Mastitis is one of the most predominant disease affecting dairy herds worldwide and happens to be the most frequent disease having a huge economic impact on diary industry
(Miller et al., 1993; Ganguly, 2018). Control of mastitis is important owing to its association with reduced production and reproductive performance in addition to the economic loss it imparts
(Wakchaure et al., 2015). Among the various bacterial pathogens implicated in mastitis,
Staphylococcus spp. is the principal etiological agent in cattle and buffaloes
(Sharma et al., 2007). Coagulase negative Staphylococci (CNS) has been considered to be minor pathogens earlier, but now it is considered as an emerging group of pathogens involved in bovine mastitis. Mastitis is one of the most significant reasons for the usage of antibiotics in animals. Antibiotic resistance, the ability of bacteria to resist the effects of antibiotic, is a major impediment for the successful control of mastitis worldwide. Among the various antimicrobial agents which have been approved for use in bovine mastitis, β-lactams, such as cephalosporins and penicillins, play an important role. Major mechanism of resistance against β-lactams in
Staphylococcus is mediated by either β-lactamases encoded by
blaZ gene or the
mecA encoded alternative penicillin binding protein, PBP2a, which exhibits a reduced binding to the β-lactam antibiotics (Aarestrup, 2006). Presence of antimicrobial resistance genes in
S. aureus and other CNS species in cattle may be significant, because it possesses a human health hazard, either due to transfer of resistance genes laterally between staphylococcal species or by direct transfer of resistant pathogens (Walther and Perreten, 2007). Under these circumstances, the present study was conducted to analyze the pattern of antibiotic resistance in
S. aureus and other CNS isolated from cases of bovine mastitis and to characterize the genes responsible for resistance against the β-lactam antibiotics, which are commonly employed for treatment of mastitis. The study will provide a first-hand data regarding the rational usage of antibiotics in field conditions against mastitis.
A total of 30 milk samples were collected aseptically from animals with clinical mastitis presented at Teaching Veterinary Clinical Complex, College of Veterinary and Animal Sciences Thrissur, Kerala, Veterinary Hospital Kokkalai and University Livestock Farm under Kerala Veterinary and Animal Sciences University and also from animals brought to the Veterinary Dispensaries under Animal Husbandry Department. The samples were plated on to blood agar (BA), brain heart infusion agar (BHIA) and were incubated at 37°C for 24 h. The isolates were subjected to identification based on colony characters, morphology on Gram’s staining and biochemical characteristics as per
Quinn et al., (2002). Antibiogram profile of the isolates were observed employing Kirby-Bauer disc diffusion method using the antibiotic discs; methicillin (5 mcg), penicillin G (10 units), cefalexin (30 mcg), cefuroxime (30 mcg), enrofloxacin (10 mcg) and tetracycline (30 mcg) as per CLSI (2011).
Template DNA was extracted from coagulase positive and CNS spp. using Genomic DNA purification kit of Origin (Kerala, India). Polymerase chain reaction assays were standardized using
in-house designed primers for
mecA, and primers for
blaZ gene were selected as per
Martineau et al., (2000). The standardized conditions for the amplification of
mecA and
blaZ genes were depicted in Table 1 and 2. The PCR amplified products were subjected to agarose gel electrophoresis and the results were documented in a gel documentation system. The PCR positive amplicons of
mecA and
blaZ genes were sequenced using respective forward and reverse primers in an automated sequencer using Sanger’s dideoxy chain termination method at SciGenom Labs Pvt. Ltd., Cochin. The obtained sequences were aligned with other sequences in GenBank using BLASTn.
Out of the 30 samples, three were confirmed as
S.aureus and remaining ten were other species of CNS
viz.,
S. hyicus,
S. sciuri,
S. haemolyticus,
S. chromogens,
S. cohnii subspp.
cohnii and
S. caseolyticus. This is in accordance with
Mohanty et al., (2013) who observed that predominant organisms responsible for bovine mastitis were
Staphylococcus followed by
Escherichia coli,
Streptococcus,
Bacillus,
Corynebacterium,
Listeria and
Kliebsiella. Taponen and Pyorala (2009) reported that
Staphylococcus spp. were the common bacteria isolated from cases of bovine mastitis and now CNS is becoming predominant over
S. aureus in most countries. All the isolates when subjected to antibiogram, showed resistance to methicillin and penicillin which are the important beta lactam antibiotics. With cefalexin and cefuroxime, the representative first and second generation cephalosporins, coming under beta lactam group, majority of the isolates showed resistance. DNA was extracted from the isolates and subsequently PCR was conducted with specific primes targeting
mecA and
blaZ gene of
Staphylococcus. Asfour and Darwish (2011) documented that
mecA and
blaZ genes are responsible for methicillin and penicillin reistance in
Staphylococcus isolates.
Paul et al., (2015) documented amplification of
mecA gene in seven out of 20 methicillin resistant
S. aureus isolates from bovine mastitis milk samples. In the present study,
mecA gene was detected in ten out of the thirteen isolates and
blaZ gene was detected in all the thirteen isolates (Fig 1 and 2). Three isolates that showed resistance to methicillin on antibiogram were found to be negative for
mecA gene amplicon in PCR. This variation in phenotypic and genotypic results may be due to the presence of isolates encoding a divergent
mecA gene, referred as
mecC (Hamid et al., 2017). The amplicons of
mecA and
blaZ genes were sequenced and the sequence was subjected to BLAST analysis with the available sequences in the data base, which revealed 100 per cent similarity. The sequences obtained from the present study were submitted to the NCBI database (GenBank Accession No. MH807092 and MH807093 respectively). Based on the results, it was concluded that CNS was now becoming the predominant pathogens involved in bovine mastitis and they were showing a high rate of resistance against beta lactam antibiotics which were the most common category of drugs employed in mastitis therapy.
