First mecC and mecA Positive Livestock-Associated Methicillin Resistant Staphylococcus aureus (mecC MRSA/LA-MRSA) from Dairy Cattle in Malaysia

Livestock associated Methicillin resistant Staphylococcus aureus (S. aureus) (LA-MRSA) was reported to be zoonotic and may transmit to farmers and veterinarians. The objectives of this study were to investigate the occurrence of LA-MRSA from dairy cattle and to evaluate the antimicrobial resistance profiles of the isolates. A total of 63 milk and 32 nasal swab samples were randomly collected from dairy cattle. The samples were processed to isolate S. aureus, MRSA and LA-MRSA using both phenotypic and molecular methods using PCR. The confirmed S. aureus isolates were cultured on oxacillin resistant screening agar base (ORSAB) to detect MRSA and the isolates were further confirmed by PCR targeting the mecA gene. Detection of the novel mecA gene, mecC gene was conducted by PCR amplification. The antimicrobial susceptibility tests were conducted using disc diffusion method. Results revealed 17/95 (17.89%) and 15/95 (15.79%) were positive for mecA and mecC genes respectively. Out of the 15 mecC positive isolates, 12 were positive for both mecA and mecC. The MRSA isolates showed multidrug resistance. The findings showed high prevalence of mecC-positive LA-MRSA in Malaysia and highlight the public health risks to people that may come in contact with the carrier animals or those who may consume unpasteurized milk products from these animals.


Introduction
Staphylococcus aureus is a normal inhabitant of the skin and mucous membrane of healthy human and animals. However, it can also be opportunistic pathogen and causes multiple infectious diseases in humans and animals [1] and the bacteria can spread through air, contaminated surfaces, animals, or human [2]. It has been widely reported that S. aureus is commonly detected in raw milk from both apparently healthy animals and those with clinical mastitis. A recent study from China reported 46.2% (90/195) of raw milk samples taken from dairy cows with mastitis were positive for S. aureus [3]. Staphylococcus aureus is also known for its multidrug resistance and MRSA is one of the most potent drug resistant bacteria that has been causing nosocomial infections and community associated infections and animal diseases. According to the centers for disease control and prevention (CDC), strains of S. aureus that are oxacillin and methicillin resistant are considered resistant to all ß-lactam agents, including cephalosporins and carbapenems. It has been reported that animal MRSA isolates were significantly more resistant to ciprofloxacin, gentamicin, and clindamycin as compared to human MRSA isolates [4]. Recently, a highly divergent mecA gene, mecC was found in S. aureus causing bovine mastitis [5]. This novel LA-MRSA was first reported from cattle in the UK and Denmark where it was reported to cause human and animal infections. In recent years, the mecC MRSA/LA-MRSA strains have been reported from a few European countries and from different host species. Molecular characterization

Isolation and Identification of Staphylococcus Aureus
The swab samples were submerged into 5 ml of tryptone soy broth (TSB) and incubated at 37 • C for 24 h for enrichment. Whereas 3 ml of the milk from each sample were mixed into 7 mL of TSB and incubated as above. The samples were then cultured on blood agar and nutrient agar and incubated at 37 • C for 24 h. After 24 h colony morphology, Gram staining and biochemical tests were used to identify S. aureus. Presumptive S. aureus were further confirmed by PCR amplification of S. aureus specific gene (nucA). The confirmed S. aureus colonies were cultured on oxacillin resistant screening agar base (ORSAB) to screen for MRSA. Blue colonies on ORSAB after incubating for 24-48 h were presumptively identified as MRSA and were cultured on nutrient agar to Further confirmation was done by PCR detection of methicillin resistance encoding gene, mecA.

DNA Extraction
Extraction of the genomic DNA was conducted by using commercial DNA extraction kit, Machery-Nagel DNA, RNA, and Protein Purification Kit (Duren, Germany) following the recommended procedures. The extracted DNA was stored in a freezer at −20 • C until used.

S. aureus-Specific Gene Amplification
Confirmation of S. aureus was done by amplifying the nucA gene using the primer sequence (Table 1) as described earlier [19]. Two microliters of samples were added to master mix consisted of 20.9 µL nuclease free water, 10 µL 5X buffer, 1.5 µL 50mM MgCl 2 , 0.5 µL 10mM dNTPs, 5 µL of each primer and 0.1 µL Taq DNA polymerase (5 u/µL). The PCR amplification was done using the following protocols, initial denaturation at 94 • C for 5 min, 35 amplification cycles consisting of denaturation at 94 • C for 30 s, annealing at 62 • C for 45 s and extension at 72 • C for 45 s, followed by final extension at 72 • C for 10 min.

Amplification of Methicillin-Resistance Encoding Gene (mecA)
Methicillin resistant S. aureus specific gene, mecA (Table 1) was amplified to confirm MRSA isolates according as described previously [20]. Two microliters of sample was added to 48µl of master mix consisted of 26.5 µL nuclease free water, 10 µL 5X buffer, 2 µL 50 mM MgCl 2 , 1 µL 10 mM dNTPs, 3.75 µL of both forward and reverse primers and 1 µL Taq DNA polymerase (5 u/µL). PCR amplification was done using the following protocol, pre-denaturation 1 min, denaturation at 94 • C for 1 min, annealing at 60 • C for 1 min, extension at 72 • C for 3 min and final extension at 72 • C for 5 min. Amplification products yielding 533 bp were considered as positive.  (Table 1) as described earlier [21] were used to identify mecC positive LA-MRSA isolates. Two microliters of sample were added to 48 µL of master mix containing 26.5 µL nuclease free water, 10 µL 5X buffer, 2 µL 50 mM MgCl2, 1 µL 10 mM dNTPs, 3.75 µL for both 10 µM mecC R and mecC F and 1 µL Taq DNA polymerase (5 u/µL). The PCR protocol was set as pre-denaturation at 95 • C for 2 min, 30 cycles of amplification with denaturation at 95 • C for 45 s, annealing at 55 • C for 1 min, extension at 72 • C for 2 min and final extension at 72 • C for 5 min. The PCR products were analyzed by gel electrophoresis using 1.2% agarose and gel imaging was done using Gel Doc TM EZ Imager (Bio-Rad, Hercules, CA, USA). The expected amplification product of 304 bp signifies a positive detection of mecC gene.

Isolationand Identification of S. aureus
The results showed that 44.4% (28/63) of the milk samples and 50% (16/32) of the nasal swabs were positive for S. aureus as confirmed by PCR. Overall, the detection rate of S. aureus was 46.3% (44/95) ( Table 2 and Figure 1). Livestock associated Methicillin resistant S. aureus harboring mecC gene were identified by conducting PCR on all phenotypically identified MRSA isolates with positive growth on MRSA selective agar, ORSAB. Specific primers for mecC genes (Table 1) as described earlier [21] were used to identify mecC positive LA-MRSA isolates. Two microliters of sample were added to 48 µL of master mix containing 26.5 µL nuclease free water, 10 µL 5X buffer, 2 µL 50 mM MgCl2, 1 µL 10 mM dNTPs, 3.75 µL for both 10 µM mecC R and mecC F and 1 µL Taq DNA polymerase (5 u/µL). The PCR protocol was set as pre-denaturation at 95 °C for 2 min, 30 cycles of amplification with denaturation at 95 °C for 45 s, annealing at 55 °C for 1 min, extension at 72 °C for 2 min and final extension at 72 °C for 5 min. The PCR products were analyzed by gel electrophoresis using 1.2% agarose and gel imaging was done using Gel Doc TM EZ Imager (Bio-Rad, Hercules, CA, USA). The expected amplification product of 304 bp signifies a positive detection of mecC gene.

Isolationand Identification of S. aureus
The results showed that 44.4% (28/63) of the milk samples and 50% (16/32) of the nasal swabs were positive for S. aureus as confirmed by PCR. Overall, the detection rate of S. aureus was 46.3% (44/95) ( Table 2 and Figure 1).

Identification and Confirmation of MRSA
Polymerase chain reaction analysis of 28 S. aureus isolates from milk samples and 16 S. aureus isolates from nasal swabs samples showed that 46.23% (13/28) and 25% (4/16) isolates were positive for mecA gene respectively (Table 3, Figure 2).

Identification and Confirmation of MRSA
Polymerase chain reaction analysis of 28 S. aureus isolates from milk samples and 16 S. aureus isolates from nasal swabs samples showed that 46.23% (13/28) and 25% (4/16) isolates were positive for mecA gene respectively (Table 3, Figure 2).

Detection of mecC Positive LA-MRSA Isolates
Presence of the mecC gene is used for confirmation of the novel MRSA strains harboring this specific methicillin resistance encoding gene [22]. Among all positive isolates on ORSAB agar, 15 isolates were positive for mecC gene. Out of the 15 mecC positive isolates, 12 were also positive for mecA gene (Table 4 and Figure 3).

Detection of mecC Positive LA-MRSA Isolates
Presence of the mecC gene is used for confirmation of the novel MRSA strains harboring this specific methicillin resistance encoding gene [22]. Among all positive isolates on ORSAB agar, 15 isolates were positive for mecC gene. Out of the 15 mecC positive isolates, 12 were also positive for mecA gene (Table 4 and Figure 3).

Identification and Confirmation of MRSA
Polymerase chain reaction analysis of 28 S. aureus isolates from milk samples and 16 S. aureus isolates from nasal swabs samples showed that 46.23% (13/28) and 25% (4/16) isolates were positive for mecA gene respectively (Table 3, Figure 2).

Detection of mecC Positive LA-MRSA Isolates
Presence of the mecC gene is used for confirmation of the novel MRSA strains harboring this specific methicillin resistance encoding gene [22]. Among all positive isolates on ORSAB agar, 15 isolates were positive for mecC gene. Out of the 15 mecC positive isolates, 12 were also positive for mecA gene (Table 4 and Figure 3).

Discussions
In this study, out of the total 95 samples, 44 samples (46.3%) were positive for S. aureus and 17 (38.6%) of these were confirmed to be MRSA. Likewise a recent study of S. aureus isolates obtained from bovine mastitic milk samples in Bangladesh reported a high prevalence of 20% (29/145) MRSA identified by the presence of the mecA gene [23]. In contrast, a more recent study from China reported 15.52% of the 219 bovine mastitis S. aureus isolates were confirmed as MRSA by cefoxitin disc diffusion test, oxacillin microdilution test, and mecA detection [24]. A relatively lower prevalence rates of MRSA were also reported from other studies conducted in in different countries. Including US and China [25][26][27]. A study from Italy reported that 3.8% (40/484) S. aureus isolates from milk and milk products were MRSA [28], whereas another study from the same country reported that out of 169 S. aureus strains isolated from Italian dairy cows, 12 (7.1%) and 157 (92.9%) were MRSA and methicillin susceptible S. aureus (MSSA) respectively [29].
Different prevalence rates of bovine MRSA has been reported in many countries. Huber et al. [30] reported a low prevalence of MRSA in bovine milk (2 out of 142 S. aureus isolates) in Switzerland. In Germany 16.7% of prevalence rate was detected [31] and 0.4% in Hungary [32]. In a recent study by Paterson et al. [33], 7 MRSA isolates were detected in 1500 bulk milk tank samples tested in UK. Whereas 4.4% of the analyzed bulk milk samples in Germany were positive for MRSA [34]. Some of the Asian countries have also reported the occurrence of bovine MRSA. Pu et al. [8] reported 47.6% prevalence in China, while others reported 6.3% in Korea [13], 13.1% in India [9] and 1.5% in Japan [35]. These reports imply that Asian countries have relatively higher prevalence rates of bovine MRSA as compared to European countries and Malaysia is among the Asian countries that has reported high prevalence of bovine MRSA and these findings might be attributed to possible imprudent usage of antibiotics.
High percentage of MRSA isolated from dairy cattle in this study might be due to the fact that antibiotics are extensively used to control and prevent bacterial infections such as bovine mastitis.

Discussions
In this study, out of the total 95 samples, 44 samples (46.3%) were positive for S. aureus and 17 (38.6%) of these were confirmed to be MRSA. Likewise a recent study of S. aureus isolates obtained from bovine mastitic milk samples in Bangladesh reported a high prevalence of 20% (29/145) MRSA identified by the presence of the mecA gene [23]. In contrast, a more recent study from China reported 15.52% of the 219 bovine mastitis S. aureus isolates were confirmed as MRSA by cefoxitin disc diffusion test, oxacillin microdilution test, and mecA detection [24]. A relatively lower prevalence rates of MRSA were also reported from other studies conducted in in different countries. Including US and China [25][26][27]. A study from Italy reported that 3.8% (40/484) S. aureus isolates from milk and milk products were MRSA [28], whereas another study from the same country reported that out of 169 S. aureus strains isolated from Italian dairy cows, 12 (7.1%) and 157 (92.9%) were MRSA and methicillin susceptible S. aureus (MSSA) respectively [29].
Different prevalence rates of bovine MRSA has been reported in many countries. Huber et al. [30] reported a low prevalence of MRSA in bovine milk (2 out of 142 S. aureus isolates) in Switzerland. In Germany 16.7% of prevalence rate was detected [31] and 0.4% in Hungary [32]. In a recent study by Paterson et al. [33], 7 MRSA isolates were detected in 1500 bulk milk tank samples tested in UK. Whereas 4.4% of the analyzed bulk milk samples in Germany were positive for MRSA [34]. Some of the Asian countries have also reported the occurrence of bovine MRSA. Pu et al. [8] reported 47.6% prevalence in China, while others reported 6.3% in Korea [13], 13.1% in India [9] and 1.5% in Japan [35]. These reports imply that Asian countries have relatively higher prevalence rates of bovine MRSA as compared to European countries and Malaysia is among the Asian countries that has reported high prevalence of bovine MRSA and these findings might be attributed to possible imprudent usage of antibiotics.
High percentage of MRSA isolated from dairy cattle in this study might be due to the fact that antibiotics are extensively used to control and prevent bacterial infections such as bovine mastitis. The indiscriminate use of antibiotics may lead to the emergence of multidrug-resistant bacterial strains and increases the risk of presence of residues of these drugs in milk [36]. Detection of high percentage of MRSA from dairy cattle also shows that there is high risk of potential zoonotic transmission especially to the farmers, veterinarians handling the livestock and to the public who may consume the dairy products that are not processed properly. This is because MRSA infected cattle can act as a reservoir of MRSA and may transmit the bacteria to other animals and humans [37,38].
In this study, out of 95 samples, a total of 15 (57.69%) livestock associated methicillin resistant Staphylococcus aureus (LA-MRSA) harboring the novel mec gene, mecC were detected by PCR. Out of the 15 positive isolates, 11 were positive for both mecA and mecC genes. This shows that LA-MRSA possessing mecC gene is not only present in European countries but also in Asian, particularly in Malaysia. To the best of our knowledge, the current finding is the first report on LA-MRSA (mecC positive) in dairy cattle in Malaysia and is among the few reports of mecC positive LA-MRSA outside Europe. This high percentage of mecC positive LA-MRSA in dairy cattle shows that there is high risk for zoonotic transmission of this pathogen to veterinarians or farmers because of its capacity to colonize a wide range of hosts [5]. Studies have reported that MRSA colonization in cattle may be an occupational risk to veterinarians, farmers, milkers, and people working at slaughterhouses [5,32]. It has also been reported that the transmission of animal MRSA to veterinary personnel can occur and such transmission commonly occurs in personnel working with large animals [11,39,40].
In this study, different groups of antibiotics were tested, and the results show that all the MRSA isolates were resistant to oxacillin, but are susceptible to imipenem and enrofloxacin. Moreover, all the isolates were resistant towards at least one type of antibiotics, showing multi-drug resistance characteristics of the MRSA isolates. Among others, the possible explanation for the MRSA isolates being resistant to penicillin and other similar antibiotics can be due to the fact that these groups of antibiotics are commonly used by farmers and veterinarians in treating dairy cattle especially for disease such as mastitis. To prevent the incidence of antibiotic resistance in dairy cattle from rising, surveillance for early identification of novel antibiotic resistant clones of S. aureus is recommended [41]. It is also important to improve biosecurity and implement good animal husbandry practices in dairy farms to prevent the spread of MRSA and other antimicrobial resistant pathogenic bacteria. Due to the zoonotic potential of LA-MRSA, veterinarians and farm workers are advised to adhere to safety procedures including usage of personal protective equipment whenever handling the animals.
In conclusion, the findings from the current study are preliminary and more studies needs to be conducted for further identification of the mecC positive isolates through additional molecular characterization and typing techniques. Nevertheless, the isolation of mecC positive LA-MRSA from dairy cattle in this study is the first such report on the detection of this MRSA strain in Malaysia and is expected to serve as a preliminary data to initiate comprehensive and large-scale research. The information generated from this study is important to understand the presence of this bacteria in dairy cattle and determine the public health risks it may pose, mainly to the animal owners, people who may come in contact with carrier animals and those who may consume unpasteurized dairy products. Moreover, the data from this research can also be used to educate the public on the potential public threat posed by LA-MRSA from dairy cattle and milk products from these animals.