**2. MRSA in food-producing animals**

#### **2.1. Porcine MRSA**

In 2005, pigs were reported as an animal reservoir of *S. aureus* in France, including MRSA [17]. Pig farmers were more frequently colonized than nonfarmers and one of the most in humans is usually divided into two groups: hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) [10]. A third group of MRSA, known as livestockassociated MRSA (LA-MRSA), now has emerged and infects livestock, pets, and wild animals. LA-MRSA was first detected in milk with bovine mastitis from Belgium in 1972 [11–13]. Thereafter, MRSA reports in various food and companion animals, such as pigs, cattle, chickens, dogs, cats, and horses, have increased [11, 14]. A novel strain of MRSA belonging to multilocus sequencing type (MLST) 398 (ST398) and related strains collectively grouped into clonal complex 398 (CC 398) have been frequently found in pigs, chickens, veal calves, dairy cattle, horses, dogs, and milk in various countries [11]. Both methicillin-susceptible *S. aureus* (MSSA) and MRSA have been associated with companion and food production animals [15–19]. The most significant of these is intramammary infection of dairy cattle leading to mastitis, which causes a substantial economic loss to the dairy industry worldwide [6, 20, 21]. The CC398 *S. aureus* isolate was more prevalent in nasal swabs of pig and cattle farmers than of nonfarming human controls [22, 23]. An examination of livestock-associated MRSA (LA-MRSA) in human case isolates in the Netherlands indicated an increase from 0% in 2002 to greater than 21% in mid-2006 [23] and 35% in 2009 [24]. In most European countries, CC398 remains the most commonly identified type of LA-MRSA [15, 25–27]. However, the epidemiology of LA-MRSA differs in other geographic areas. A different strain of LA-MRSA, CC9, appears to be the prominent type in several Asian countries [28–32]. Poultry may harbor CC398 strains [16, 33, 34] but CC5 [33, 35] and other types unrelated to CC398 have also been reported [36]. The diversity of LA-MRSA in the USA appears to be higher than that identified in Europe or Asia, with reports of both CC398 as well as a variety of "human″ types of *S. aureus* in livestock. LA-MRSA infections among livestock animals and associated farmers are of great concern as these sources could potentially serve as reservoirs for zoonotic infections [14]. Contamination of food with enterotoxin producing *S. aureus* leads to over 240,000 cases of food-borne illness in the United States annually. Although most *S. aureus*–related food-poisoning incidents are self-limiting and go away within 2 days, some serious infections have been reported as well [4, 5]. A large number of the reported staphylococcal food-poisoning outbreaks can be traced back to a human source harboring *S. aureus* producing certain staphylococcal enterotoxins (SEs) [1, 37]. Most of the LA-MRSA strains, particularly the ST398 group, do not appear to code for any of the known SEs [11, 38–42]. However, genes for SEs B, K, and Q have been detected in MRSA CC398 strains isolated from geographically diverse pig farms in Germany [43]. The acquisition of enterotoxin genes along with the virulence factors, such as Panton-Valentine leukocidin (*pvl*) genes by LA-MRSA may eventually pose a threat to humans, suggesting that

animals have the potential to be a source of primary contamination as well [16, 44].

In 2005, pigs were reported as an animal reservoir of *S. aureus* in France, including MRSA [17]. Pig farmers were more frequently colonized than nonfarmers and one of the most

**2. MRSA in food-producing animals**

**2.1. Porcine MRSA**

48 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

prevalent strains of *S. aureus* in pig farmers was CC398 [45–49]. Forty-five percent of veterinarians attending pig farms in the Netherlands were positive for MRSA [48]. In Belgium and Denmark, the prevalence of MRSA in veterinarians was 9.5% and 1.4%, respectively [47]. German researchers reported that MRSA ST398 that carried SCC*mec* IV or V, accessory gene regulator type I and capsule type 5 [25] in pig primary production herds was higher in their country (45–70%) than in the rest of the European Union [45, 46]. In an interesting report, 12.5% of attendees at an international meeting concerning pig health carried MRSA and 91.2% (31/34) of them CC398 [50]. While early studies on farms and of meat identified CC398 strains in animals, farm workers, and meat products [51, 52], other studies also documented CC398 in populations with no obvious livestock contact [53–55]. The emergence of this strain was also reported in pigs and pig farmers in the Netherlands [22, 56, 57], Denmark [58], Germany [59], and Canada [60]. Detection rates of MRSA in breeding and production herds were 46%, 43.5%, and 40% in Spain, Germany, and Belgium, respectively, but no single strain of MRSA was found in Finland and Denmark in 2008 [61]. The majority of LA-MRSA lineage belonged to ST398, accounting for 92.5% of the MRSA isolates. Other ST types, human-associated MRSA ST1 and cattle-associated ST97 in finishing holdings, and ST9 in the same animal species in Europe [62], CC9, and CC49 in Switzerland [63] have also been reported.

Various farm types in the Netherlands were reported to have MRSA in 23–71% of their pigs, and it was especially high in farms with finishing pigs (pigs are almost ready to be sent to market) [49, 57, 64]. The presence of MRSA is dependent on pig production type and herd size and increases from 31 to 86% depending upon small-, medium-, and large-sized farms carrying <250, >500, and >1000 animals, respectively [45, 62, 64, 65]. MRSA prevalence also varied with farm type, e.g., fattening and closed (farrow-to-finish) farms exhibited 94 and 56% MRSA, respectively [66]. Transportation from farm to slaughterhouse [38, 67], lairage [38], national and international trade [57], and slaughter house employees all have been reported to enhance MRSA contamination and may play important roles in transmission of the bacteria [68]. It was proposed that MRSA contamination in piglets is dependent on the status of sows [69]. When a sow was colonized with MRSA, 100% piglets were MRSA-positive. However, 84% piglets were MRSA-positive when there was no MRSA contamination in a sow. Higher numbers of MRSA were isolated in suckling (52.9%) and weanling piglets (53.4%) than sows (38.3%) [64]. Prevalence of MRSA in pigs has been linked to their age but the data are not conclusive. MRSA were identified in 100% of 9–12-week-old pigs, whereas in adult animals it decreased to 36% [51]. On the other hand, Weese et al. [69] reported that MRSA was more prevalent in post-weaning (85%) than preweaning pigs (34.5%) in pig farms without antimicrobial treatment. Percent of MRSA colonization in Canadian piglets on days 1, 28, 56, and 70 were 1, 34, 50, and 42%, respectively [69]. Khanna et al. [60] reported no variation in MRSA prevalence based on age groups. MRSA does not seem to cause serious infection in pigs, but there have been a few reports of MRSA from exudative epidermitis lesions of piglets on a breeding farm [70] and in pigs suffering from infection of the urinary-genital tract, skin infection, and metritis-mastitis-agalactia syndrome [71]. While MRSA ST398 isolated from diseased pigs did not carry the major virulence genes, such as toxic shock syndrome toxin 1, *pvl*, and exfoliative toxins, they carried some virulence genes, such as α- and δ-hemolysins, proteases, capsule type-specific genes, microbial surface components recognizing adhesive matrix molecules, biofilm-associated, and enterotoxin genes [44]. Their MLST, *spa*, and SCC*mec* types were identified as ST398, t011, and IV, respectively.

The epidemiology of livestock-associated *S. aureus* in the USA appears to be notably different than that in European countries. Most of the porcine MRSA isolates in Canada, Europe, Peru, and USA were CC398 [65]. A human epidemic clone, Canadian MRSA-2 (CMRSA-2, USA100, CC5), was found in nasal and rectal swabs of pigs in Canada [60]. This isolate was the most common cause of health care-associated infections in Canada. CMRSA-5 (USA500, ST8) also isolated from retail pork in Canada, is a human epidemic strain that also has been documented in horses and horse personnel [72]. Three *spa* types (t011, t034, t108) within CC398 were the most frequent in breeding and production herds in Europe, and t108 was most popular only in the Netherlands among European countries. On the contrary, prevalence of t899 in Italian breeding and production herds was between 24% and 27% [62, 65]. In Italy, CC1 and CC97 lineages among MLST types that do not belong to CC398 were significantly high in the primary production of pigs [62]. In one study carried out in a jail setting in Texas, CC398 isolates made up of 13.2% of all MSSA identified within this population. Apart from CC398, other human strains of *S. aureus* have also been found in USA livestock. Studies carried out on swine farms in the USA have identified human strains within the noses of live animals [55, 73, 74] and farm dust [75]. Several papers have reported CC5 strains rather than CC398 associated types to be the dominant strain isolated from pig farms in both Iowa and Ohio [75, 76], whereas others have found CC398 to be the most common molecular type [51, 76]. Studies on workers on pig farms and in processing plants found substantial diversity within *S. aureus* isolates, including CC398, CC5, and CC8 strains, among others [77–79]. MRSA attributed to ST5 was recently reported in pigs in the USA [38]. A different swine-associated MRSA strain, CC9, is circulating among pigs and pig farmers in China and Malaysia [29, 30, 80, 81]. MRSA ST22, known as human epidemic clone EMRSA-15 in the UK, was also discovered in pigs and in hospitalized patients in Singapore with an elevated frequency [82, 83]. High frequency of MRSA CC9 with *spa* types t899 and t4358 was reported in porcine samples in Asian countries, whereas it was not found as much in Europe or the USA [28–30, 84]. In Thailand, porcine MRSA ST9 isolates had a unique *spa* type (t337) and SCC*mec* type (SCC*mec* IX) that were different from other LA-MRSA ST9 strains found in Asian countries [28]. In 2012, Lim et al. [85] reported two ST types, livestock-associated ST398 and human-associated ST72, from pigs, which was the first finding of ST398 in Korean pigs.

#### **2.2. Bovine MRSA**

The first report of MRSA in farm animals was published in the early 1970s, when the bacteria were isolated from the milk of dairy cows with mastitis in Belgium [86] and clustered in the CC398 group [87]. Devriese and Hommez [88] suspected that these samples were most likely contaminated by humans. In the past few years, MRSA has been isolated from cows or their milk in Korea [89–91], Hungary, Mexico, and the Netherlands [89, 92, 93]. There have also been numerous reports of MRSA from cows or their milk in Brazil, Italy, Pakistan, Nigeria, Turkey, and the USA [94–96]. Subsequently, several reports have described bovine udder infections caused by LA-MRSA CC398 [97]. In Dutch farms, MRSA was detected in 18–31% of veal calves [98]. In 2010, the European Union reported that 20% of veal calves in Germany molecules, biofilm-associated, and enterotoxin genes [44]. Their MLST, *spa*, and SCC*mec* types

The epidemiology of livestock-associated *S. aureus* in the USA appears to be notably different than that in European countries. Most of the porcine MRSA isolates in Canada, Europe, Peru, and USA were CC398 [65]. A human epidemic clone, Canadian MRSA-2 (CMRSA-2, USA100, CC5), was found in nasal and rectal swabs of pigs in Canada [60]. This isolate was the most common cause of health care-associated infections in Canada. CMRSA-5 (USA500, ST8) also isolated from retail pork in Canada, is a human epidemic strain that also has been documented in horses and horse personnel [72]. Three *spa* types (t011, t034, t108) within CC398 were the most frequent in breeding and production herds in Europe, and t108 was most popular only in the Netherlands among European countries. On the contrary, prevalence of t899 in Italian breeding and production herds was between 24% and 27% [62, 65]. In Italy, CC1 and CC97 lineages among MLST types that do not belong to CC398 were significantly high in the primary production of pigs [62]. In one study carried out in a jail setting in Texas, CC398 isolates made up of 13.2% of all MSSA identified within this population. Apart from CC398, other human strains of *S. aureus* have also been found in USA livestock. Studies carried out on swine farms in the USA have identified human strains within the noses of live animals [55, 73, 74] and farm dust [75]. Several papers have reported CC5 strains rather than CC398 associated types to be the dominant strain isolated from pig farms in both Iowa and Ohio [75, 76], whereas others have found CC398 to be the most common molecular type [51, 76]. Studies on workers on pig farms and in processing plants found substantial diversity within *S. aureus* isolates, including CC398, CC5, and CC8 strains, among others [77–79]. MRSA attributed to ST5 was recently reported in pigs in the USA [38]. A different swine-associated MRSA strain, CC9, is circulating among pigs and pig farmers in China and Malaysia [29, 30, 80, 81]. MRSA ST22, known as human epidemic clone EMRSA-15 in the UK, was also discovered in pigs and in hospitalized patients in Singapore with an elevated frequency [82, 83]. High frequency of MRSA CC9 with *spa* types t899 and t4358 was reported in porcine samples in Asian countries, whereas it was not found as much in Europe or the USA [28–30, 84]. In Thailand, porcine MRSA ST9 isolates had a unique *spa* type (t337) and SCC*mec* type (SCC*mec* IX) that were different from other LA-MRSA ST9 strains found in Asian countries [28]. In 2012, Lim et al. [85] reported two ST types, livestock-associated ST398 and human-associated ST72, from pigs,

The first report of MRSA in farm animals was published in the early 1970s, when the bacteria were isolated from the milk of dairy cows with mastitis in Belgium [86] and clustered in the CC398 group [87]. Devriese and Hommez [88] suspected that these samples were most likely contaminated by humans. In the past few years, MRSA has been isolated from cows or their milk in Korea [89–91], Hungary, Mexico, and the Netherlands [89, 92, 93]. There have also been numerous reports of MRSA from cows or their milk in Brazil, Italy, Pakistan, Nigeria, Turkey, and the USA [94–96]. Subsequently, several reports have described bovine udder infections caused by LA-MRSA CC398 [97]. In Dutch farms, MRSA was detected in 18–31% of veal calves [98]. In 2010, the European Union reported that 20% of veal calves in Germany

were identified as ST398, t011, and IV, respectively.

50 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

which was the first finding of ST398 in Korean pigs.

**2.2. Bovine MRSA**

carried MRSA [99]. A survey of 51 veal calf farms in the Netherlands indicated that an average of 38% of farmers and 16% of family members were colonized with LA-MRSA [19, 100]. Recently, another group of LA-MRSA strains (CC130, CC425, and CC1943) that were initially thought to be bovine-specific lineages emerged in humans [101]. A number of multidrugresistant MRSA were isolated from bovine mastitis in Germany [97, 102], the majority of which were MRSA ST398 related to animal strains, but an isolate of the clonal complex group CC8 was identified as a human epidemic MRSA strain Irish-01 [103]. In 2010, Hata et al. first discovered MRSA in cow's milk in Japan and the genotypes (ST5-SCC*mec* II) were the same as or similar to human strains [104]. There has been a dramatic surge in human CC398 infection and colonization in the Netherlands, increasing from 0% in 2002 to more than 21% in 2006 [23]. A recent Dutch study indicated that the annual incidence of MRSA in humans more than tripled from 2001 to 2006 where 23%, 26%, 16%, and 10% of the patients acquired MRSA from a foreign hospital, animals, nosocomial transmission, and the community, respectively [105]. The presence of MRSA CC398 in pig farms with a concomitant increase in CC398 infections in humans clearly suggests that pigs or cattle are specifically a risk factor and CC398 MRSA colonization and prevalence in humans is associated with animal contact [23, 46, 106, 107]. The above argument is further strengthened by the findings that the CC398 carrier status of farm workers decreased dramatically when they took a break from direct animal care duties [19, 100]. People who visited farms to collect samples for a shorter duration carried MRSA transiently as compared to those who had prolonged visits, suggesting that a prolonged contact with animals is probably an important factor for higher rates of colonization [49].

While a majority of the MRSA collected from dairy cattle belonged to ST398 [89], other ST types, such as ST1-t286-SCC*mec* IV, ST72-t324 [108], ST59-t437–V [91], ST10-t127-SCC*mec* IVa genotype [92], SCC*mec* types IVg [109], CC97, t4795, and t1730 [110], and a *mecA* variant (*mecA*LGA251) known as *mecC*, are also reported from MRSA CC130 and ST425 isolates [111]. The *mecC* type was also detected in Danish MRSA CC130 isolated from a cow and the genotypic characteristics, such as *spa* type (t843), MLVA (MT429) and PFGE profiles of bovine isolates were the same as the human isolates, implying transmission between humans and ruminants [112]. The geographic variation in the prevalence and origin of CC398 colonization and incidence of infection in humans is quite interesting. MRSA has been detected in retail beef, but nasal and fecal sampling of nearly 500 Canadian feedlot cattle, shortly before slaughter, detected no MRSA [113]. Moreover, while CC398 MRSA infection in humans is a leading cause of CA-MRSA infection in some European countries, it is rare in North America despite the presence of CC398 in livestock [114]. The reasons for the low incidence of CC398 infections in the USA may include differences in direct and indirect contact with food animals, much lower population density in North American pig-rearing regions, and the common presence of other competing MRSA strains in people in the general population. Although some studies suggested that the MRSA present in cattle is bovine-specific, most of the reports indicated that MRSA found in cattle were derived from humans [89, 92, 115, 116]. Bovine, porcine, canine, feline, and equine MRSA isolates containing the *pvl* gene and other virulence factors, such as *chp, scn, seb, sek*, and *seq*, toxic shock syndrome toxin 1 (*tsst1* or *tst*) gene [91, 117], hemolysin, protease, superantigen-like protein, capsule, and biofilm-associated genes [118–121], may pose a potential threat to public health.

#### **2.3. Poultry MRSA**

CC398 is not limited to large livestock animals alone; it has also been reported in poultry [18, 122], manure from chicken farms and soil fertilized with this manure [123]. However, the numbers of MRSA ST398-t011-SCC*mec* V isolated from chickens are lower (0–28%) than in pigs (82–92%) on the same farm [124]. In another study from Belgium [109], MRSA present in 0.8–1.8% layers and broilers (chickens raised for meat) was clustered into two ST types, ST398 (t011, t899) and ST239 (t037). Two other studies from Belgian broiler farms [18, 125] reported 12.8–14.3% of randomly selected Belgian broiler farms to be positive for CC398. In 2010, the Federal Institute for Risk Assessment reported a contamination rate of 32% in turkey meat in Berlin, Germany [126]. Similar contamination frequencies were reported from Canada and the USA [72, 127], as well as from Taiwan [128]. Moon et al. [91] reported that the *S. aureus* isolated from chicken carcasses contained 1.3% MRSA, which was more than the MRSA isolated from other animal carcasses (0.3%) in Korea. Poultry-associated *S. aureus* isolates belonging to genotypes other than CC398 have also been reported from different geographic regions [36, 129–131]. Mulders et al. [132] reported that 6.9% of MRSA present in broiler chickens in the Netherlands represented the ST9-t1430 genotype. A single *spa* type, t1456, in poultry was seen and distinguishable from the *spa* types of ST398 observed in other animals in Belgium [125]. Genotypic and antimicrobial patterns between 14 MRSA isolates from broilers and pigs were identical [133]. The MRSA isolates from Korea had the genotype of ST692-t2247–III [91] and the MRSA isolates from Hong Kong exhibited the genotypes ST9-t899–IV [81] and CC9 (t899, t1234) [134]. A study from Denmark analyzed the isolates from infected poultry and detected a predominant common human epidemic clone CC5 [129]. Using a population genomics approach, Lowder et al. [36] examined the origin of *S. aureus* isolates from diseased and healthy poultry from four continents and found that the majority of isolates belonged to a single clonal complex CC5 belonging to a known human-associated lineage. The poultry isolates were more closely related to each other than to human CC5 isolates, but were most similar to a subclade of CC5 that was circulating in Polish hospitals in the 1980s. In a study conducted in Korea, 930 food samples were collected, and four strains of the CA-MRSA CC5 human clone were identified [135]. In a human case study, a 63-year-old Dutch woman who owned a chicken farm developed a life-threatening endocarditis; the infecting MRSA isolate was identified as CC398 [136], similar to an isolate found in a pig farm nearby and to MRSA isolates previously found in other pig farms in the Netherlands.

#### **2.4. Other meat products**

*S. aureus* is found frequently in a variety of retail meat products. A Dutch Food Safety Agency analyzed 2217 samples of various kinds of meats from the retail stores and found that 11.9% of 2217 samples had MRSA [137]. The distribution of MRSA within various meat types was listed as follows: beef, 10.6%; veal, 15.2%; lamb and mutton, 6.2%; pork, 10.7%; chicken, 16.0%; turkey, 35.3%; fowl, 3.4%; and game, 2.2%. Of all the MRSA isolates, 85% of the isolates belonged to ST398; the other STs were possibly of human origin [137]. Another Dutch survey found that 46% of retail meat samples contained *S. aureus* strains, of which two (2%) were MRSA: one was CC398 and the other was USA300 [138]. Studies in Switzerland and Japan showed the prevalence of *S. aureus* in meat products to be 23 and 65%, respectively [122, 139]. A USA survey of 120 retail meat samples indicated that 39.2% contained *S. aureus* strains, 5% of which were MRSA of the types USA100 (ST5) and USA300 (ST8) [127]. A Canadian survey [72] found that 7.7% of retail meat samples harbored MRSA; 30% belonged to the clonal complex CC398, 40% were CC8, and 30% were CC5, a strain commonly found in humans in both the USA and Canada.

#### **2.5. Milk**

**2.3. Poultry MRSA**

52 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

CC398 is not limited to large livestock animals alone; it has also been reported in poultry [18, 122], manure from chicken farms and soil fertilized with this manure [123]. However, the numbers of MRSA ST398-t011-SCC*mec* V isolated from chickens are lower (0–28%) than in pigs (82–92%) on the same farm [124]. In another study from Belgium [109], MRSA present in 0.8–1.8% layers and broilers (chickens raised for meat) was clustered into two ST types, ST398 (t011, t899) and ST239 (t037). Two other studies from Belgian broiler farms [18, 125] reported 12.8–14.3% of randomly selected Belgian broiler farms to be positive for CC398. In 2010, the Federal Institute for Risk Assessment reported a contamination rate of 32% in turkey meat in Berlin, Germany [126]. Similar contamination frequencies were reported from Canada and the USA [72, 127], as well as from Taiwan [128]. Moon et al. [91] reported that the *S. aureus* isolated from chicken carcasses contained 1.3% MRSA, which was more than the MRSA isolated from other animal carcasses (0.3%) in Korea. Poultry-associated *S. aureus* isolates belonging to genotypes other than CC398 have also been reported from different geographic regions [36, 129–131]. Mulders et al. [132] reported that 6.9% of MRSA present in broiler chickens in the Netherlands represented the ST9-t1430 genotype. A single *spa* type, t1456, in poultry was seen and distinguishable from the *spa* types of ST398 observed in other animals in Belgium [125]. Genotypic and antimicrobial patterns between 14 MRSA isolates from broilers and pigs were identical [133]. The MRSA isolates from Korea had the genotype of ST692-t2247–III [91] and the MRSA isolates from Hong Kong exhibited the genotypes ST9-t899–IV [81] and CC9 (t899, t1234) [134]. A study from Denmark analyzed the isolates from infected poultry and detected a predominant common human epidemic clone CC5 [129]. Using a population genomics approach, Lowder et al. [36] examined the origin of *S. aureus* isolates from diseased and healthy poultry from four continents and found that the majority of isolates belonged to a single clonal complex CC5 belonging to a known human-associated lineage. The poultry isolates were more closely related to each other than to human CC5 isolates, but were most similar to a subclade of CC5 that was circulating in Polish hospitals in the 1980s. In a study conducted in Korea, 930 food samples were collected, and four strains of the CA-MRSA CC5 human clone were identified [135]. In a human case study, a 63-year-old Dutch woman who owned a chicken farm developed a life-threatening endocarditis; the infecting MRSA isolate was identified as CC398 [136], similar to an isolate found in a pig farm nearby and to MRSA

isolates previously found in other pig farms in the Netherlands.

*S. aureus* is found frequently in a variety of retail meat products. A Dutch Food Safety Agency analyzed 2217 samples of various kinds of meats from the retail stores and found that 11.9% of 2217 samples had MRSA [137]. The distribution of MRSA within various meat types was listed as follows: beef, 10.6%; veal, 15.2%; lamb and mutton, 6.2%; pork, 10.7%; chicken, 16.0%; turkey, 35.3%; fowl, 3.4%; and game, 2.2%. Of all the MRSA isolates, 85% of the isolates belonged to ST398; the other STs were possibly of human origin [137]. Another Dutch survey found that 46% of retail meat samples contained *S. aureus* strains, of which two (2%) were MRSA: one was CC398 and the other was USA300 [138]. Studies in Switzerland and Japan showed the prevalence of *S. aureus* in meat products to be 23 and 65%, respectively [122, 139].

**2.4. Other meat products**

In general, the occurrence of MRSA in bovine mastitis isolates is well studied and its prevalence seems to be very low [140]. Following the initial reports of isolation of MRSA from mastitic cows [86], sporadic cases of MRSA in dairy cattle were detected among *S. aureus* isolates from clinical or subclinical mastitis. In one of the studies from Korea [109], MRSA were isolated from the milk of cows with an isolation ratio of 0.18%. In one report on dairy farms in Belgium, a high percentage (15%) of MRSA was found in lactating cows [140]; these cows had a previous history of MRSA. The long-term low prevalence of MRSA mastitis is quite surprising, given the number of years since the first identification of MRSA in cattle and the close contact of humans with the udders of dairy cattle. In Germany, the highest proportion of positive samples (45%) was found in nasal swabs from veal calves at slaughter and the lowest rate was 4.1% in bulk tank milk. Most isolates, irrespective of origin, were from *spa* types t011 and t034 belonging to the clonal complex CC398 [141]. The finding of LA-MRSA CC398 in tank milk suggests udder colonization and possibly cases of subclinical mastitis in dairy cattle in Germany [141]. Close contact of dairy cattle with humans could lead to a transfer of strains between them. In one of the reports from Hungary, MRSA isolates from mastitic cows and a worker were found identical by phenotypic and genotypic analysis indicating a transfer between cows and human [92].

#### **2.6. Fish**

Fish is not a normal host for staphylococci and its presence on fish is either due to disease in the fish, contamination, or poor personnel hygiene. The first report of the isolation of MRSA from Tilapia was published in 2010, where 559 *S. aureus* isolates from the brain, eyes, and kidneys of tilapia from 11 farms collected for a period of 2 years were analyzed and 50% were identified as MRSA [142]. In another study [143] from Korea that analyzed 165 *S. aureus* strains isolated from different food samples between 2003 and 2006, four were identified as MRSA. Two of these were from beef and two from fish. The two fish isolates, one from sea bass and other from rockfish, were identified as ST1 and ST72, respectively. An analysis of 200 ready to eat (RTE) fish samples collected from 10 shops belonging to four supermarket chains in Japan, 5 were MRSA and 5 others were identified as coagulase-negative MRSA [144]. Molecular typing of two MRSA isolates by *spa* sequencing and MLST typing identified t1767 and ST8, respectively. Interestingly, MRSA ST8 strains have been predominantly isolated from humans in the USA and Europe but are of rare occurrence in foods in Japan. It is not certain if the MRSA in fish was from human or fish origin. In another report from Greece, one hundred samples from RTE fish products were examined and two were reported to have MRSA belonging to the *spa* types t316 (ST359) and t548 (ST5) [145]. In a recent report, a patient developed foot infection with MRSA after a fish pedicure [146], but the origin of the MRSA could not be determined in this case. Reports of fish from Egypt, India, and Yemen have also been reported to harbor as much as 3.5% MRSA [147]. In this report, two of the MRSA isolates were found to harbor the enterotoxin genes *seg* and *sei*. Since the global spread of multi-drugresistant bacteria has increased in the past decade, the finding of enterotoxigenic MRSA in fish should be of concern. The global trade of fish increases the possibility of intercontinental transmission of multidrug-resistant and enterotoxigenic *S. aureus* and its potential influence on consumer health worldwide should be monitored.
