A total of 237 milk samples were collected from cows and she buffaloes with different parity, age and postpartum period affected with mastitis, randomly from different regions of Andhra Pradesh during the period from April 2019 to September 2019. Milk samples were streaked on Mannitol salt agar (MSA) and Mac Conkey agar (MAC) plates and incubated at 37ºC for about 48 hr. Single colonies were identified based on Gram’s staining, colony morphology and catalase test. The Staphylococcus species were further differentiated by tube coagulase test.

Genomic DNA was isolated using lysis method (boiling method). Three ml of overnight broth culture was taken and cells were pelleted by centrifugation at 10000 rpm for 10 min. The pellet was washed with PBS by centrifugation at 10000 rpm for 10 min. The pellet was resuspended in 200 µl of TE buffer by gentle mixing and boiled at 95 °C for 20 min. This was followed by snap chilling at−20 °C for 10 min.

The suspension was centrifuged at 10000 rpm for 10 min and the resultant supernatant containing DNA was stored at−20 °C for use in PCR protocols.

All the Staphylococcal isolates were checked for their ability to produce biofilm using modified Congo red agar (Mariana et al., 2009). Congo Red Agar plates were inoculated with the Staphylococcal isolates and incubated at 37 °C for 48 hr initially and subsequently at room temperature for 2-4 days. The slime producing strains form strong black pigmentation to slightly black colonies, which indicates positive for biofilm production, where as non-slime producers develop red colonies which indicates negative for biofilm production.

The Staphylococcal isolates that were found to form biofilm on Congo Red agar were screened for the presence of biofilm genes viz., icaA, bap, icaAB and aap, which are majorly associated with the biofilm production. The oligonucleotide primers, PCR cycle conditions and the corresponding amplicon sizes for the biofilm genes were mentioned in Table 1.

First the isolates were screened for icaA and bap genes in duplex PCR as per the method of Janeczko et al. (2014). Further, the Staphylococcal isolates that showed positive biofilm phenotype were also screened for the presence of icaAB gene (Iorio et al., 2011) and aap gene (Srednik et al., 2017) in two separate PCR tests.

For all the Staphylococcal isolates, antimicrobial susceptibility test was performed by the standard Kirby Bauer disc diffusion method on Muller Hinton agar according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2014). The isolates were tested for susceptibility to the following antibiotics viz., cefoxitin (CX30), ceftriaxone (CTR30), ciprofloxacin (CIP5), gentamicin (GEN10), oxacillin (OX1) and penicillin G (P10). CoNS isolates were tested for susceptibility to novobiocin (NV5) also. Cefoxitin (CX30) is used as a surrogate for mecA mediated methicillin (oxacillin) resistance.

For Staphylococcal isolates, mecA and mecC (mecA homologue) genes that confer resistance to the b- lactam antibiotics were targeted. The oligonucleotide primers, PCR conditions and the corresponding amplicon sizes for the antibiotic resistance genes were mentioned in Table 1. All the Staphylococcal isolates were screened for the presence of mecA gene as per the method of Vishnu priya et al. (2014). The isolates were further subjected to mecC PCR as per the method of Paterson et al. (2012).

Biofilm formation on Congo red agar was observed in 44.8 per cent (56/125) of the Staphylococcal isolates from the present study. The incidence of biofilm phenotype was relatively higher in CoNS isolates (37/80, 46.25%) than CPS (19/45, 42.22%) (Table 2). In contrast to this, Arciola et al. (2001) reported that the incidence of biofilm formation tested with CRA was higher in S. aureus (61%) than S. epidermidis (49%) isolates.

Out of the 56 Staphylococcal isolates that were found to form biofilm on Congo red agar only four (7.2%) were found to possess the bap gene as they showed the amplified product of 971 bp (Fig. 1). The other biofilm genes icaA, icaAB and aap were not detected in any of these 56 isolates. Although high percentage of the Staphylococcal isolates were phenotypically positive for biofilm production on CRA, biofilm gene (bap) is detected only in four of these isolates.

Among the CPS isolates (45), resistance was high to gentamicin (45, 100%) followed by penicillin (13, 28.8%), cefoxitin (10, 22.2%) and oxacillin (4, 8.8%) and all of them were susceptible to ceftriaxone and ciprofloxacin. Among the CoNS isolates (80), resistance was observed to gentamicin (74, 92.5%), novobiocin (43, 53.8%), cefoxitin (39, 48.8%), penicillin (36, 45%), oxacillin (35, 43.8%). Most of the CoNS isolates were found susceptible to ceftriaxone and ciprofloxacin (Table 3). Resistance to novobiocin was observed in 53.8% (43/80) of the CoNS isolates. Among 45 CPS and 80 CoNS isolates, 10 CPS and 39 CoNS showed resistance to cefoxitin and hence they were regarded as methicillin resistant (MRSA and MRCoNS) as per CLSI recommendations.

Majority of the CPS and CoNS isolates from our study (around 96%) were susceptible to ciprofloxacin and ceftriaxone, but most of them were resistant to gentamicin (100% of CPS and 92.5% of CoNS). Resistance to cefoxitin was observed to be high among CoNS isolates (48.8%) when compared to CPS isolates (22.2%). Similarly, high percentage of CoNS isolates (43.8%) were oxacillin resistant than CPS isolates (9%). In contrast, Frey et al. (2012) observed low resistance to gentamicin (2.4%) and penicillin (23%) among CoNS isolated from bovine mastitis in Switzerland.

A previous study on bovine mastitis from our laboratory reported high sensitivity of Staphylococcus (CPS and CoNS) to ciprofloxacin, ceftriaxone, gentamicin and 50 per cent resistance to cefoxitin (Usharani, 2016). Contrastingly, Sumathi et al. (2008) observed that gentamicin was the most effective drug against Staphylococci, followed by ciprofloxacin. Chandrasekharan et al. (2014) also observed high sensitivity of Staphylococcal isolates to enrofloxacin followed by gentamicin and ceftriaxone. Development of resistance against a particular antibiotic in a specific region might be due to frequent and long term use and under dosage (Sabour et al., 2004, Moon et al., 2007 and Kumar et al., 2010).

In PCR for mecA and mecC genes, only eight isolates (8/125, 6.4%) of Staphylococci were found to possess mecA gene with amplicons of 310 bp (Fig. 2) and they were confirmed as methicillin resistant. Out of these 8 mecA positive isolates, 3 were methicillin resistant S. aureus and the remaining 5 were methicillin resistant CoNS isolates. Out of these 8 isolates, 3 were coagulase positive and 5 were coagulase negative and in none of the isolates, the expected amplicon product of 188 bp was found and hence it is considered that they were all not carrying mecC gene.

Out of the 49 Staphylococcal isolates that were phenotypically identified as positive for mecA mediated oxacillin resistance in the cefoxitin disc test, mecA gene was detected only in 6 isolates and other 2 isolates that were found to carry mecA gene were phenotypically susceptible to cefoxitin. None of the Staphylococcal isolates were positive for mecC gene indicating no prevalence of methicillin resistance by harboring mecC gene. All the eight mecA positive isolates were resistant to gentamicin and only six of them were resistant to cefoxitin and 5 to penicillin. But only 2 isolates were resistant to oxacillin, the other 6 mecA positive isolates (3 CPS, 3 CoNS) were found susceptible to oxacillin (1 mcg) disc.

In a similar study, Piessens et al. (2012) reported low incidence of mecA gene (11.7%) from 366 CoNS isolated from milk. Srednik et al. (2017) also reported low incidence of mecA gene (4) compared to blaZ gene (21) among CoNS (87) isolates from bovine mastitis. Srednik et al. (2015) also reported low incidence of mecA gene (6) when compared to blaZ gene (19) in CoNS (93) isolates from bovine mastitis.

According to the results of the present study, the prevalence of methicillin resistant phenotype was considerably high among CoNS (39/80, 49%) though only 6.25 per cent (5/80) of them carried mecA gene and could be confirmed as MRCoNS. As indiscriminate use of antibiotics was one of the reasons for development of resistance, there is necessity for the implementation of in vitro antibiotic susceptibility test prior to the use of antibiotics in the treatment and prevention of intra mammary infections. Further investigation to establish the molecular basis of antibiotic resistance and biofilm potential is required.