Background
Staphylococcus aureus, a Gram-positive bacterium, is a causative agent of a variety of infections in humans and animals [
1]. Many of the illnesses of humans, such as pneumonia and endocarditis, are related to
S. aureus [
2]. In animals,
S. aureus is associated with bovine mastitis, one of the most cost-intensive diseases in the food industry. Bovine mastitis is an infectious disease responsible for significant financial losses to dairy and food farmers worldwide [
3]. Since methicillin-resistant
S. aureus (MRSA) was first reported in the United Kingdom, it has become a particular public threat to human health, and various hospital-associated MRSA (HA-MRSA) clones have been disseminated worldwide [
4]. Since the 1990s, community-associated MRSA (CA-MRSA) has emerged as a serious health problem worldwide [
5], first in communities and later in healthcare facilities. However, livestock-associated MRSA (LA-MRSA) clones,such as LA-MRSA ST398 may be transmitted to humans and have posed public health concerns [
6]. Therefore, it is imperative to perform surveillance at the interface between human and animal hosts to explore human health risks [
7].
The emergence of multidrug resistant (MDR) strains poses several challenges to the clinical facilities [
8]. In particular, the increasing prevalence of antimicrobial-resistant
S. aureus serves as a threat to the healthcare system [
9,
10]. On the other hand, most
S. aureus strains are able to produce a large number of virulence factors, including
staphylococcal enterotoxins (SEs), exfoliative toxin (ET) and toxic shock syndrome toxin-1 (
TSST-
1) genes. Moreover, the production of SEs is particularly significant, as the ingestion of the preformed toxins is a major cause of foodborne poisoning wordwide [
11].
S. aureus has not only has been isolated from raw milk, a potential reservoir of
S. aureus, but also from the environments and workers of dairy farms [
12,
13]. In addition, studies have reported that some
S. aureus strains persist in powdered infant formula [
1]. However, reports on
S. aureus isolation from raw milk of healthy animals are relatively scanty. Very little is known about the antimicrobial susceptibility, resistance genes of enterotoxigenic
S. aureus strains, and the prevalence and molecular characterization of
S. aureus isolates.
The aim of the present study was to investigate the prevalence and perform molecular characterization of S. aureus isolates from healthy animals and patients in Henan Province, China. Furthermore, we evaluated the antimicrobial susceptibility, resistance genes, virulence genes of these isolates and characterize the molecular types by pulsed-field gel electrophoresis (PFGE), spa, and SCCmec.
Discussion
In this study, we investigated the prevalence and performed molecular characterization of
S. aureus isolated from Henan province, China, to facilitate better understanding of the epidemiology of
S. aureus. We found that the prevalence of
S. aureus strains was 22.3%, consistent with the previous reports [
14], but was significantly higher than that reported in other countries [
15,
16]. Unlike a previous study [
1] where in all
S. aureus samples were collected from animals and humans, more than 90% of the 143
S. aureus isolates in the present study were recovered from healthy animals. The percentage of animal samples positive for
S. aureus (23.7%) in our study was similar to that previously reported [
17]. However, some previous studies have detected higher percentages [
18], while others have reported lower percentages [
11,
12]. The percentage of human samples detected positive for
S. aureus (n = 92, 13 isolates, 14.1%) in our study was similar to that reports in a previous study [
19], but other studies have detected higher percentages [
20] and some studies have reported lower percentages [
21]. In addition, none of the 44 samples from the Henan Province People’s Hospital was found positive, probably owing to the difference in the nature or source of samples. Hence, further studies are warranted.
In the present study, the prevalence of MRSA was only 5.59%. MRSA is known to cause a wide variety of infections in humans and animals. Very little is known about the frequency of MRSA transmission between animals and humans,but MRSA transmission from healthy animals poses a great threat to medical science and veterinarian clinic. As observed with
S. aureus, MRSA may cause some infections in humans and animals. The percentages of MRSA detected in other studies have been variable [
20‐
22]. Studies on the prevalence of MRSA in China have detected MRSA isolates in animal and human samples [
1,
23]. MRSA prevalence in China was 27.5% in 1999 and rapidly reached 60.7% in 2009 [
24]. Other studies have reported 69.5% and 78.5% MRSA prevalence in Shanghai and Guangzhou, respectively. Inland cities such as Chongqing have been reported to exhibit a prevalence of 45.0% [
25]. In the present study,
Cap5 (56.64%) was detected as the dominant serotype. However,
Cap8 has been reported as the dominant serotype in other studies [
26].
In this study, we observed high resistance rates. The resistance rates to penicillin (96.50%) and tetracycline (68.53%) detected in this study were similar to those observed in previous studies [
15,
27]. The high resistance rates may be related to the use of antimicrobials treating mastitis in cattle farm and growth promotion or prophylaxis in swine and chicken. The prevalence of resistance to multiple antibiotics detected in our study was similar to that reported in previous studies [
28]. In addition, the antibiotic resistance patterns of
S. aureus strains recovered from raw milk and dairy products were reported [
3]. In our study, oxacillin resistance was detected in strains isolated from raw milk, chicken, and human isolates, in contradiction with the previous reports [
1,
29]. These studies reported that MRSA are only found in pig farms but not in raw milk, chicken, and human isolates. Furthermore, 8
mecA-positive isolates were resistant to oxacillin. Previously reported a significant correlation between oxacillin resistance and resistance to ciprofloxacin, clindamycin, gentamycin, and erythromycin [
30], consistent with the results of our study. In addition, some
S. aureus isolates were resistant to methicillin, but lack of
mecA gene, as reported in another study [
31]. Therefore, phenotypically resistant MRSA could be misdiagnosed using molecular methods alone, suggestive of the multiple mechanisms in MRSA. In addition, tigecycline, showed good activity against
S. aureus, thereby ensuring effective control efforts in humans. Tigecycline resistance was recently identified for different pathogens, especially in MDR strains [
32]. Hence, further studies are needed to evaluate the transmission between animals and humans.
Identification of the
mecA gene in
S. aureus is a gold standard for the detection of MRSA, which exhibits low affinity for β-lactam antimicrobials [
18]. The
blaZ gene confers penicillin resistance. We found that 96.50% of strains were resistant to penicillin, but only eight (4.90%) strains harbored
blaZ. This observation is in line with the previously reported results [
33]. In our study, both
tetM and
tetK were detected in tetracycline-resistant strains, whereas approximately 80% of tetracycline-resistant strains carried
tetM, as previously observed [
33]. In addition, the majority of the strains carried
tetM as well as
blaZ, consistent with the results of a previous study [
34]. Macrolide resistance genes
ermA,
ermB, and
ermC were present either alone or in combination with
erm(
A)+
erm(
B),
erm(B) +
erm(C), or
erm(A) +
erm(B) +
erm(C), which was in accordance with a previous study [
35]. In this study, 22 (15.38%) isolates carried florfenicol resistance gene
fexA, and the number was significantly higher than that reported in a previous study [
36]. Gentamicin resistance was associated with the aminoglycoside resistance genes
acc(6′)-
aph(2″),
ant(4′)-
Ia, or
aphA, and these three genes co-existed in the majority of isolates, as observed in a previous study [
35]. The location of
vga(A) or
vga(C) on a plasmid may play an important role in its persistence and dissemination [
22,
37]. Bacitracin, a polypeptide antibiotic, is used as an animal growth promoter for prophylaxis and therapy of consumable animals in China. In the present study, the prevalence of the
bcrB gene in swine strains (59.1%) was higher than that in chicken strains (46.7%); this observation may be largely related to the wider use of bacitracin as a feed additive in swine than in chicken. To the best of our knowledge, this is the first report to demonstrate
bcrB gene in
S. aureus isolates from animals in Henan province, China. Some recent reports have shown the widespread of the high-level bacitracin resistance (MIC ≥ 256 μg/mL) in enterococci [
38,
39]. In the present study, the prevalence of linezolid resistance gene
optrA in chicken strains (26.7%) was higher than that in swine strains (13.6%). This is the first report to detect
optrA gene in
S. aureus isolates from animals in Henan province, China. The gene
optrA was recently detected in
S. sciuri from swine [
40]. Although linezolid is not approved for use in animals, selective pressure from other antibiotics such as florfenicol, tiamulin, and lincomycin that are widely used in animals may promote the spread of
optrA. Thus, more attention needs to be paid to the possibility that
optrA may find its way through the food chain or pathogenic bacteria of humans. In addition, the emergence and dissemination of these MDR isolates in animals pose a threat to public health, given that
optrA-mediated linezolid resistance may rapidly spread among different bacterial species. Therefore, the surveillance of
optrA gene in China is very important to limit its dissemination to prevent the potential threat to animal and human health.
The existence of SEs in
S. aureus isolated from animals and humans vary from our reported. The results of the present study on the existence of
sea,
seb, and
sec in all
S. aureus isolates are contradictory to those previously reported, wherein
sea was observed in 45.2% of isolates and
seb was detected in 18.5% of isolates [
41]. In another study,
sea was reported in 26.2% of isolates and
seb in 39.3% of isolates [
23]. However, the detection rate for
sec gene was low in 6% of the isolated
S. aureus strains [
42]. The high occurrence of β-hemolysin genes
hla (48.95%) and
hlb (41.26%) among
S. aureus isolates is in line with the results of other reports [
43]. In our study, the
TSST virulence gene associated with
TSST-
1 was detected in only 4 (2.80%) isolates, consistent with the results of another study [
41]. Moreover, more than half of the strains carried
lukED gene (57.34%), as previously reported [
19]. In addition, a small number of isolates carried
pvl gene (8, 5.59%), consistent with a previous report [
23], and this number was lower than the occurrences of
pvl-positive
S. aureus in a previous study [
44]. In the present study, four of
pvl-positive isolates were MRSA, while the remaining four isolates showed different molecular types.
The results of PFGE analysis showed that a part of the isolates were identical and showed the same PFGE patterns (P1). These were derived from swine, chicken, and raw milks of different animals. This observation is in line with that reported in a previous study, wherein same PFGE patterns were observed for strains from goat milk powders at different processing stages [
3]. Thus, cross contamination of
S. aureus may occur in different animals. A previous study showed that each region had its own predominant PFGE pattern [
22]. Identical the same PFGE patterns were observed for strains from swine, chicken, and raw milks of different animals, supporting our hypothesis that the infection may be probably acquired through the spread of
S. aureus. Strains isolated from swine and chicken or raw milks were present in these clusters, suggestive of the possible transmission during the slaughtering procedure as previously reported [
15]. As observed in the
spa typing results, t15075 and t189 were the most common
spa types. However,
t899 was the most prevalent
spa types in
S. aureus [
22,
45]. Although PFGE patterns of the isolates showed more variations than those observed by
spa typing, new technologies such as next generation sequencing may provide better understanding of the origin, transmission, and evolution of MRSA. These advanced technologies would be included in our further studies on the origin and spread of MRSA in China [
22]. In this study, PFGE and
spa typing were used in combination with
SCCmec typing. The majority of the strains were assigned to ten major PFGE types, and P1 and P2 were the two most common clusters containing 17 and 15 isolates, respectively. Moreover, PFGE type P1 among three
spa types (t15075, t189, and t2646),
spa types t189 among main three clusters (P1, P2, and P3). In this study, MRSA-SCC
mec Iva-t437 was observed in human isolates, as previously reported [
23,
46].