose public health hazard from MRSA. This study also determined the prevalence, the related risk factors, antimicrobial resistance, molecular characteristics and genetic relationships of MRSA isolates. A total of 93 CMT positive composite milk and 240 quarter milk and 384 nasal swab samples were examined. Gram staining and biochemical tests were used to identify S. aureus. Cefoxitin disc diffusion test and PCR detection of mecA gene were used to determine methicillin resistance. Antimicrobial resistance was tested using the disc diffusion method. The molecular characteristics of MRSA isolates were determined using SCCmec, spa and MLST typing and genetic relationships among MRSA isolates was identified based on spa sequences. Data related to cow-level and herd-level factors were analyzed to determine the associated risk for the occurrence of MRSA in mastitis infected dairy buffalos.

The prevalence of S. aureus on composite milk, quarter milk and nasal swab samples were 41.94% (39/93), 12.92% (31/240) and 7.03% (27/384), respectively. The phenotypic prevalence of MRSA was 25.81% (24/93) for the composite milk, 6.67% (16/240) for the quarter milk and 4.17% (16/384) for the nasal swab samples. However, mecA gene was only detected on 37.50% (9/24) of the composite milk, 6.25% (1/16) of the quarter milk and 18.75% (3/16) of the nasal swab samples. All MRSA isolates from composite milk samples were resistant to penicillin while majority (62.50%) exhibited resistance to erythromycin. A total of 9 resistance patterns were observed for composite milk isolates with 37.50% (9/24) considered MDR. All quarter milk isolates were resistant to penicillin and 81.25% showed intermediate susceptibility to erythromycin. These isolates displayed 4 resistance patterns and 25% (4/16) were MDR. The nasal isolates showed 100% resistance to penicillin, 81.25% resistance to clindamycin and 56.25% resistance to rifampicin. Multidrug resistance was observed on 87.50% (14/16) of the nasal MRSA isolates with a total of 14 resistance patterns recorded.

The cow-level MRSA prevalence was 25.81%. Cows having a history of mastitis were found to have a significant association with the occurrence of MRSA in dairy buffalos. The odds of having MRSA infection was 3.18 times more likely to those with previous history of mastitis compared to those without record (CI=1.03-9.79,p=0.040). Herd-level MRSA prevalence was recorded to be 35.29%. Herd size was considered to have a significant association (p<0.05) and the presence of other animals in a herd was considered to have highly significant association with MRSA infection (p<0.01). Herds with more 6 animals were 4.24 times more likely to have MRSA as compared to herds with less than or equal to 6 animals (OR=4.24, CI=1.05-17.07, p=0.042). Dairy herds that kept other animals were less likely to have MRSA compared to those without other animals in the herd (OR=0.15, CI=0.04-0.58, p=0.006). The SCCmec types of the 13 MRSA isolates were SCCmec types IVc (6), type II (1), type I (1) and untypeable (5). The spa and ST types identified for the milk samples were t019 (ST30), t1939 (ST12), t701 (ST1649), t311 (ST5), t657, t7867 (ST1148), t015 (ST508), t138 (ST5991) and t1642 (ST5992) while nasal carriage were t800 (ST9) and t091 (ST2454). Two novel ST types (ST5991 and ST5992) were identified in this study. The phylogenetic analysis showed a related spa sequences on nasal and milk isolates suggesting that nasal carriage is a possible transmission pathway for milk contamination in dairy buffalos.

There is relatively high prevalence of MRSA observed in mastitis infected composite milk samples but low prevalence from quarter milk and nasal carriage of dairy buffalos and with low detection rate of mecA gene. Although resistance to antibiotics was observed, there is still a wide range of antibiotic options for the treatment of mastitis in dairy buffalos. The relatively higher multidrug resistance rate from nasal carriage could pose zoonotic threat from possible shedding of resistant MRSA in the dairy farm environment. Significant associated risk factors should be addressed with proper herd and cow management to reduce mastitis infection cause by MRSA. The identification of CAMRSA and HA-MRSA strains from molecular characterization of MRSA isolates in dairy

buffalo production suggests a possible human to animal transmission.