2.1 Semen collection for bacteriological study
Total 48 semen ejaculates were collected from different goat breeds (Barbari, n= 12; Jamunapari, n= 12; Jakhrana, n= 12; Bundelkhandi, n=12) with good health condition, which were randomly selected during the breeding season, and maintained under semi-intensive system of rearing with same managemental conditions throughout this study. The whole experiment is carried out as per the guidelines of the Institute Animal Ethics Committee (IAEC).
2.2 Microbiological evaluation of semen
The fresh semen samples were inoculated into nutrient broth separately, and incubated at 37ºC overnight. The broth culture was inoculated on the nutrient agar and mannitol salt agar according to the method described by Mishra et al. (2014).
For identification of bacteria (Staphylococcus aureus), Gram’s stain was performed according to the method described by Merchant and Packer (1967). For identifying coagulase positive staphylococci, coagulase test was performed using rabbit plasma as per the standard protocol. For coagulase test, a well isolated staphylococcal colony is to be emulsified in a drop of water on a clean and grease free glass slide with minimum of spreading. A flamed and cooled straight inoculating wire is dipped into the undiluted plasma at room temperature, and the adhering traces of plasma (not a loopful) is mixed into the staphylococcal suspension on the slide. In positive case, a coarse clumping of cocci is visible to the naked eye within 10 seconds.
2.4 Molecular characterization of Staphylococcus spp.
All the 36 isolates of Staphylococcus spp. were subjected to DNA extraction using Nucleo-pore Fungus Bacteria genomic DNA kit (Cat#NP-7006D) following the manufacturer’s instructions. The DNA was checked for quality and quantity using QuantusTM fluorometer using the Quanti Fluor® ONEdsDNA system (E4871) following the manufacturer’s instructions. Multiplex PCR was performed as described by Shome et al. (2011) with the oligonucleotide sequences and other details as described in the Table 1.
The primers are used in the specified concentration as mentioned above along with 2x Emerald GT amp master mix (TAKARA, Japan), along with 1μl template DNA (equivalent to approx. 2-3ng of bacterial genomic DNA) to make up the total volume of reaction to 25μl. The PCR was set-up with the following thermal conditions:
2.5 Anti-microbial resistance studies for Staphylococcus spp. isolates from buck semen
The confirmed coagulase negative Staphylococci (CoNS) and coagulase positive Staphylococci (CoPS) isolates were then subjected to β-Lactamase production and Methicillin resistant Staphylococcus aureus (MRSA) typing using both phenotypic and genotypic tests. All the antimicrobial tests were performed according to CLSI standards (CLSI, 2013) and their break-points.
2.5.1 β-Lactamase production in Staphylococcus spp by Penicillin zone edge test
This test was performed by using disk diffusion test by placing penicillin (10U) disk, and incubating at 35°C for overnight (16-18 hours) incubation. The tests are interpreted by observing the edges of inhibition zones which appear as sharp edge (cliff-like) as β-Lactamase positive and fuzzy edge (beach-like) as β-Lactamase negative.
2.5.2 Methicillin resistant Staphylococcus aureus (MRSA) typing using phenotypic tests
The phenotypic MRSA test was performed using the disk diffusion test employing cefoxitin disk (30 μg) for S. aureus, and CoNS. The inoculum was prepared, and spread on the Mueller Hinton agar (Oxoid, Thermofisher), and the antibiotic disk was placed over the inoculated plate after 15 min, and incubated at 35°C for 18 hours (S. aureus), and 24 hours (for CoNS). The cefoxitin disk (30 μg) with a minimum zone of inhibition (ZoI) of 22 mm and above is indicative of MRSA, and less than 22 mm is not indicative of MRSA. Similarly, for CoNS (except S. pseudointermedius and S. schleiferi) the ZoI should be ≥25mm for MRSA.
2.5.3 Methicillin resistant Staphylococcus aureus (MRSA) typing using Molecular methods
All the CoPS and CoNS isolates were subjected to genotypic MRSA detection using the genes viz., mecA and mecC (Table.2.). The primers used and their thermal cycling conditions are mentioned below.
The primers were used at a volume of 1μl (10pMol/μl) each of the forward and reverse primer along with 1μl template DNA (equivalent to approx. 2-3ng of bacterial genomic DNA), in 12.5μl of 2x Emerald GT amp master mix (TAKARA, Japan), and nuclease free water added in rest of volume to make up the total volume of reaction to 25μl. The PCR was set-up with the following thermal conditions illustrated below for both the MRSA genes.
2.5.4 Vancomycin resistant Staphylcoccus aureus (VRSA) detection by genotypic test.
All the isolates of Staphylococcus (n=36) were subjected to VRSA detection using two sets of genes viz., VanA and VanB. The details of the primers and their conditions are described in Table.2. The concentration and volume of the primers and reagents including the thermal conditions were similar to the previous section