Isolation and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) from Milk in Shire Dairy Farms, Tigray, Ethiopia

Antibiotic-resistant Staphylococcus aureus isolates pose a severe challenge to both veterinary and health professions and dairy cattle producers. Cross-sectional study was conducted from November 2014 to May 2015 to isolate and identify S. aureus from mastitic cows' milk and estimate the occurrence of MRSA in the dairy farms of Shire. Physical examination and California mastitis test were performed on a total of 220 dairy cows. Bacteriological isolation and identification and antibiogram testing were performed. Furthermore, multiplex polymerase chain reaction (PCR) was done for the detection of mec A and fem A genes. Out of the 220 dairy cows, 64 (29.09%) were positive for bovine mastitis, and of these, 32.81% were coagulase-positive S. aureus (CoPS). Antibiogram test results showed 100% of the isolates were resistant to penicillin G, nalidixic acid, and ampicillin, and 33.33% of the CoPS showed resistance to oxacillin (phenotypically MRSA positive). But 38.09% of the CoPS were found to be resistant and susceptible to vancomycin. PCR amplification of the seven phenotypically MRSA isolates revealed that 42.9% and 71.4% of them were found to carry fem A and mec A genes, respectively. The current study revealed the existence of alarming level of CoPS and development of multidrug resistance.


Introduction
Roughly 75% of the infectious diseases in humans over the past two decades have originated from animals and can transmit directly or indirectly from animals to humans [1]. Staphylococcus aureus (S. aureus) is recognized worldwide as a leading pathogen causing many serious diseases in dairy and healthcare surroundings [2]. In the last few decades, Staphylococcal food poisoning has been reported as the third cause of food-borne illnesses in the world [3]. Moreover, antibiotic-resistant S. aureus isolates pose a severe challenge to both veterinary and health professions and dairy cattle producers because they have a negative impact on therapy [4]. In the 1960s, approximately 80% of all clinical isolates of S. aureus were β-lactamase producers [5]. In the early 1960s, the first isolates of MRSA were detected in the United Kingdom (UK) [6]. e antibiotic resistance mechanism of MRSA is mediated through expression of mec A, encoding a penicillinbinding protein PBP2a that has a low affinity for β-lactam antibiotics [7]. e mec A gene is a DNA segment of 2.1 kb that is non-native to S. aureus and is inserted in a large block of exogenous DNA, known as the staphylococcal cassette chromosome mec (SCCmec) [8].
Infections due to MRSA have increased worldwide during the past twenty years [9]. Traditionally, two groups are distinguished among its strains: hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) [10]. However, MRSA infection and colonization have also been reported in horses, dogs, cats, birds, dairy cows, and chicken, and this new type of isolate emerged into the S. aureus population, belonging to clonal complex 398 (CC398) and is known as livestock-associated MRSA (LA-MRSA) [11,12]. Hence, besides its importance as a hospital and community pathogen, MRSA has also been considered as an emerging problem in veterinary medicine in recent years [13,14].
In recent years, MRSA strains have been recovered from several animal-source foods, such as poultry, pork, and beef, suggesting that foods may serve as the reservoir and source of CA-MRSA [15]. Milk has been reported to be contaminated with MRSA, which is suspected to have been acquired from dairy animals or from postmilking contamination and poor sanitary practice [16]. In addition, S. aureus is considered as one of the most important causative agents of mastitis in dairy cattle [17]. Hence, determination of antimicrobial resistance profile of LA-MRSA isolates is crucial for empirical treatment of infections associated with LA-MRSA. Moreover, there was no published and/or accessible research work in the current study area, Shire, as well as in the region. us, the objective of this study was to isolate and identify S. aureus from mastitic cows' milk and estimate the occurrence of MRSA.

Study Area.
e study was conducted from November 2014 to May 2015 in Shire, Ethiopia. Shire is located at about 1087 km north of Addis Ababa and 304 km from Mekelle at an altitude of 1984 meter above sea level. It is located between 14°6′N 38°17′E/14.100°N 38.283°E latitude and longitude, respectively. e average range of temperature is 25°C-35°C, with 900-1100 mm of annual average range of rainfall. ere are four milk shades (dairy farms) containing about 1500 dairy cattle kept in these farms (shades), of which 579 were crossbred dairy cows and the remaining were calves and heifers [18].

Study Design.
A cross-sectional type of study was conducted from November 2014 to May 2015 in the Shire town, Northwest of Tigray, Ethiopia, to isolate and identify S. aureus from mastitic cows' milk and estimate the occurrence of MRSA strains.

Sample Size and Sample Collection.
A total of 220 dairy cows from the four milk shades were selected purposively on the bases of the availability of dairy cows and the willingness and cooperation of the farm owners during sampling. About 10 mL of the milk sample was aseptically collected from each of clinically and subclinically (CMT positive) mastitis nonblind quarters of the selected cows using sterile universal bottles for bacterial isolation according to the study by Quinn et al. [19]. en, the collected samples were transported in an ice box to the Mekelle University, College of Veterinary Sciences, Microbiology Laboratory for microbiological examination. If immediate inoculation was not convenient, the samples were kept at 4°C for a maximum of 24 h until cultured on standard bacteriological media.

Bacteriological Isolation and Identification.
A loop of milk sample was streaked on 5% Sheep Blood Agar (Oxoid, UK), and the plates were incubated aerobically at 37°C and examined after 24 h of incubation for growth. e colonies were provisionally identified on the basis of staining reaction with Gram's stain, cellular morphology, and hemolytic pattern on blood agar. e representative colonies were subcultured on Mannitol Salt Agar (MSA) (Oxoid, UK) and incubated at 37°C. en, the colonies that grew on MSA were subcultured on nutrient media, and the cultures were preserved and maintained for characterizing the isolates. Catalase test, oxidase test, tube coagulase test, DNase test, and growth on MSA were performed according to the study by Quinn et al. [19], and samples that were considered as positive for coagulase-positive S. aureus (CoPS) were further characterized.

Antimicrobial Susceptibility Testing.
e S. aureus isolates were screened for in vitro antimicrobial susceptibility using the agar disk diffusion method according to the procedure given by Kirby et al. [20] on Mueller-Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, England). It is stated that in the absence of methicillin, the best alternative is to use cefoxitin or oxacillin for MRSA identification [21]. e following eleven different antibiotic discs, with their concentrations given in parentheses, were used in the anti-

Polymerase Chain Reaction (PCR) for Detection of mec A and fem A Genes.
e S. aureus genomic DNA extraction and purification were performed as per the protocol given by ermo Scientific, GeneJET Genomic DNA Purification Kit for gram-positive organisms.
en, seven of the phenotypically MRSA isolates were screened for the presence of mec A and fem A genes by multiplex PCR according to the procedure given by Johnson et al. [23] using the following specific primers: F-5′GTA GAA ATG ACT GAA CGT CCG ATA A3′ and R-5′CCA ATT CCA CAT TGT TTC GGT CTA A3′ for mec A gene (having a band size of 310 bp) and F-5′-AAA AAA GCA CAT AAC AAG CG-3′ and R-5′GAT AAA GAA GAA ACG AGC AG-3′ for fem A gene (S. aureus species specific and encoding a factor responsible for methicillin resistance and has an amplicon size of 132 bp). Each PCR reaction mixture (50 μL) was prepared from 5 μL of 10X reaction buffer, 5 μL of template DNA, 1 μL of each primer, 3 μL of 10 mM dNTP mixture, and 1 μL of Taq polymerase. e remaining volume had nuclease-free deionized water. Amplification was carried out in a Tianlong PCR thermocycler with thermal cycling conditions of an initial denaturation at 94°C for 6 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 30 s and extension at 72°C for 45 s, and with final extension at 72°C for 6 min. Finally, PCR products were separated by running on 1.5% (w/v) agarose gel containing 0.5 g/mL ethidium bromide. Electrophoresis was conducted in a horizontal equipment system for 55 min at 110 V using 1X TAE buffer (40 mM Tris, 1 mM EDTA and 20 mM glacial acetic acid, pH 8.0). e amplicons were visualized under UV-light gel doc, and their molecular weights were estimated by comparing with 100 bp DNA molecular weight marker (Solis BioDyne, Tartu, Estonia) [24].

Data Management and Analysis.
All collected data were entered into Microsoft Excel Sheet and analyzed through Statistical Package for Social Sciences, version 16. Accordingly, descriptive statistics such as percentages and frequency distribution were used to determine the prevalence.

Results
e physical examination of the udder and CMT test results indicated that out of the 220 dairy cows, 64 (29.09%) were positive for bovine mastitis. Bacteriological characterization of these 64 mastitis-positive samples revealed that 21 (32.81%) were found CoPS. e antimicrobial sensitivity test results of the 21 CoPS isolates showed their susceptibility to kanamycin and streptomycin (42.85%). However, 100% of the isolates were resistant to penicillin G, nalidixic acid, and ampicillin, 85.71% to amoxicillin, 47.61% to trimethoprim and oxytetracycline, and 57.14% to compound sulfonamides, and 33.33% of the CoPS were resistant for oxacillin (phenotypically MRSA positive). But 38.09% of the CoPS were found to be resistant and susceptible to vancomycin. Moreover, all the 21 (100%) isolates developed multidrug resistance as it is shown in Table 1.
e PCR amplification of the seven phenotypically MRSA isolates was revealed that 3 (42.9%) of them were found to carry fem A gene and 5 (71.4%) of them were found to carry mec A genes, which had a molecular weight of 132 bp and 310 bp, respectively.
ese differences in the prevalence of S. aureus from the different studies might be due to variations in sample size, isolation techniques, husbandry practices, awareness and skills of the farm owners, animal health delivery systems, and geographic regions of the sampled area. e antimicrobial sensitivity test results of the current CoPS isolates was almost in agreement with the findings of Veterinary Medicine International Asmelash et al. [34] who reported 100% resistant to penicillin G and amoxicillin and 42.7% to cefoxitin; Abera et al. [33] who reported 94.4% resistant to penicillin G and 58.3% to trimethoprim-sulfamethoxazole; Rgbe et al. [29] who reported 82.4% of resistant to ampicillin and 52.9% to trimethoprim-sulfamethoxazole; Abebe et al. [47] who observed 92.2% resistant to penicillin G and 33.3% to oxacillin; Abo-Shama [62] in Sohag Governorate, Egypt, who reported 43.1% resistant to oxacillin and 83.7% to one or more antimicrobial agent; Mueena et al. [53] who reported 100% resistant to penicillin and amoxicillin; Fitsum [40] who reported 93.3% resistant to penicillin G, 53.3% to streptomycin, and 40% to tetracycline; Tilahun et al. [35] who observed 100% resistant to penicillin G and amoxicillin and 42.7% to cefoxitin; Muyiwa et al. [41] who illustrated a large proportion (60%-100%) of resistant to penicillin G, ampicillin, and streptomycin; Ananya and Pranab [56] who reported 87.5% resistant to penicillin G; Pati and Reena [31] who reported 96% resistant to penicillin G and 93% to ampicillin; and Jibril and Huruma [60] who reported 71.74% of resistant to penicillin G and 41.30% to tetracycline.
Moreover, the current result was in line with that of Indu and Brintya [28] [63]. e resistance of S. aureus to penicillin and closely related antibiotics might be attributed to the production of β-lactamase, an enzyme that inactivates penicillin and closely related antibiotics. Around 50% of mastitis-causing S. aureus strains produce β-lactamase [64]. Moreover, the development of antimicrobial resistance might be as a result of repeated therapeutic and/or indiscriminate use of them in the dairy farms, particularly penicillin and oxytetracycline for the treatment of mastitis cases in the study area at large in the country.

Conclusion and Recommendations
e current study revealed that there is a high prevalence of CoPS as well as MRSA as a cause of bovine mastitis. ough nearly half of the CoPS isolates were susceptible to kanamycin and streptomycin, the high percentage were resistant to penicillin G, nalidixic acid, ampicillin, amoxicillin, trimethoprim, oxytetracycline, compound sulfonamides, oxacillin, and vancomycin. Moreover, all of the CoPS isolates developed multidrug resistance. is higher percentage of multidrug resistance pattern indicates alarming situation for designing prevention and control measures. However, the current study was only targeting mec A and fem A genes for a few of the phenotypically MRSA-positive isolates for molecular characterization. In general, the detection of CoPS in the milk samples and development of drug resistance indicate that the product is unwholesome for human consumption. Hence, it has a serious economic, animal welfare, food safety, and public health problem. erefore, strict hygiene should be implemented in the farms by creating awareness among the farm workers, managers, and attendants regarding transmission, zoonotic importance, and control and prevention strategies of the disease; and dispensing of non-prescribed drugs and indiscriminate use of antibiotics should be avoided. Moreover, further studies on molecular characterization and sequencing of MRSA should be conducted by targeting other important genes in addition to the targeted genes in the study area at large in the country. Data Availability e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors state that there are no conflicts of interest.