The yaks (Bos grunniens) are part of the genus Bos within the family Bovidae. As an iconic symbol of high altitude and of Tibet, more than 14 million yaks reside in high-altitude environments of China, Nepal, India, Kyrgyzstan, Pakistan, Russian Federation and Mongolia, mainly across the Qinghai-Tibet Plateau (above 2500–6000 m) in China (Mi et al., 2013). Yaks are essential animals for Tibetans and other nomadic pastoralists in high-altitude environments owing to their provision of the basic necessities (such as milk, meat, transportation, hides for tented accommodation and dung for fuel) (Qiu et al., 2015). However, frequent outbreaks of bacterial diseases in yaks cause serious economic losses and healthy concern to local people (Bandyopadhyay et al., 2012).

Enterococci are the members of Gram-positive lactic acid-producing bacteria widely distributed in the gut microbiota of humans and other animals (García-Solache and Rice, 2019). Enterococci belong to facultative anaerobes and are highly tolerant to diverse environmental conditions, such as extreme PH, salt concentrations and a wide range of temperature (from 10 to >45°C) (Arias and Murray, 2012). The genus Enterococcus includes Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus mundtii, and Enterococcus casseliflavus, etc. (Ahmed and Baptiste, 2018). Both E. faecium and E. faecalis are the predominant species that are easily isolated from feces of healthy organisms (Landete et al., 2018). Commensal enterococci are generally not pathogenic in healthy hosts; however, in susceptible hosts, they can cause infections in the wounds, dysbiotic gastrointestinal tract, urinary tract, and endocarditis. More importantly, these infections are associated with biofilms (Ch’ng et al., 2019). Enterococcal biofilms increase their hardiness and contribute to both persistence during infection and contamination of the food industry and the environment (Piggot et al., 2012; Da-Silva-Fernandes et al., 2017). The ability of enterococci to form biofilms also increases their intrinsic tolerance to antibiotics and thus is a serious obstacle for the treatment of infections (Ch’ng et al., 2019).

Global public health is severely threatened by antimicrobial resistance (AMR), the major source of which is composed of the “ESKAPE” pathogens (E. faecium, Staphylococcus aureus, Klebsiella spp., Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) (Woksepp et al., 2014). Among them, E. faecium is considered as a typical multidrug-resistant (MDR) pathogen owing to both its enormous capacity to develop acquired resistance during antimicrobials chemotherapy and its intrinsic resistance to various antibiotics such as aminoglycosides and β-lactam-based antibiotics (Dale et al., 2015). The World Health Organization (WHO) recently released a list of drug-resistant “priority pathogens” that pose a significant threat to public health and urgently need new therapies (World Health Organization [WHO], 2017). Vancomycin-resistant E. faecium is classified as a Priority 2 pathogen in this list. Other enterococcal species that frequently occur in animals, such as E. durans, E. hirae, and E. casseliflavus, are also appropriate indicators of AMR in Gram-positive bacteria and their appearance can constitute a ‘pool’ of resistant genes, which may be transferred either to other commensal bacteria or even to pathogenic bacteria (Hamed et al., 2018).

As enterococci potentially transfer resistant genes from enteric bacteria in animals to humans via the food chain and they are potential pathogens, they have become a global public health concern. In previous reports, available data showed MDR Enterococcus with a variety of virulent determinants isolated from commensal animals, such as chickens, pigs and cattle (Diarra et al., 2010; Beshiru et al., 2017; De Jong et al., 2019; Igbinosa and Beshiru, 2019). However, there has been no available data on enterococci isolated from yaks. During the sampling process, we consulted local Tibetans and teachers of the Sichuan Academy of Grassland Sciences about the use of antibiotics in yaks. Some Tibetans used tetracycline antibiotics and cephalosporins, many Tibetans did not know the specific drug used to treat yak diseases. The level of antibiotic resistance and virulence determinants in enterococci isolated from yaks can reflect the selection pressure as a result of the use of antimicrobials and the potential pathogenesis, respectively.

Some enterococcal infections are associated with the production of biofilms, which increase enterococci’s intrinsic resistance to antimicrobials. Also, enterococci have the ability to horizontally transfer antibiotic resistance genes in biofilms at high rates, and the transfer is promoted by Epa, Ebp, PrgABC (Bhatty et al., 2015; Dale et al., 2015; La-Rosa et al., 2016). Both antibiotic resistance and biofilms of enterococci can increase the difficulty to treat bacterial diseases in yaks. It is therefore important to monitor AMR, virulence genes, and biofilm formation capacity of enterococci in yaks. In this study, we provided the first comprehensive analysis of antibiotic resistance, virulence genes and biofilm formation of Enterococcus species isolated from yaks.

Compared with findings about enterococci of other commensal animals, available data on enterococci isolated from yaks is still inadequate. The yak is vital to the production system of the Tibetans and other nomadic herders in high-altitude regions (Xiao-Yun et al., 2006). Here, we provide the comprehensive analysis of the antimicrobial susceptibility, biofilm formation, and virulence genes of several enterococcal species isolated from yaks. We observed a high proportion of multidrug-resistant enterococci with virulence factors in yaks.

In current study, 381 Enterococci were isolated. Unlike previous studies, the isolation rate of E. faecalis was slightly higher than that of E. faecium (De Jong et al., 2018, 2019; Lei et al., 2019). Moreover, the isolation rate of E. mundtii was 7.61% (n = 29), which contradicts the findings from previous studies that show a very low isolation rate of E. mundtii in other commensal animals (Kim et al., 2016; De Jong et al., 2018; Osman et al., 2019). These results indicate that Enterococcus species composition varies in different host environments.

Enterococcal resistance and tolerance to antimicrobials is a serious threat to global health that needs to be addressed as a priority. Enterococci are intrinsically resistant to various antibiotics (for example, aminoglycosides, clindamycin, and β-lactam-based antibiotics). Due to the low permeability of enterococcal cell wall to large aminoglycoside molecules, enterococci are moderately resistant to aminoglycosides and are more prevalence in E. faecium than E. faecalis (Aslangul et al., 2006; Abat et al., 2016). Enterococci have the ability to overexpress penicillin-binding proteins with low affinity for β-lactams, which allows enterococci intrinsically resistant to penicillin (Murray, 1997; Duez et al., 2001). Compared with E. faecalis, E. faecium is more intrinsically resistant to antimicrobials. However, E. faecalis can show tolerance to antibiotics by forming a thicker biofilm. Biofilms of E. faecalis isolated from clinic show increased tolerance to vancomycin and tigecycline (Hashem et al., 2017). In the present study, we found that 15 E. faecalis strains were resistant to vancomycin. However, vancomycin-resistant genes were not detected in these strains. It may be due to the formation of biofilms that enhance tolerance to vancomycin.

Enterococci can readily acquire resistance to antimicrobials during antibiotics chemotherapies. In this study, 60.37% of the Enterococcus isolates were MDR. We observed that erythromycin and tetracycline had a combined resistance trend, which was in line with the findings of previous study (Kim et al., 2019). High antibiotic resistance in the study area may be a marker for differences in multiple factors such as antimicrobials use, disease control measures, or genetic mutations leading to multidrug-resistant phenotypes (Jadhav et al., 2011). Our study reveal that local Tibetan people’s inappropriate use of antibiotics caused MDR enterococci to appear in yaks. Furthermore, yak is a free-grazing animal that can move around in a large geographic area, and thus yak feces containing MDR bacteria can also cause pollution to the local ecological environment. Therefore, robust monitoring programs are needed to control the spread of AMR in yaks.

The association among enterococcal species and resistance phenotypes varied in this study. Overall, E. faecium strains had a higher rate of MDR, whereas most of E. mundtii isolates were sensitive to antibiotics. Similarly, the higher prevalence of MDR E. faecium among Enterococcus spp. isolated from other animals has been documented previously (Novais et al., 2013; De Jong et al., 2018). Moreover, enterococcal species also had significant differences in antibiotic resistance among various antibiotics, for we observed that E. faecium was more resistant to penicillin and ampicillin than E. faecalis. This was in line with the findings of other study (Novais et al., 2013). Since enterococci that produce β-lactamase are extremely rare, the acquired resistance of E. faecium to ampicillin is mainly due to the penicillin-binding protein5 (pbp5) mutation, which has a lower affinity for β-lactam antibiotics, while the fact that E. faecalis has acquired resistance to ampicillin is mainly due to pbp4 mutation (Rice et al., 2004; Ono et al., 2005). Therefore, our results indicate that pbp5 of E. faecium may be more susceptible to mutations than pbp4 of E. faecalis under antibiotic stress.

According to the results of the antibiotic resistance phenotype, various resistance genes were detected in Enterococcus strains. Among the tetracycline resistance genes, tetM and tetL were significantly present in tetracycline-resistant enterococci. Similarly, ermB was more than ermA present in erythromycin-resistant enterococci. These results were in line with the previous studies (Schwaiger et al., 2009; Kim et al., 2019). We also detected oxazolidinone resistance genes optrA and poxtA. Oxazolidinones (tedizolid and linezolid) are effective antibiotics for the treatment of multidrug resistant Gram-positive bacterial (including vancomycin-resistant Enterococcus) infections, and these resistant genes can usually be linked with mobile genetic elements for horizontal transmission (Hao et al., 2019; Lei et al., 2019), so we should standardize the use of oxazolidinone and strengthen the detection of oxazolidinone-resistant enterococci to prevent the spread of resistant genes to the environment and humans. Enterococci resistance to ciprofloxacin is usually due to the mutations in the gyrA and parC quinolone resistance-determining region (Onodera et al., 2002; Leavis et al., 2016), so we did not detect plasmid-mediated quinolone resistance genes in isolated strains.

Enterococcal biofilms have been observed in many infections. Moreover, Biofilm is an important source of pollution in the food processing industry and enterococci isolated from food have the ability to form biofilms (Ch’ng et al., 2019; Igbinosa and Beshiru, 2019). Therefore, we determined biofilm formation capacity among Enterococcus species from yaks and its correlation with virulence genes. We found that many of the E. faecalis isolates showed higher-level biofilm formation and virulence genes, whereas most of the tested E. mundtii strains were negative to biofilm and all tested of virulence genes. Our findings corroborate the results reported that E. faecalis was the first-most frequent Enterococcus species associated with disease (Gawryszewska et al., 2017), while E. mundtii was low in virulence and infrequently associated to infections (Repizo et al., 2014).

We found that ace had a correlation with biofilm formation in E. faecium. Compared with many factors involved in E. faecalis biofilm formation, several E. faecium genes are involved in the development of biofilm, including esp, ebpABC, and ace (Sava et al., 2010; Lim et al., 2017). However, we observed the low prevalence of ebpA, ebpB, ebpC, and esp genes in E. faecium. Hemolysin–cytolysin (Cyl) and the proteases gelatinase (GelE) are important secreted factors and play a key role in enterococcal pathogenesis. These genes (cylA and gelE) were more often found in E. faecalis strains. Similar findings were also reported that E. faecalis more often carried cylA and gelE genes among enterococcal species (Golob et al., 2019; Stępień-Pyśniak et al., 2019).

Most biofilm-associated infections are caused by a combination of multiple microorganisms, and usually two or more of species can be found at the site of infection. Enterococci can account for a large proportion of the population in wound infections identified by next generation sequencing technology (Holá et al., 2010; Dworniczek et al., 2012). Isolation of E. faecalis is often accompanied by other bacteria. For example, Proteus mirabilis was recovered from nearly 40% of E. faecalis biofilms (Macleod and Stickler, 2007). It was also found on catheters together with Klebsiella pneumoniae and Escherichia coli (Galvan et al., 2016). Meanwhile, enterococci can act as gene pool to spread antibiotic resistance within and between species. This may lead to the aggravation of clinical symptoms and increase the difficulty of diagnosis and control of yak diseases. For the local Tibetans, yak milk and meat are not only the main protein food, but also an important economic source by exporting for high-end consumption (Yue et al., 2013), and thus AMR genes of enterococci in yaks can transfer to humans via the food chain. Therefore, it is altogether fitting and proper that we analyze the antibiotic resistance and virulence of enterococci isolated from yaks.

The results of this study reveal that 60.37% isolated strains were MDR and 9 antibiotic-resistant genes were detected. Likewise, great frequency of biofilm formation and virulence genes were observed among the isolated enterococci. The emergence of AMR genes and virulence genes in enterococci from yaks is a serious concern because they could be transmitted to humans through the food chain and the spread of these genes could significantly limit the treatment options for MDR bacteria. Finally, the study demonstrate that yaks are reservoirs of antimicrobial resistant enterococci with potential virulence. Therefore, we should initiate robust surveillance programs to control and monitor the use of antibiotics.

All datasets generated for this study are included in the article/Supplementary Material.

The animal study was reviewed and approved by the College of Life Science, Sichuan University affiliation ethics committee, and all efforts were made to minimize animal suffering.

PC designed the study and wrote the manuscript. LF, XF, TA, XZ, HuL, and XL collected the samples. PC, JH, LZ, CLi, YZ, HaL, and WY performed the experiments. HW, CLe, and XY supervised the research.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.