Staphylococcus aureus is a common facultative pathogenic bacterium that has long been recognized as a challenge in both human and veterinary medicine (Nemeghaire et al., 2014). In cattle, it is responsible for approximately one-third of cases of clinical and subclinical mastitis (Bradley et al., 2007; Botrel et al., 2010), a disease that causes major economic loss in the dairy industry worldwide. Since MRSA was first reported in the United Kingdom in 1961, it has become a global cause of hospital-associated (HA) and community-associated (CA) infections (Uhlemann et al., 2014). In the past few decades, a new subgroup of S. aureus, so-called livestock-associated S. aureus (LA-SA) has been described. There have been several reports of MRSA colonization and/or infections in dairy cattle since the first report of MRSA in mastitis in 1972 (Devriese et al., 1972; Monecke et al., 2007; Fessler et al., 2010; Huber et al., 2010). LA-SA ST398 was originally reported to have emerged in the Netherlands among pigs and pig farmers in 2003 (Voss et al., 2005), and was later found in Austria, Germany, and Denmark (Van Cleef et al., 2011; Bal et al., 2016). It developed into an overwhelmingly dominant lineage in Europe and North America (Vanderhaeghen et al., 2010; Cuny et al., 2013). Unlike those in Europe and North America, the epidemic LA MRSA clones in Asian countries have their own characteristics. LA MRSA ST9 has been predominantly isolated from pigs in China, Hong Kong, Taiwan, Thailand, and Malaysia. LA MRSA ST221 has been reported in Japan, and LA MRSA ST398 has emerged among pigs in Singapore and South Korea (Cui et al., 2009; Guardabassi et al., 2009; Neela et al., 2009; Baba et al., 2010; Ho J. et al., 2012; Larsen et al., 2012). Although the sequence type of LA MRSA isolates was mainly ST9 in most Asian countries, these isolates harbored different SCCmec elements in different areas, such as SCCmec III in China, SCCmec IVb, or V in Hong Kong, SCCmec V in Malaysia, SCCmec IX in Thailand and non-types I–VII in Taiwan (Larsen et al., 2012; Fang et al., 2014; Chuang and Huang, 2015). ST9 strains were usually associated with the MDR phenotype, with high resistance rates (>80%) to erythromycin, clindamycin, tetracycline, ciprofloxacin and gentamicin (Wan et al., 2013). To date, most studies on LA S. aureus have been performed in pigs, mainly with MRSA strains, even though the vast majority of S. aureus strains are host-specific. The data on molecular typing and antibiotic-related studies of bovine derived S. aureus are very limited. A relatively higher incidence of S. aureus mastitis has been reported in China than in other countries, which may cause a particular public threat (Li et al., 2011; He et al., 2014). According to previous studies performed in six Chinese provinces from March 2010 to August 2013, the most popular S. aureus clones causing bovine mastitis were primarily ST97 (51.9%), ST398 (13.6%) and ST2154 (8.6%), with ST97-MRSA-SCCmec IV representing the most common clone (50%; Wang et al., 2015). However, the characteristics of S. aureus from dairy cows in eastern regions of China have not yet been thoroughly discussed. To provide elementary evidence for developing the appropriate treatment and control measures for bovine mastitis, it is essential to understand the molecular epidemiology and the antibiotic resistance of S. aureus infections locally. Therefore, the present study was designed to analyze the molecular characteristics of S. aureus strains in bovine mastitis isolated from 2014 to 2015 in the Shanghai and Zhejiang areas.

S. aureus is the most common causative organism of bovine mastitis, which is considered a common, complicated and economically unbearable disease in dairy animals worldwide (Wyder et al., 2011). Dairy animals with mastitis frequently shed S. aureus into the milk supply, which can contribute to food poisoning in humans. This study intended to investigate the molecular profile and antimicrobial resistance of S. aureus isolates recovered from bovine mastitis from 2014 to 2015 in eastern regions of China.

In this study, we characterized 212 S. aureus isolates from bovine mastitis milk samples collected from farms in Shanghai and Zhejiang areas and identified 6.6% as MRSA strains, which was lower than western countries and Hong Kong (16–21.3%; Guardabassi et al., 2009), but higher than Korea (21/657, 3.2%; Lim et al., 2012), Malaysia (1.4%; Neela et al., 2009), and Japan (0.9%; Baba et al., 2010). These different frequencies may be due to the different animal populations studied or the implemented methodologies, among other factors.

Our study identified 14 bovine MRSA strains, including nine ST9-SCCmecXII-t899 strains, four belonging to SCCmec IV type (two ST965-t062, one ST59-t437, one ST537-t2755), and one SCCmecV strain (ST630-t4549). The MRSA isolates from eastern regions of China belonged to different spa and SCCmec types, suggesting their genetic diversity. In those previous studies, CC9 was identified as the predominant MRSA associated with pig farming and harbored various SCCmec types in different areas. For example, SCCmec was type III in mainland China, types IVb or V in Hong Kong, non-types I–VII in Taiwan, type V in Malaysia and type IX in Thailand (Cui et al., 2009; Ho J. et al., 2012; Larsen et al., 2012). In addition, SCCmec XII was a new SCCmec element that was first identified in two ST9-MRSA-t899 strains from pigs in China (Ho P. L. et al., 2012; Wang et al., 2015; Wu et al., 2015). It included a rare class C2 mec gene complex and a ccrC gene complex. The newly identified SCCmec was 48.575 kb in length and contained 45 predicted open reading frames. Therefore, the emerging ST9-MRSA-SCCmec XII strains can be isolated in both pigs and bovines in China, suggesting the potential possibility of its cross-species transmission. Notably, the CC9 MRSA isolates in the current study were shown to be multidrug-resistant, with high resistance rates to β-lactams and eight other antibiotics widely used in chemotherapy. The presence of multidrug resistant MRSA clone CC9 suggests an alarming situation and could be a serious challenge to therapy. The PFGE result in our study showed that the restriction profiles of 10 CC9 strains were identical with only one fragment difference between MRSA CC9 and MSSA CC9 (Supplementary Image 1). These observations support the notion that CC9 MSSA lineages may provide a stable genetic environment for the integration of SCCmec in favor of their infection and transmission in different areas.

Among the 212 S. aureus isolates, the prevalence of MSSA in this study was 93.4%. In addition, our study revealed more heterogeneous MSSA lineages, 16 distinct STs were identified in MSSA, but only five STs were found in MRSA. The most frequently represented lineage in MSSA was CC97 (39.2%, 83/212), which accounted for more than one third of all S. aureus isolates, followed by CC520 (16%, 34/212), CC188 (10%, 21/212), CC398 (7.1%, 15/212), and CC7 (5.2%, 11/212). CC97 was found to be the most predominant clone, which is consistent with a recent study on bovine mastitis infection that also identified CC97 as the dominant strain type among six provinces of China from March 2010 to August 2013 (Wang et al., 2015). However, the difference is that all CC97 isolates identified in our study were MSSA strains. In particular, CC97 strains have become the dominant lineage in Chile (Smith et al., 2005), Brazil (Aires-de-Sousa et al., 2007; Rabello et al., 2007), Japan (Hata et al., 2010), the Netherlands (Ikawaty et al., 2009), and the United States (Smith et al., 2005). We found that CC97 isolates were more resistant to gentamicin and penicillin than other MSSA isolates. This implies that appropriate drug selection based on different MSSA types may reduce the reservoir of drug-resistant bacteria.

The second most frequent MLST type shown here was CC520 (16%), which was only reported in a few studies and could indicate the emergence of a new epidemic clone. Compared to other lineages, CC520 carried more virulence elements but presented a lower drug resistance rate, which may facilitate its success as a pathogen spread in bovines. Moreover, in our study, 21 MSSA isolates belonged to CC188, which is a double locus variant (DLV) of CC1 that includes MW2/USA400, the highly virulent and first-known Panton-Valentine leukocidin (PVL)-positive MRSA strain (Centers for Disease Control and Prevention, 1999). In a previous study of human infection, CC188 was the predominant clone in community-onset MSSA infections (Chen et al., 2012). However, the contamination of bovine mastitis samples with S. aureus by humans cannot be excluded. We propose that these results represent a probable transient acquisition from human handling, which will require more attention in further studies.

LA MRSA was originally reported to have emerged in 2003 among healthy pigs and pig farmers in France and the Netherlands (Witte et al., 2007). In European countries and in North America, CC398 was the overwhelmingly dominant LA MRSA, whereas CC9 predominated in most Asian countries (Cortimiglia et al., 2016; Sharma et al., 2016). In our study, all CC398 strains were identified as MSSA strains. Additionally, the observed spa type distribution of MSSA CC398 mirrored the most frequently detected spa types for MRSA CC398: t034. In contrast to the large number of reports on MRSA CC398 (Fessler et al., 2010; Schijffelen et al., 2010; Smith and Pearson, 2010; Crombé et al., 2012; Price et al., 2012), limited attention has been given to the ecology of MSSA CC398 in livestock farms (Hasman et al., 2010). MSSA CC398 is a clone of clinical importance that affects humans and livestock in different geographic regions. Price et al. analyzed 89 isolates and concluded that LA-MRSA likely originates from humans as MSSA. They theorized that the recent emergence of LA-MRSA can be seen as a reintroduction of MSSA that acquired methicillin-resistance to the original host (Price et al., 2012). Therefore, CC398-MSSA strains in this study may be a risk factor for the transmission of MRSA to people in close contact with the infected animals. Additionally, the present study showed that CC398 was more resistant to clindamycin, erythromycin, and ciprofloxacin. Our results may contribute greatly to the understanding of the emergence and transmission of MRSA CC398 among livestock because they may indicate the existence of a reservoir from which MRSA CC398 strains can emerge.

S. aureus is the most common causative organism of bovine mastitis. However, the treatment and control of this infection often fails due to the complex nature of the S. aureus strains. The development of drug resistance is a significant feature of these organisms. The results of this research revealed that all MRSA isolates were resistant to oxacillin, penicillin and cefoxitin. All MSSA isolates were susceptible to cefazolin, cefuroxime, and oxacillin. In general, MRSA strains showed much higher resistance rates to all the tested antibiotics than MSSA strains (Table 3). All the MRSA strains were found to be resistant to at least four antibiotics. It was apparent that CC9 strains had significantly higher multiple antibiotic resistance profiles compared with other lineages. The widespread resistance of cattle-derived S. aureus will be a serious challenge to bovine mastitis therapy. The ability of bacterial pathogens to produce biofilms is regarded as a major cause of resistance to antibiotics and as responsible for persistent infections and innate immune defense for both animal and human infectious diseases (Cucarella et al., 2004; Bjarnsholt, 2013). The biofilm formed in multilayer growth of bacteria, avoiding bacterial clearance by the host immune system. In our study, we detected biofilm formation in different lineages. The results showed that the biofilm formation ability of CC7 and CC188 strains was significantly higher than other lineages. However, the resistance rates to different antibiotics in CC7 and CC188 were not higher than other lineages. This may indicate that the mechanism of drug resistance in bovine mastitis-associated S. aureus may depend on other factors than biofilm formation. While biofilm formation increases drug resistance and infection time, it can reduce bacterial virulence. Hemolysis (lysis of erythrocytes) is a significant virulence determinant of S. aureus, representing a crucial way for the bacteria to acquire iron. As shown in our study, lysis of red blood cell capacity was relatively strong in all S. aureus strains of animal origin, except CC7 and CC188. Therefore, different types of S. aureus have different potential risks to human beings.

In conclusion, the prevailing S. aureus strains in bovine mastitis were mainly MSSA strains in eastern regions of China. However, the presence of the multidrug-resistant and toxigenic MRSA clone ST9-t899 SCCmec XII suggests an alarming situation and might be a serious challenge to therapy, creating a public health concern. There were differences in the resistance genes, virulence genes and biological activities of the different molecular types of strains. As S. aureus from livestock may emerge as a threat to public health, comprehensive surveillance should be taken against S. aureus infection in the management of animal husbandry products.

Blood of healthy individuals for the lysis of erythrocytes by bacterial culture was collected using a standard method in accordance with a protocol approved by the ethics committee of Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China. All individuals provided written informed consent prior to donating blood.

ML, JS, and XW planned and supervised the experiments and wrote the paper. TL, HL, XW, QG, and YD performed the experiments and/or analyzed the data.

This work was supported by the National Natural Science Foundation of China (grants 81371875, 81671975, and 81601737), the Shanghai Committee of Science and Technology, China (grant 15411960500) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (grant 81421001).