1. IntroductionMastitis is one of the most common emerging infections of milking animals, and affects the quantity and quality of milk. It involves the inflammation of mammary glands, the etiology of which can be infectious or non-infectious [1]. It causes irritation in the parenchyma of the mammary organs, typically caused by bacteria attacking the udder, multiplying there, and delivering harmful toxins to the mammary organs [2,3]. Mastitis has challenged economies worldwide, directly or indirectly, including Pakistan. In Pakistan, it has caused an alarming situation, and significant attention needs to be given to its control [4,5]. In dairy cows, mastitis leads to financial crises due to decreased milk production, treatment costs, including antibiotic treatments, and pre-emptive culling [2].Of the hundreds of causes of mastitis known today, the most typical etiological agents reported are Staphylococcus aureus (S. aureus), streptococcus agalactiae, and others [6]. In buffalo, 70–80% cases of mastitis are due to S. aureus [7]. S. aureus is a pathogenic bacterium that is considered a threat to both animals and humans [8,9]. It causes approximately one-third of buffalo mastitis cases. In Pakistan, studies have shown that S. aureus is the primary infectious agent that causes mastitis [10]. For years, erythromycin has been used in treatment for various infections, and has been elected as an alternative to penicillin, cephalosporin, and other beta lactams for Gram-positive microbes [11,12]. However, resistance to erythromycin in S. aureus has prevailed, as its activity has resulted in the production of methylase encoded with the erm genes. Reports demonstrate that the genes ermA, erm B and erm C in bovine mastitis isolates are responsible for erythromycin resistance [13].Antibiotic therapies are frequently ineffective in treating S. aureus infections, due to some of the unique features of the pathogen such as: the ability of the organism to colonize and produce micro-abscesses in the mammary gland which leads to be protected from normal defense mechanisms, the potential of invading bovine mammary epithelial cells, the switching to the small-colony variant (SCV) phenotype and biofilm formation which are relevant to chronic and recurrent infections, the capability of persisting within phagosomes, the ability to convert to L-form when exposed to antibiotics, and the ability to produce toxins [14]. Because of the 2low clearance rate in S. aureus associated mastitis, procedures have been developed to continue treatment for 6 to 8 days to maintain therapeutic levels of antibiotics [15]. Therefore, the overuse of antibiotics has resulted in the rise of multidrug-resistant S. aureus. It is well-known that excessive antimicrobial use is the main driver of antibiotic resistance [3].S. aureus usually causes subclinical mastitis, which is very difficult to treat (due to high seeding rate of infected animals and chronic recurrent infections) [10]. This is probably related to the numerous virulence factors present in this pathogen, such as the ability to produce biofilm, or the production of toxins, or various enzymes designed to damage and better occupy the infected area [16]. On the other hand, it is also related to the occurrence of antibiotic resistance among these bacteria, which has arisen from the overuse of these active substances in veterinary medicine and in agriculture. Although the problem of S. aureus in dairy cattle has been known for years, a perfect preventive therapy or treatment has not yet been developed. This is due to the rapid genetic variability of this pathogen and the lack of knowledge about the interaction between the bacterium and the host. Studies from around the world have shown that there is no clear pattern in the distribution of virulence genes (adhesins or toxins) among bovine isolates [3]. Depending on the area from which the strains originate, a different antibiotic resistance profile can be observed. Such a situation significantly complicates the results of mastitis prevention research and the development of new therapies, and further research towards a better understanding of these bacteria should be conducted [17].The prevalence of S. aureus in bovine mastitis in Pakistan is very high, and the problem is affecting the dairy industry. Many therapeutic strategies to treat mastitis have failed due to the antibiotic resistance shown by S. aureus [18]. Due to the high concern given to zoonotic infection, determination of the phenotypic and genotypic antimicrobial resistance profile of S. aureus recovered from mastitis-infected milk and the nasal carriages of farm personnel is important. Published data in antibiotic resistance-related studies on mastitis-derived S. aureus and on the molecular characterization and phylogenetic analysis of erythromycin resistance in S. aureus recovered from bovine mastitis and farm personnel in Pakistan are sparse. This study investigated strain-relatedness, the presence of genetic determinants of antibiotic resistance, and potential sources of zoonotic infections by applying phenotypic and genotypic methods. This will give an insight into the zoonotic characteristics, sources of origin, and antibiotic resistance profiles of S. aureus in Pakistan, as well as the control methods required. 4. DiscussionBovine mastitis is a clinically important disease found in dairy animals. Pakistan has an abundance of small household farms where buffaloes are the major producers of milk [18]. Interaction between buffaloes and humans can lead to the transfer of pathogens. Antibiotic-resistant S. aureus is the most pathogenic and contagious pathogen causing bovine mastitis [4,27]. Thus, this study was conducted with the objective of evaluating the association between phenotypic and genotypic erythromycin antibiotic resistance in S. aureus isolated recovered from mastitis-infected buffaloes and their milkers in dairy farms of Lahore, Pakistan. Another objective was to analyze the genetic similarities between the ermA, ermB and ermC genes in S. aureus isolates from bovine mastitis cases and farm personnel. The phylogenetic analysis of antibiotic resistance genes ermA, ermB and ermC in S. aureus isolates was also performed.In total, 30 of the 40 samples considered were positive for S. aureus. Of the 30 samples, 14 were milk samples and 16 were nasal samples from milkers. The overall prevalence of S. aureus in mastitis-affected buffaloes was found to be 75%. This prevalence is similar to that reported in a recently published study by [8]. However, this prevalence is higher than reported by Ashfaq and Muhammad in 2008 [28]. According to their study in Pakistan, S. aureus was found to be positive in 48.08% of isolates from dairy buffaloes.The resistance of S. aureus to antimicrobial agents is an increasing global concern. The determination of antimicrobial resistance profiles is necessary to develop effective therapeutic strategies [29]. The monitoring of resistant strain spread should also be considered important. In this study, the antimicrobial susceptibility profiles of S. aureus were determined and high levels of resistance to erythromycin (52.1%) followed by clindamycin (42.8%) were detected. This resistance rate is comparable to one found by another study Mahdavi and colleagues in which they reported 48% and 46% resistance to erythromycin and clindamycin [30].Results of our study are similar to the study conducted in China, where they concluded that most of the common drugs such as Erythromycin, Tetracycline, Clindamycin, penicillin and cotrimoxazole are ineffective against S. aureus isolated from bovine mastitis samples. Our study also showed higher resistance of S. aureus against these drugs [31]. Antibiotic resistance was determined in 14 milk samples from mastitic buffaloes, and 47% of S. aureus isolates were resistant to erythromycin. Clindamycin resistance was shown in 33% of isolates. It was also found that genotypic and phenotypic erythromycin resistance are associated. The genotypic antibiotic resistance profiles of 14 milk samples from mastitic buffaloes were determined. Overall, 52.1%, 21.4% and 35.7% of S. aureus isolates were positive for the ermA, ermB and ermC genes, respectively. Sixteen milker nasal S. aureus isolates were also tested for the presence of ermA, B and C genes, and these genes were observed in 56.2%, 31.3% and 43.8% of the samples, respectively. This finding is in correspondence to another study conducted in China, according to which highest number of genes detected from S. aureus was ermA gene [32].In small household farms where there is interaction between animals and humans, there is suitable opportunity for microbial organisms to colonize humans [33]. However, some reports have noted that S. aureus is host-specific, but this applies to some specific lineages [16,26]. Most of them do not show host-specificity. Apart from these characteristics, S. aureus is an adaptive pathogen, and can adapt to many unfavorable circumstances [14]. Some genetic adaptions and mutations have helped it to become a successful pathogen [34].To investigate the transmission of antibiotic resistance genes between buffaloes and humans via S. aureus transfer, the ermA, B and C genes were analyzed. This showed that there is little or no difference between S. aureus isolates of buffaloes and humans who live in close contact. Another study conducted on erythromycin resistant S. aureus isolated from environmental samples also reported the comparable results in sensitivity pattern of S. aureus, indicating that origin of isolates is independent of the sensitivity pattern [35]. These bugs can be acquired from environment by both milkers and buffaloes, which ultimately leads to infection [16]. This study reports that there is a transmission of antibiotic-resistant S. aureus between buffaloes and closely related humans, as the ermA, ermB and ermC gene sequences from buffaloes and their milkers from same farm were closely related, but distantly related to those isolated from other farms.

Study Limitations: The current study did not report the data on clinical and subclinical mastitis because of the restriction in ethical approval from the target institutions. Furthermore, because of the financial issues, the sequencing analysis of PCR products could not be done in order to further identify the isolates as well as to check their phylogenetics.