Prevalence, molecular characterization and antimicrobial resistance pattern of Staphylococcus species isolates from buck semen

Staphylococcus aureus is one of the most prevalent pathogens, and a causative agent of a variety of infections in humans and animals. A total of 48 semen samples were collected from healthy bucks of different breeds to investigate the prevalence of S. aureus . Antimicrobial resistance and virulence of the Staphylococcus isolates were determined to assess the adverse effects of them on buck fertility. The bacterial isolates were tentatively conrmed as Staphylococcus spp. based on the Gram’s staining, growth on Mannitol salt agar and catalase test. Overall, 75% (n = 36) of the samples were positive for Staphylococcus spp. from the total 48 buck semen ejaculates from different breeds. Out of 36 staphylococcal isolates, 23 (47.92%) were coagulase negative (CoNS) and 13 (27.08%) were coagulase positive Staphylococcus (CoPS) based on the slide coagulase test. In the current study, on the basis of molecular characterization, we identied S. aureus , S. chromogenes, S. haemolyticus, S. sciuri, S. simulans and S. epidermidis amongst the staphylococcal isolates in the buck semen. This study revealed a high prevalence of Staphylococcus species in semen of the healthy bucks. The isolates exhibited varying degrees of multidrug resistance genotypically as well as phenotypically. The presence of antibiotic resistance and virulence genes may pose a potential threat to reproductive health of animals, highlighting the need for vigilant monitoring of these isolates at the time of semen cryopreservation. out in goats. In the current study, the analysis showed, 75% (n = 36) of the samples were positive for Staphylococcus spp. from the total 48 buck semen ejaculates from different breeds. Out of 36 staphylococcal isolates, 23 (47.92%) were coagulase negative (CoNS) and 13 (27.08 %) were coagulase positive Staphylococcus (CoPS) based on the slide coagulase test. In the current study, on the basis of molecular characterization, we identied S. aureus, S. chromogenes Findings of this study throw light in the darkness of pathogenicity of Staphylococcus spp. present in buck-semen.


Introduction
Bacteria are present in every ejaculate of semen, and the aim to achieve sterility is paramount nonachievable task. Bacteria may gain access to the semen as a result of systemic infections due to bacteraemia, or due to local infections in parts of the genital tract, and in ammation genito-urinary tract.
Another possible source can be due to the bacterial contamination during collection of ejaculate that gets contaminated from the preputial saprophytic ora, external sources like fecal bacteria, environment and other substances of animal origin present in the diluents and buffers or equipment, human personnel etc.  (Fitzgerald 2012). Therefore, it is imperative to perform surveillance at the interface between human and animal hosts to explore human health risks (Cuny et al. 2015). Nowadays, the emergence of multidrug resistant (MDR) strains poses several challenges to the clinical facilities. In particular, the increasing prevalence of antimicrobial resistant S. aureus serves as a threat to the healthcare system (Hammad et al. 2012). On the other hand, most of S. aureus strains are able to produce a large number of virulence factors, including staphylococcal enterotoxins (SEs), exfoliative toxin (ET) and toxic shock syndrome toxin-1 (TSST-1). Moreover, the production of SEs is particularly signi cant, as the ingestion of the preformed toxins is a major cause of foodborne poisoning worldwide (Basanisi et al. 2017).
These pathogens, in one way or other, may infect inseminated females or contribute to a rapid deterioration in sperm quality (Al-Kass et al. 2019). Nevertheless, scientific evidence based on published data concerning the major risk pathogens or bacterial contamination of the germplasms used in goat AI operations in India, has been lacking until recently. The aim of the present study was to investigate the prevalence, and perform molecular characterization of Staphylococcus isolates from the semen samples of bucks. Furthermore, we evaluated the antimicrobial susceptibility, resistance genes and virulence genes of these isolates. Moreover, this type of work has not been carried out in goats. Findings of this study throw light in the darkness of pathogenicity of Staphylococcus spp. present in buck-semen.

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).

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 identi cation 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 emulsi ed in a drop of water on a clean and grease free glass slide with minimum of spreading. A amed 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.

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 Quantus TM uorometer 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 speci ed 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 con rmed 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.

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.

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, Thermo sher), 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.

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.

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

3.1: Isolation of Staphylococcus from Buck semen
The bacterial isolates were tentatively con rmed as Staphylococcus isolates based on the Gram's staining, growth on mannitol salt agar (Fig.1)  After identi cation of bacteria as Staphylococcus spp., further classi cation of bacteria was done on the basis of molecular characterization (Fig.2). In the current study, we isolated S. aureus, S. chromogenes, S. haemolyticus, S. sciuri, S. simulans and S. epidermidis in the buck semen as given in the Table.5.

Detection of β-Lactamase producing Staphylococcus isolates by penicillin zone edge test
The Staphylococcus isolates were initially screened for the beta-lactamase activity using the penicillin zone edge test (Fig.3). Out of the 36 isolates tested, 7 (19.44%) produced cliff like zone edges suggestive of β-Lactamase positive isolates. Among the β-Lactamase producing Staphylococcus isolates, 2 were coagulase positive and 5 were coagulase negative. A total of 13 S. aureus and 23 CoNS were tested for methicillin resistance using the MecA (Fig.4A) and MecC gene (Fig.4B) conventional PCRs. Of the 13 isolates of S. aureus, 5 were positive for MecA, and 4 were positive for MecC. So, overall the MRSA isolates were 5 as per the MecA gene which was on the higher side. As far as CoNS is concerned, 5 isolates and 2 isolates were detected for MecA and MecC, respectively, and the overall MRCoNS isolates were 5 as per the MecA which was on the higher side (Table.5).
Among the S. aureus isolates, 4 and 3 isolates were found positive for VanA and VanB genes (vancomycin resistant), respectively whereas, among the CoNS, 5 and 6 isolates were positive, respectively (Table.5 and Fig.4C).

Discussion
Infections of the male genitourinary tract may contribute to infertility by adversely affecting sperm function, causing in ammatory disorder, anatomical obstruction, alarmingly initiating leucocyte response with its concomitant oxidative stress (Gangwar et al. 2020). The effects of these conditions may be sperm damage, elevated leucocyte response, poor motility and immature sperms compromising the semen quality. Consequently it leads to compromise of sperm cell function and the whole spermatogenesis (Gangwar et al. 2021). In the present study, we investigated the prevalence and performed molecular characterization of Staphylococcus spp. isolated from buck semen samples to facilitate better understanding of its prevalence in male genital tract and threat associated to it in buck fertility. The current study was conducted to assess the presence of Staphylcoccus species in the semen of bucks which were used for breeding. The semen analysis showed that the CoNS isolates were more in number (47.92%) compared to CoPS (27.08%). Among the Staphylococcus spp. isolates, both coagulase and non-coagulase producing bacteria are of equal importance with the growing pathogenic nature of the later (Zong et al. 2011).The CoNS are opportunistic in nature with all the pathogenic elements (Nasaj et al. 2020) and moreover the MRCoNS isolates are equally important in creating AMR hazard due to the harbouring of SCCmec elements in clinical isolates.
Staphylococcus aureus can colonize the reproductive tract affecting its functions (Kačániová et al. 2020) and the reproductive potential of spermatozoa. Most strains isolated from bull semen were S. aureus and S. epidermidis, and the metabolic products derived from these species were reported to have deleterious effects on the acrosomal integrity and motility (Meyer al. 1980). Earlier a study conducted on ejaculates of ram semen on bacteriological quality yielded Staphylococcus aureus and Staphylococcus epidermidis along with E. coli, Enterobacter spp., Proteus mirabilis in 97% of the samples when stored at 15°C (Yániz et al. 2010). In buffaloes, a similar study was conducted in the past revealed eleven different bacterial species in its semen including S. aureus, S. intermedius, S. epidermidis and coagulase negative Staphylococcus besides other gram-negative bacteria like E. coli, Pseudomonas etc. with the predominant bacteria being S. aureus. S. aureus affects the sperm function by hay wiring the metabolic processes (Boryczko, 1982), thereby reducing its viability. We found that the prevalence of S. aureus strains (Table. 1.) at 75 %, which was higher as compared to the previous reports (Liu et al. 2018). In the present study, S. aureus, S. chromogenes, S. haemolyticus, S. sciuri, S. simulans, S. epidermidis were isolated from the buck semen (Table. 3.). The in uence of gram-positive uropathogenic bacteria on sperm morphology and function has been poorly investigated until now. A previous nding reported that aerobic cocci are present in about 50% of semen samples of male partners in infertile couples (Mehta et al. 2002). One of the most frequently isolated microorganisms from male animals with genital tract infections or semen contamination is Escherichia coli (Diemer et al. 2003) and Staphylococcus spp. (Enwurua et al. 2016). In the current study, besides S. aureus, we have obtained considerable number of isolates of S. chromogenes, S. hemolyticus, S. simulans and S. sciuri based on the multiplex PCR.
Staphylococcus aureus is amongst the most versatile and successful of the human pathogens and is capable of a wide spectrum of infections in the host. It colonizes the mucosal cavities, external ori ces and skin surfaces and causes a variety of suppurative infections and toxic syndromes. Besides this, S. aureus is invariably involved in infertility among humans (Momoh et al. 2011). S. aureus has been observed as causative organism accounting for 68.2% of seminal fluid infections (Emokpae et al. 2009). Similar reports from Okon et al. (2005), where S. aureus was isolated from 62.5% of the seminal fluids. S. aureus has also been reported to be commonly isolated microorganism from cervical samples (Okonofua et al. 1995). Huwe et al. (1998) studied the influence of diferent uropathogenic microorganisms on human sperm motility parameters by means of CASA, and reported that S. aureus retards the sperm motility. Similar study was also done previously by Liu et al. (2002) where significant decrease in sperm motility was found when spermatozoa were co-incubated with S. aureus. The reason behind the retarded motility is due to the inhibition of Mg++ ATPase activity of spermatozoa (Gupta and Prabha 2012). Some authors have suggested that direct interaction between bacteria and spermatozoa facilitates immobilization of spermatozoa (Nunez-Calonge et al. 1998), while others have reported evidence for soluble spermicidal factor produced and secreted by bacteria in the extracellular medium.
AMR is a very important aspect that requires immediate attention in any farm set up. Hence, in the current study, we screened all the isolates of Staphylococcus from buck semen for beta-lactamase production, methicillin resistance and vancomycin resistance. Among the coagulase positive isolates, none of them were MRSA positive, which was an interesting nding. On the other hand, among coagulase negative S. aureus isolates, a total of 6 isolates were found MRSA positive based on the cefoxitin disk diffusion phenotypic test. Similar patterns of MRSA in CoNS have been earlier reported in many instances (Li et al. 2021) and the horizontal transfer of AMR genes from less pathogenic S. epidermidis to S. aureus has been identi ed in certain studies during co-colonization (Li et al. 2021). But the genotypic test has revealed a totally different picture with good number of S. aureus (CoPS) carrying both MecA and its homologue MecC gene, besides the CoNS having equal number of MRSA isolates by the same test. Usually, the genes for resistance and other pathogenic traits of Staphylococcus aureus are present in the mobile genetic elements like pathogenicity islands, plasmids, transposons and staphylococcal chromosomal cassettes (SCCs) which may be exchanged in nature through various phenomena like conjugation, phage-aided transduction, transformation etc. (Wan et al. 2021). Similarly, methicillin resistance in S. aureus is coded in mobile genetic elements called staphylococcal cassette chromosome mec (SCCmec); where the methicillin resistant gene MecA is present that code for the penicillin binding protein 2a. In the current study, as it can be seen that good number of isolates from buck semen were of coagulase negative origin, and a considerable proportion carried the MecA as well as MecC gene with some of them detected as phenotypic MRSAs. On these lines, the SCCs are transferred between staphylococci and there is a huge possibility that MecA positive coagulase negative staphylococci may be a good source for exchange of these elements with pathogenic S. aureus as reported elsewhere (Hanssen and Ericson Sollid 2006). Although its effect on spermatozoa has been well established with spermicidal activity, reduction in sperm quality etc., but the post thaw effect of these MRSA especially during arti cial insemination on breeding does is unknown. Moreover, the animal handlers who collect the semen, the arti cial vagina and other equipments including the vaginal speculum need to be assessed for the presence of MRSA and MRCoNS, including the possibility of horizontal transmission of AMR and other mobile genetic elements from human-animal interaction.
MecC is another important homologue of MecA gene and found associated with several MRSA lineages including the most common lineage 'CC130' (Gómez et al. 2021). MecC has been often associated with domestic animals (Gómez et al. 2014) including livestock like cattle and lactating cows (Schlotter et al. 2014) as well as wild animals (Loncaric et al. 2013), was considered to be zoonotic (Becker et al. 2014). Moreover, the presence of MecC positive isolates in the current study especially in the reproductive tract of small ruminants could be treated as a concern that needs immediate control measures.
Notwithstanding, it is also important to study the lineages of these current isolates bearing the MecC or MecA to associate their origin and their transmission dynamics in the future.
Another important resistance is the vancomycin resistance, which was typed using the VanA and VanB genes. Vancomycin, a glycopeptide antibiotic is the drug of choice for treating patients complicated by MRSA infection, but the last few decades witnessed the development of intermediate (McGuinness et al. 2017) as well as completely resistant strains to vancomycin (Chang et al. 2003;Mukherjee et al. 2021). The current ndings were similar to the MRSA genotyping with almost equal number positive for VanA and VanB in both S. aureus and CoNS isolates. The genotyping PCR gives rst-hand information regarding the potential AMR characteristics of the isolates in a given entity. Also, these special genes might express given the apt environment leading to more damage by horizontal transfer of these genes to highly pathogenic isolates of Staphylococcus.
Generally, the prevalence of these bacteria is noteworthy as they may adhere to spermatozoa and decrease sperm motility (Prieto-Martínez et al. 2014). They can also damage acrosomes by producing toxin and cause DNA damage by producing reactive oxygen species (Morrell 2006). Eventually, this could result in decreased fertility rates and culling of breeding animals, and thus causing considerable financial losses to dairy industry (Moustacas et al. 2010). Hence, it is imperative to meticulously preclude the microbial contaminants from entering semen processing and storage facilities and AI operations (Eaglesome and Garcia 1997).

Conclusion
Arti cial insemination is a very important tool in assisting reproductive technologies in livestock. The quality of the semen with respect to phenotypic and other functional analyses is well taken care of, while the microbial quality is a very important aspect that needs to be thoroughly addressed. Hence in this current study, we did the speciation and Anti-microbial resistance (AMR) analysis of staphylococcus isolates from buck semen. The AMR patterns could help decipher important information about the selection of antibiotics in semen dilutor and in turn could help assess the public health risk due to antimicrobial resistant isolates and their in uence on personnel assisting the semen collection and processing for AI.