Molecular characteristics of Staphylococcus aureus strains isolated from subclinical mastitis of water buffaloes in Guangdong Province, China

Intramammary infections (IMI) in animals reared for milk production can result in large economic losses and distress to the animals. Staphylococcus aureus is an important causative agent of IMI in dairy cows, but its prevalence in water buffaloes has not been determined. Therefore, the current study was conducted to investigate the prevalence of subclinical mastitis in water buffaloes and the antimicrobial susceptibility, virulence genes and biofilm formation abilities of Staphylococcus aureus isolates recovered from water buffaloes in Guangdong, China. Staphylococcus aureus strains were isolated from milk samples of water buffaloes with subclinical mastitis, and twofold microdilution, PCR and crystal violet staining methods were used to determine antimicrobial susceptibility, distributions of virulence and antimicrobial resistance genes and biofilm formation ability, respectively. Our results indicated that 29.44% of water buffaloes were diagnosed with subclinical mastitis, and the most prevalent pathogens were Escherichia coli (96.17%), coagulase-negative staphylococci (CoNS) (67.60%) and S. aureus (28.57%). Most S. aureus isolates showed resistance to bacitracin, doxycycline, penicillin, florfenicol, and tetracycline but were susceptible to ciprofloxacin, ceftizoxime, cefoquinoxime, and ofloxacin. Moreover, 63.72% of S. aureus isolates were positive for tetM, and the prevalence of msrB, blaZ, mecA, fexA, and tetK ranged from 21.24 to 6.19%. All S. aureus isolates harbored clfB and icaA genes, and the virulence genes hla (93.8%), hld (91.15%), clfA (90.27%), fnbA (86.73%), and hlb (83.19%), and tsst, icaD, sec, see, fnbB, and sea showed a varied prevalence ranging from 3.5 to 65.49%. All S. aureus isolates possessed the ability to form biofilms, and 30.09% of isolates showed strong biofilm formation abilities, while 19.47% of isolates were weak biofilm producers. Our results indicated that subclinical mastitis is prevalent in water buffaloes in Guangdong, China, and S. aureus is prevalent in samples from water buffaloes with subclinical mastitis. Most S. aureus isolates were susceptible to cephalosporins and fluoroquinolones; thus, ceftizoxime and cefoquinoxime can be used to treat subclinical mastitis in water buffaloes.


Introduction
Water buffalo (Bubalus bubalis) is of importance in the milk industry and contributes to approximately 15% of milk production (1).Many parts of the world have traditionally produced buffalo milk, including Asia, Egypt, and Europe.China produces approximately 5% of global buffalo milk, and Guangdong, Guangxi and Hunan are its primary producers (2).Italy is the main producer of buffalo milk in Europe because of the popularity of buffalo mozzarella cheese (3), which retails for twice the price of bovine milk cheese (4).
Mastitis is one of the most prevalent diseases among dairy animals, causing economic losses to milk producers due to a decrease in milk quality and production, an increase in veterinary and labor costs and an increased rate of culling (5).Bacteria are the primary causative agents of mastitis, although physical trauma and mechanical injury also contribute (1).The primary bacterial causes of mastitis in both dairy cows and water buffaloes are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli.Staphylococcus aureus is one of the primary pathogens causing mastitis in dairy cows at a level of approximately 40% in China (6).
Among antimicrobial agents, antibiotics are normally used to treat infections caused by bacteria, including intramammary infections (IMI) in domestic animals.In dairy animals, mastitis and reproductive diseases often require prolonged use of antimicrobial agents (7).Unfortunately, the widespread use of antimicrobial agents has resulted in high levels of antimicrobial resistance, leading to clinical treatment failures.Therefore, monitoring systems for antimicrobial resistance of bacterial pathogens can provide essential information for the rational use of antimicrobial agents when treating infections (8).Knowledge regarding the prevalence of mastitis and the knowledge of pathogens causing mastitis is critical in preventing the occurrence of mastitis and can provide effective measures for control and appropriate therapeutic protocols (9).
There are two groups of virulence genes in S. aureus, including surface-localized structural components serving as virulence factors and secreted virulence factors involved in evading host defenses and colonizing mammary glands (10).Surface-localized structural components include membrane-bound factors (fibrinogen binding protein, collagen binding protein and elastin binding protein), cell wall-bound factors (lipoteichoic acid, peptidoglycan, protein A and protease) and cell surface-associated factors (capsule).Secretory factors include toxins (staphylococcal enterotoxins, leucocidin, toxic shock syndrome toxin, and hemolysins) and enzymes (staphylokinase, coagulase, lipase, DNase, and hyaluronidase).Moreover, biofilm formation also contributes to adhesion and invasion into mammary epithelial cells and thus provides an escape from the host immune system.Enterotoxins often lead to food poisoning and include staphylococcal enterotoxins A to F and G to Q (11).Enterotoxins G to Q are more prevalent in isolates from dairy cows with mastitis than in isolates from cows without mastitis; this has implicated these virulence factors in the occurrence of mastitis (12).However, the clear mechanisms of virulence in mastitis of dairy cows need further study.
In China, the number of water buffalo farms is increasing, and thus, mastitis is occurring more frequently in these animals.Mastitis can be divided into two forms: clinical and subclinical.In clinical mastitis, clots and flakes can be observed in milk, and the quarters become swollen with severe conditions leading to the formation of lacerations, necrosis and cord formation of the teat.While no clinical signs or symptoms can be seen in subclinical mastitis, there is a reduction in milk production and deterioration of milk quality (13).Subclinical infection is 15-40 times more prevalent than clinical infection and can rapidly spread on a farm (14).
To reduce the prevalence of subclinical mastitis in water buffaloes, it is essential to have knowledge of prevalence data and understand the antimicrobial susceptibility profiles of subclinical mastitis caused by S. aureus.Therefore, this study was conducted to investigate the prevalence of subclinical mastitis in water buffaloes in Guangdong, China, and to determine the antimicrobial susceptibility of S. aureus isolates and their antimicrobial resistance and virulence determinants.

Sample size
Ausvet epidemiological calculators 1 were used to calculate the sample sizes according to the method from Charan and Biswas at a 95% confidence level (15) as follows: where z 1 2 D / equals 2.58 with a 1% type error (p < 0.01).The expected proportion of water buffaloes in Guangdong Province is 0.05, and the expected precision (d) is 0.01.

Sample collection
The milking of animals on all farms was performed twice a day, and the sample collection procedure consisted of fore-stripping (3-5 squirts of milk) followed by teat disinfection with 0.25% iodine solution and drying with a clean towel.The milking clusters were then attached and removed automatically when finished, followed by postmilking teat disinfection with 0.5% iodine.Duplicate milk samples for each quarter were aseptically collected according to standard protocols of the National Mastitis Council (16).Briefly, milk samples (3 mL) were collected from all quarters of each animal after the first 3 streams of milk were discarded and placed in an ice box and transferred to the laboratory within 6 h.Presumptive evidence of subclinical mastitis (17) was determined using a commercial California Mastitis Test kit (CMT) (ImmuCell, Portland, ME, United States) following the recommendations of the manufacturer.Briefly, 2 mL of milk sample was mixed with an equal amount of CMT solution in the paddle and stirred for 30 s. Thickening indicated elevated somatic cell counts (SCC), and these samples were then used for bacterial isolation and identification.

Bacterial isolation
Identification of S. aureus from milk samples was carried out as previously described (18).Briefly, a 0.1 mL milk sample was inoculated into 3 mL tryptic soy broth (TSB; AoBox, Beijing, China) containing 10% NaCl and incubated at 37°C for 24 h.A loopful of enrichment broth culture was streaked onto a Baird-Parker agar plate (AoBox) and incubated at 37°C for 24 h.The suspected S. aureus colonies had a black and shiny appearance with a thin white border surrounded by a light area.The suspected colonies were streaked onto chromogenic S. aureus agar plates (CHROMagar, Paris, France).At least 3 positive colonies per sample were then confirmed as coagulase-positive S. aureus using commercial API STAPH test strips (bioMérieux, Marcy-l'Ѐtoile, France).Staphylococcus aureus isolates were stored at −80°C in cryogenic vials (Biologix, Shandong, China) containing 1 mL TSB and 30% glycerin.Coagulase-negative staphylococci were isolated as previously described (19).In brief, milk samples were cultured on blood agar plates (AoBox) at 37°C for 48 h.Typical colonies were selected and identified using classical biochemical methods, including Gram staining and oxidase, catalase and DNase tests, and the ability to coagulate rabbit plasma using commercial kits (Sigma Chemical, Shanghai, China).If the suspected strains failed biochemical identification, molecular identification using PCR amplification and sequencing of the sodA gene was performed as previously described (19).
Escherichia coli isolation utilized a 0.1 mL milk sample inoculated in 3 mL Mueller-Hinton broth (MHB; Aobox), which was incubated at 37°C for 24 h.Samples were then streaked onto MacConkey Agar plates, and plates were kept at 37°C for 24 h.Presumptive identification of E. coli were pink colonies that were then subjected to matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a Microflex LT instrument (Bruker Daltonics, Bremen, Germany).
Streptococcus agalactiae and S. dysgalactiae were identified as previously described (20) using EN medium, and positive isolates were then transferred to Columbia Blood Agar Base Medium containing 5% sheep blood (Hope Bio-Technology, Qingdao, China) and incubated at 37°C for 24 h.Colonies with typical Streptococcus morphologies were then subjected to catalase and 6.5% NaCl tests.Isolates were further identified as S. agalactiae and S. dysgalactiae according to reactions using the sodium hippurate test, esculin hydrate, and CAMP tests.

Antimicrobial susceptibility testing
Antimicrobial resistance phenotypes of S. aureus isolates were determined using the microdilution method in MH broth according to the Clinical Laboratory and Standards Institute guidelines (CLSI) (21).A loopful of each S. aureus isolate preserved in glycerinated TSB was streaked on a Baird-Parker agar plate and incubated at 37°C for 24 h.The colonies were inoculated in MH broth, and the cultures were diluted in sterile normal saline and adjusted to a turbidity of 0.5 McFarland standard (10 5 -10 6 colonyforming units (CFU)/mL).The suspension was then swabbed on Muller-Hinton agar plates and incubated at 37°C for 24 h as previously described.Staphylococcus aureus ATCC 25923 was used as the reference strain.Each experiment was repeated at least three times.Breakpoints for different antimicrobial agents were based on CLSI guidelines (21).

Antimicrobial resistance and virulence gene detection
Strains used for testing were taken from frozen stocks, cultures were streaked onto TSA plates containing 5% sterile defibrinated sheep blood, and the plates were incubated at 37°C for 24 h.Single colonies were inoculated into 3 mL TSB and cultured with shaking at 37°C for 24 h.The cultures were centrifuged at 3000 × g, and cell pellets were suspended in phosphate buffered saline (PBS, pH 7.4; Solarbio, Beijing, China) containing 20 mg/mL lysostaphin (Meilunbio, Dalian, China).The mixture was kept at 37°C for 30 min, genomic DNA was extracted using a TIANamp bacterial DNA extraction kit (TianGen, Beijing, China), and DNA quality was evaluated by UV spectroscopy with a NanoDrop-2000 instrument (Thermo Fisher, Shanghai, China).The extracted DNA was diluted to 50 mg/L in sterile deionized water for PCR assays (see below).
Virulence genes (hla, hlb, hld, sea, seb, sec, sed, see, tst, and lukF), biofilm-related genes (bap, icaA, and icaD) and adhesionrelated genes (fnbA, fnbB, clfA, and clfB) were detected using PCR as previously described (23).The primers were provided by Sangon Biotech (Shanghai, China), and water rather than DNA template was added as a contamination control.DNA from isolates that harbored virulence genes or ARGs was used as a positive control.These were included in all PCRs.Gene amplifications were performed using a commercial PCR instrument (Bio-Rad, Hercules, CA, United States) as previously described (24).Briefly, the PCR mixture contained DNA (1 μL), 0.2 μL of each primer, Prime STAR Max DNA polymerase (12.5 μL), and ddH 2 O (11.1 μL).The PCR cycling conditions consisted of an initial denaturation at 95°C for 10 min, followed by 30 cycles of 95°C for 30 s, annealing at appropriate temperatures for 30 s (Supplementary Table S1) and extension at 72°C for 1 min.

Biofilm formation
Biofilm formation was determined in 96-well microtiter plate assays using minimal medium M9 (Sigma Chemical) as previously described (25).Briefly, the overnight cultures in TSB were diluted

Statistical analysis
Pearson correlation analysis was applied to differences in antimicrobial resistance rates in correlation to antimicrobial resistance genes harbored by S. aureus isolates.T tests were used to analyze the significance of biofilm formation between S. aureus isolates.All analyses were carried out using Prism 8 (GraphPad, Boston, MA, United States).

Discussion
Mastitis is a disease that is globally prevalent in dairy animals (1), and water buffaloes are generally less susceptible to this infection in comparison with dairy cows because of strong muscles at the opening of the teat canal (27).In the current study, 29.44% of water buffaloes were diagnosed with subclinical mastitis, which was consistent with a previous report for these animals (28).Previous studies have indicated that the prevalence of subclinical mastitis in water buffaloes ranges from 6.0 to 87% (27).It seems that factors such as animal age, stage of lactation, management style and farm environment may have contributed to these variations (13).To our knowledge, few studies have been carried out to investigate the bacteriology of subclinical mastitis in water buffaloes.In this study, we found that CoNS, E. coli and S. aureus were the dominant bacterial pathogens and that S. agalactiae and S. dysgalactiae were present to a lesser degree.Escherichia coli infections often lead to severe systemic clinical symptoms, and this was the most prevalent pathogen in our study.Similarly, E. coli was the most prevalent pathogen in subclinical mastitis infections in a Nepal water buffalo study (28), while Streptococcus (39.2%) was the most prevalent pathogen in mastitis dairy cows, and only 8.4% were present as E. coli (29).Therefore, it seems that other factors (see above) may influence the occurrence of mastitis.Moreover, S. aureus (61%) was the dominant pathogen in cattle from Jammu and Kashmir Distribution of antimicrobial resistance genes among S. aureus isolates.

FIGURE 2
The detection rate of virulence genes among S. aureus isolates.with mastitis, and E. coli (13%), CoNS (13.04%),Streptococcus uberis (4.35%) and S. dysgalactiae (8.69%) were also isolated from samples (30).Similar results were reported by other researchers (27); the reasons for this might be topographical and management conditions and the difference between dairy cows and water buffaloes.
Antimicrobial susceptibility can provide important information in choosing antimicrobial agents when treating infections.In this study, S. aureus isolates showed high resistance to penicillin, florfenicol, erythromycin, tetracycline, doxycycline and bacitracin.Similar results were observed in S. aureus isolates from dairy cows with mastitis in northern China (31).However, other researchers reported lower rates of antimicrobial resistance in S. aureus isolates in Pakistan (13).Cephalosporins are important antimicrobial agents, and S. aureus isolates resistant to ceftiofur have been reported (23).Similarly, antimicrobial resistance to cephalosporins, including cefoquinoxime, ceftizoxime, and ceftiofur, was detected in our study.β-lactams, fluoroquinolones, and aminoglycosides are commonly used to treat dairy mastitis (31), and this most likely contributed to the high levels of resistance we found to these agents.Saini and his colleagues also found that the herd level of antimicrobial agents used when treating mastitis in bovines was positively correlated with antimicrobial resistance among isolates from mastitic animals (32).Unfortunately, the use of antimicrobial agents in these farms was not documented in our study, so we cannot directly correlate the use of antimicrobial agents and antimicrobial resistance.Moreover, we detected antimicrobial resistance genes among isolates, although we found no significant correlation between phenotype and genotype.For example, only 22.92% (22/96) of isolates resistant to penicillin carried the blaZ gene, similar to previous findings (33,34).These inconsistencies indicated that the presence of a particular ARG was not an indicator of phenotypic resistance, and this can be influenced by numerous genetic and environmental conditions (35).
MRSA is a global health concern since it is not only resistant to β-lactams but also nonsusceptible to other commonly used antimicrobial agents (36).In the Philippines and Pakistan, the MRSA prevalence was 25.81 and 19.6%, respectively, in water buffaloes with mastitis (13,37), while a much lower rate (2.2%) of MRSA was detected in water buffaloes with mastitis in Iran (38).Several factors, such as age, feeding status, body conditions, and hand or machine hygiene on the farm, may contribute to this phenomenon.Several technologies, such as nanoparticles and antibiotics combined with plant extracts or microparticles, are widely used in food, veterinary and animal science.For example, a report indicated that antibiotics coupled with zinc oxide nanoparticles can significantly increase the zone of inhibition; similarly, amoxicillin showed the highest increase in inhibitory effects against MRSA when combined with Calotropis procera extract (39).These technologies are believed to be promising methods for treating infections caused by MRSA.
Biofilms can increase the resistance of S. aureus to antimicrobial agents and are responsible for persistent infections (40).Biofilms are composed of multiple layers of bacteria, which prevents the permeability of antimicrobial agents and thus increases tolerance.In our study, we investigated the biofilm formation ability of S. aureus isolates grouped by area.Interestingly, S. aureus isolates from Guangzhou showed significantly lower levels of biofilm formation in comparison with isolates from other areas (p < 0.01).However, the antimicrobial resistance of S. aureus isolates did not differ by region (data not shown).Similarly, a previous study indicated that the biofilm formation ability of ST7 and ST188 strains was much higher than that of other lineages even though their phenotypic antimicrobial resistance was comparable with that of other lineages (41).These data indicated that gene mutations, horizontal gene transfer and modifications of antibiotic molecules are the primary modes of antimicrobial resistance in S. aureus isolates from water buffaloes and that biofilm formation plays only a secondary role (42).
Virulence genes contribute to the pathogenesis of S. aureus infections.Adhesion is the first step for S. aureus to invade host cells and immune responses (43) and involves clfA, clfB, fnbA, and fnbB.In our study, all isolates carried clfB, and most isolates carried clfA, fnbA, and fnbB.These results were similar to previous reports where the clfB gene was detected in all isolates from bovine mastitis samples, and fnbA and clfB were comparable with the levels we found (44,45).In contrast, much lower detection levels were reported for fnbB in S. aureus isolates from Algeria and Australia (43,46).
Hemolysins are also involved in invasion and the host immune response (44,46).In our study, over 80% of our total isolates carried hla, hlb, and hld, consistent with previous reports (41,44).Toxic shock syndrome toxin, a superantigen encoded by the tsst gene, can lead to toxic shock syndrome in humans (47).and the tsst prevalence in S. aureus isolates ranged from 2.1 to 40.0% (10,44) and was 25% in our study.It is therefore important to monitor the epidemiology of such super antigenic toxin genes to protect public health from this threat.

Conclusion
In conclusion, subclinical mastitis was prevalent among water buffaloes in Guangdong, China, and S. aureus was identified as a significant pathogen associated with subclinical mastitis of water buffaloes.The majority of S. aureus isolates exhibited resistance against bacitracin, doxycycline, penicillin, florfenicol, and tetracycline while maintaining susceptibility to other antimicrobial agents, including ciprofloxacin, ceftizoxime, cefoquinoxime, and ofloxacin.Furthermore, the S. aureus isolates harbored various virulence genes, such as hla, hld, clfA, fnbA, and hlb.Notably, all S. aureus isolates showed the ability to form biofilms, with nearly one-third of the isolates possessing strong biofilm formation abilities.Given these findings, antibiotics should be cautiously used when treating subclinical mastitis in water buffaloes within this region.Additionally, the impact of biofilm formation on the transmission of antibiotic resistance must be investigated in further studies.

FIGURE 3
FIGURE 3Biofilm formation of S. aureus isolates in different areas of Guangdong.Note: There were no significant difference between S. aureus isolates from Qingyuan, Jiangmen, and Zhaoqing.But S. aureus isolates from Guangzhou were significantly lower (p < 0.01) in comparison with isolates from Qingyuan, Jiangmen, and Zhaoqing.** means p < 0.01.

TABLE 1
Antimicrobial susceptibility of S. aureus isolates from subclinical mastitis of buffaloes.
1:100, and 200 μL was transferred into each well of the microtiter plate that was incubated at 37°C for 72 h.Each well was washed with 200 μL PBS after the supernatant was discarded and fixed with 200 μL methanol for 20 min and washed again with PBS 3 × and then

TABLE 2
Biofilm formation ability of S. aureus isolates from water buffaloes.