Identification of Methicillin-Resistant Staphylococcus aureus in Bulk Tank Milk

Foodborne outbreaks caused by milk and dairy products have led to hospitalizations and deaths for human beings (Painter et al., 2013). Staphylococcus aureus is an important causative agent of toxin-mediated food poisoning, invasiveness, and antibiotic resistance (Le Loir et al., 2003; Gundogan & Avci, 2014). Ot can cause skin and wound infections or subclinical mastitis in dairy animals (Kluytmans et al., 1997; Kreausukon et al., 2012; Lee et al., 2014). The presence of biofilm producing ability of S. aureus in milk and milking environment is a public health concern for the consumers (Lee et al., 2014; Lee et al., 2016). Also, thermostable enterotoxins prior to pasteurization of raw milk produced by S. aureus may cause staphylococcal food poisoning in humans (Hein et al., 2005; Lee et al., 2012).


Introduction
Foodborne outbreaks caused by milk and dairy products have led to hospitalizations and deaths for human beings (Painter et al., 2013).Staphylococcus aureus is an important causative agent of toxin-mediated food poisoning, invasiveness, and antibiotic resistance (Le Loir et al., 2003;Gundogan & Avci, 2014).Ot can cause skin and wound infections or subclinical mastitis in dairy animals (Kluytmans et al., 1997;Kreausukon et al., 2012;Lee et al., 2014).The presence of biofilm producing ability of S. aureus in milk and milking environment is a public health concern for the consumers (Lee et al., 2014;Lee et al., 2016).Also, thermostable enterotoxins prior to pasteurization of raw milk produced by S. aureus may cause staphylococcal food poisoning in humans (Hein et al., 2005;Lee et al., 2012).
Antibiotic resistance of bacteria has become a major public health problem all over the world owing to the massive use of antibiotics in feed to promote growth in both agriculture and livestock animals (Normanno et al., 2007;Dniciuc et al., 2017).The antibiotic resistance of methicillin and other beta lactam groups result from a modified penicillin binding protein (PBP 2a) which has a low affinity for the beta lactams.This protein is encoded by mecA and mecC genes which are localized in a mobile genetic element called Staphylococcal Casette Chromosome mec (SCCmec) (Paterson et al., 2014).Recent studies reported that the mecC gene is required for confirmation of methicillin-resistant S. aureus (MRSA) because 70% identity to mecA gene (Garcia-Alvarez et al., 2011;Shore et al., 2011;Stegger et al., 2012;Oto et al., 2012).Also, mecC-MRSA has been reported in the likelihood of zoonotic transmission (Petersen et al., 2013;Harrison et al., 2013).
MRSA is an emerging pathogen in livestock animals that can infect humans and has become a growing concern for public health.MRSA has been isolated as a mastitis pathogen in bulk tank milk (Moon et al., 2007;Nam et al., 2011;Spohr et al., 2010;Doulgeraki et al., 2017).Hospital-associated MRSA (HA-MRSA) and communityassociated MRSA (CA-MRSA) infections have been reported in initial studies.Another group called livestock-associated MRSA (LA-MRSA) was first isolated in dairy cattle (Paterson et al., 2012) On recent years, MRSA has become a major concern as an emerging pathogen in livestock that can transfer the methicillin resistance to humans via food or milk (Nemati et al., 2008;Pereira et al., 2009;Wendlandt et al., 2013;Vincze et al., 2014;Wang et al., 2014;Kraushaar & Fetsch 2014;European Food Safety Authority, 2015;Lozano et al., 2016;Asiimwe et al., 2017;Aqib et al., 2017;Can et al., 2017a;Tenhagen et al., 2018).
Even though many researchers have reported the isolation of MRSA from livestock animals and foods of animal origin, the effect of MRSA in food-related problems is very rare; however, there are some concerns about foodborne MRSA infections (EFSA, 2009;Doyle et al., 2011;Herrera et al., 2016).The aims of this study were to evaluate the prevalence of S. aureus, antibiotic resistance profiles, and related mecA genes among these isolates of S. aureus from bulk tank milk samples in Turkey.

Milk samples
On this study, a total of 120 bulk tank milk samples were obtained between Dctober 2016 and September 2017 in Burdur province, located on the southern side of Turkey.The samples 151/156 151 were taken in sterile plastic collection tubes and transported to the laboratory under refrigeration (4-8 °C), and the samples were directly processed for further analyzes.

Isolation and identification of S. aureus
Bulk tank milk samples (0.1 mL) were plated on rabbit plasma fibrinogen agar medium (BP-RPF, Dxoid, Otaly) and incubated at 37 °C for 24-48 hours.Colonies developing a typical coagulase halo on BP-RPF agar were suspected of S. aureus.Two suspected colonies from samples were grown in Brain Heart Onfusion broth (BHO, Dxoid, CM1135) at 37 °C for 24 hours.Presumptive colonies of S. aureus were confirmed with some properties (Gram staining, catalase reaction, ß hemolysis, DNase, and the ability to coagulate rabbit plasma) (Onternational Drganization for Standardization, 1999).

DNA isolation
Dvernight cultures in Brain Heart Onfusion broth were used for the DNA isolation.For this purpose, 2 ml of broth cultures were centrifuged at 5 000 g. for 10 minutes and the supernatant were discarded.Bacterial pellets were washed twice with 1 ml of saline solution and centrifuged again.Bacterial pellets were resuspended in 180 µl Tris EDTA buffer (Sigma-Aldrich, 93283) containing 18 µl of lysostaphin (0.5 U/µl, Sigma, L7386) and incubated at 37 °C for 1 hour (Akineden et al., 2008).Genomic DNA was extracted according to GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA) manufacturer's protocol.A nano-drop (NanoDrop2000-Thermoscientific ™ ) technique was used to define the quantification of DNA.

PCR analysis
On the current study, suspected S. aureus isolates were detected by species specific nuc gene for confirmation.Then, confirmed S. aureus isolates were analyzed for detection of the mecA gene.Extracted DNA was subjected to real time polymerase chain reaction (RT-PCR) using the nuc and mecA gene primers in Table 1.Suspected S. aureus isolates were analyzed using the LightCycler  480 System with a 96-well format (Roche Diagnostics, Tokyo, Japan).The extracted 5 µl DNA was added to 15 µl of LightCycler  480 SYBR Green O Master (Roche Diagnostics).Thermocycling conditions using the LightCycler system were completed according to the amplification program consisting of an initial denaturation at 95°C for 10 minutes followed by a 45-cycle amplification program heated to 95 °C with a 15-seconds hold, annealing for nuc gene at 55 °C, for mecA gene at 57 °C with a 10-seconds hold, and extension at 65 °C with a 15-seconds hold.

Statistical analysis
The differences between the seasons and total mesophilic aerobic bacteria (TMAB) and Staphylococcus spp.levels was determined by one-way analysis of variance (ANDVA) and Tukey's test using the SPSS software package version 15.0 for Windows, P < 0.05 was considered statistically significant.

Prevalence of S. aureus
On this study, the prevalence of nuc and mecA genes in S. aureus isolated from bulk tank milk samples are given in tables 2 and 3.

Discussion
Milk contains many bacteria that affect the quality and safety of dairy products (Porcellato et al., 2018).Researches have showed that dairy products such as milk can cause foodborne infections and intoxications.For instance, a study from Malaysia reported that milk samples were contaminated with coliform bacteria (90%), Eschericia coli (65%), S. aureus (60%), E. coli 0157:H7 (33.5%), and Salmonella (1.4%) (Chye et al., 2004).Total mesophilic aerobic bacteria in bulk tank milk may indicate the hygienic quality of milk.Milking conditions, seasonal changes, and moisture affect the presence of microorganisms in milk (Elmoslemany et al., 2009).The levels of total bacteria in bulk tank milk may be higher in summer seasons in comparison to winter seasons (Elmoslemany et al., 2010).On our study, TMAB levels of bulk tank milk samples in the winter were higher than those in the summer and the effects of seasonal differences on microorganisms are statistically important.Because of the effective cooling conditions of bulk tank milk in Burdur province, it can be said that seasonal changes have a limited effect on the development of microorganisms because milk hygiene and equipment cleaning are more effective.
Although S. aureus is a pathogenic bacterium, it can be found in the normal mucosa of healthy humans and animals, and may cause mastitis in dairy cows.(Kluytmans et al., 1997;Kreausukon et al., 2012).On our study, S. aureus was detected in 37.32% of the isolates.The presence of S. aureus in bulk milk tank in the current study was reported to be higher by 75%, 70.4% 55.7%, 55.26%, and 39.8%, in comparison to previous study from Norway, Brazil, Czech Republic, Algeria, and Kosova, respectively (Jørgensen et al., 2005;Rall et al., 2008;Zouharova & Rysanek, 2008;Chaalal et al., 2016;Mehmeti et al., 2017).By contrast, researchers from Switzerland, Oran, and Brazil reported lower levels of S. aureus than the current study (Muehlherr et al., 2003;Fagundes et al., 2010;Lee et al., 2012;Jamali et al., 2015).This indicates that the milking conditions and the hygienic quality of bulk tank milk may cause differences between levels of S. aureus isolates in different countries.
Mastitis is capable of affecting the mammary glands and it changes milk composition (Korhonen & Kaartinen, 1995).Ot is a disease with high treatment costs which makes mastitis an economical concern for dairy farmers (Duarte et al., 2015).S. aureus is one of the main pathogens causing mastitis in dairy animals, which leads to overuse of antimicrobial agents (Roberson et al., 1998;Peles et al., 2007;Dlde Riekerink et al., 2006;Barkema et al., 2009;Gomes & Henriques, 2016).The overuse of β-lactam group antibiotics for prophylactic and mastitis treatment in dairy cows may cause MRSA in milk and dairy products (Levy, 1992;Sawant et al., 2005).Previous studies have reported MRSA in mastitis: 48.3% by Guimarães et al. (2017), 15.5% by Wang et al. (2015), 11.6% by Jamali et al. (2014), and 2.5% by Moon et al. (2007).Ot is also possible for MRSA to contaminate food products without any changes in the milk (Parisi et al., 2016).Transmission of the MRSA to humans through the consumption of milk with mastitis or direct contact with dairy cows may generate serious risks to food safety and public health.
Antibiotic resistant bacteria has become an important public health problem all over the world (Normanno et al., 2007) On this study, according to disk diffusion test results, S. aureus isolates were detected to be resistant to a majority of antibiotics, such as oxacillin, penicillin, clindamycin, and cefoxitin at 71.1%, 69.8%, 67.9%, and 67.3%, respectively.Tenhagen et al. (2018) reported that 100% of MRSA isolates are resistant to cefoxitin and penicillin in Germany.On contrast to this study, Jamali et al. (2015) reported the levels of antibiotic resistance were lower for oxacillin, penicillin, clindamycin, and cefoxitin at 13%, 44.4%, 13.6%, and 4.9%, respectively.On Jordan, the levels of oxacillin, penicillin, clindamycin, and gentamicin resistance were observed to be lower than this study (Dbaidat et al., 2018).Can et al. (2017b) reported the resistant levels of oxacillin (46.4%), cefoxitin (50%), penicillin (60.7%), and chloramphenicol (5.3%) lower than this study in Turkey.By comparing the results from other studies, it seems that the type of antibiotics used for the treatment of animals may cause different results between countries.
On conclusion, the transmission of MRSA to milk and dairy products poses the risk of spreading the antimicrobial resistant bacteria to the general population.The industry needs to apply measures to ensure contamination is minimized.Multi-drug resistant bacteria identified in milk and dairy products should be monitored.Uncontrolled antibiotic use in dairy cattle should be avoided for healthy milk production.The correct application of antibiotics should be evaluated in the udder infections of dairy cows.These are just some of the ways that the spread of MRSA can be alleviated.

Table 3 .
Prevalence of MRSA in bulk tank milk.

Table 2 .
Prevalence of S. aureus in bulk tank milk.