Prevalence, Antimicrobial Resistance Profiles, Virulence and Enterotoxins-Determinant Genes of MRSA Isolated from Subclinical Bovine Mastitis in Egypt

Subclinical mastitis caused by Staphylococcus aureus has worldwide public health significance. Here, we aimed to determine the prevalence of S. aureus, antimicrobial resistance profiles, and the virulence and enterotoxins determinant genes of MRSA strains that caused subclinical bovine mastitis. Milk samples were collected from 120 lactating animals (50 buffaloes and 70 dairy cattle) from different farms located in Ismailia Province (Egypt). The collected samples were investigated for subclinical mastitis using a California mastitis test. The total prevalence of S. aureus was 35.9% (84/234) with 36.3% (53/146) in cattle and 31% (31/88) in buffaloes. Antimicrobial susceptibility testing showed that 35.7% (30/84) of the isolated strains were resistant to cefoxitin, defined as methicillin-resistant S. aureus (MRSA), with 37.7% (20/53) in cattle and 32.2% (10/31) in buffaloes. Using PCR, 100% of the tested strains harbored coa and mecA genes, while 86.6% were positive for spa gene, with remarkable gene size polymorphism. Additionally, 10% of the tested strains contained the pvl gene. Further, using multiplex PCR, 26.6% of the tested samples had sea gene, two strains had sec gene and only one strain had sea and sec genes. The seb and sed genes were absent in the tested strains. In conclusion, mecA, coa and spa virulence genes were widely distributed in MRSA strains isolated from bovine milk, whereas the sea gene was the most predominant enterotoxin gene. Notably, this is the first report that emphasizes the prevalence of pvl gene of MRSA isolated from bovine milk in Egypt.


Introduction
Staphylococcus aureus is a significant public health bacterial pathogen, causing mastitis in dairy animals including cattle, buffalo, sheep and goats [1]. S. aureus mastitis and its produced toxins lead to great economic losses in dairy farms due to: (1) reduction in the milk production, (2) alteration in the composition and quality of the produced milk, (3) the need to discard the produced milk, (4) early from each quarter into sterile screw-capped McCartney bottles (Thermo Fisher Scientific, Waltham, MA, USA). Milk samples were immediately transported to the laboratory in an ice container [16].

California Mastitis Test (CMT)
In order to determine the milk samples infected with subclinical mastitis, CMT (screening test) was used. The test procedures were conducted as previously mentioned [17,18]. The test depends on the interaction of the reagent with DNA of somatic cells present in milk. Briefly, in a cup of the white plastic paddle, 2 mL of scam reagent was added to an equal volume of milk sample from each quarter of the udder and mixed by a gentle circular motion. The test results were evaluated visually within 20 s and interpreted as; − (0), ± (T), + (1), + + (2), and + + + (3) based on the amount of gel formation. Milk samples from individual quarters with positive CMT scores were subjected to bacteriological examination.

Isolation and Identification of S. aureus
The screened positive milk samples were incubated at 37 • C for 24 h, and then centrifuged at 3000 rpm for 5 min. The cream layer was discarded and sediments were streaked onto blood agar, nutrient agar, and mannitol salt agar plates (Oxoid, Hampshire, UK). The streaked plates were then incubated at 37 • C for 24-48 h. The suspected grown colonies were identified morphologically and biochemically as previously mentioned [17]. S. aureus circular convex golden-yellow colonies were collected and preserved at −80 • C in media containing 10% glycerol (v/v) for further analysis. The confirmation of the retrieved colonies was performed using PCR for 16Sr RNA gene identification as previously described [19].
The selected antimicrobials are representative of the drugs used for humans and in the animal industry and were chosen according to the National Antimicrobial Resistance Monitoring System (NARMS) records. The reference strain (S. aureus ATCC 25923) was used as a control for the disc diffusion technique. The test was conducted on Muller Hinton agar plates (MH, Oxoid) and the plates were incubated at 37 • C for 24 h. The test was performed in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI) criteria using the available CLSI interpretive criteria (Table 1). DNA of MRSA strains was extracted using the boiling method. Briefly, a half loopfull from S. aureus plate cultures were suspended in 100 µL of DNase-free water, heated at 95 • C for 10 min, cooled, and then centrifuged at 5000× g for 10 min. The supernatant containing the genomic DNA was collected in a new tube and stored at −20 • C for further use. DNA was quantified using a Nanodrop 1000 instrument (Thermo Scientific, Loughborough, UK).

Polymerase Chain Reaction (PCR)
The extracted DNA from MRSA strains were screened for virulence genes (coa, spa, pvl and mecA) and enterotoxins genes (sea, seb, sec and sed) detection. Amplification was performed in PCR tubes, in a 25 µL reaction volume, containing 200 µM of dNTPS buffer (dATP, dGTP, dCTP and dTTP), 50 picomol of each forward and reverse primers, and 0.5 units of taq DNA polymerase (NZYtech, Lisbon, Portugal). The PCR reaction mixtures were amplified in the MJ MiniTM Gradient Thermocycler apparatus (Biometra, Göttingen, Germany). Primers sequence, expected amplicon size, and annealing temperature are described in Table 2. Nuclease-free water was used as a negative control. Positive controls DNA were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Suez Canal University, Egypt. PCR products were visualized on a 1.5% agarose gel containing ethidium bromide under UV light and 100 bp ladder (Fermentas, Thermo Scientific, Darmstadt, Germany) was used.

Statistical Analysis
The Chi-square test was used for the analysis of the recovered frequencies using SAS ® software (version 9.4, SAS Institute, Cary, NC, USA) to test the null hypothesis of various treatments. A p-value of <0.05 was considered statistically significant.

Prevalence of S. aureus Subclinical Bovine Mastitis
The prevalence of subclinical mastitis in the collected milk samples from individual quarters using CMT was 44% (88/200) and 52.1% (146/280) in buffaloes and cattle, respectively (Table 3). There was no significant differences between the prevalence of subclinical mastitis between buffaloes and cattle (p > 0.05). Additionally, the total prevalence of S. aureus in the collected milk samples from individual quarters using CMT was 35.9% (84/234) with 36.3%% (53/146) in cattle and 31% (31/88) in buffaloes and no significant difference between cattle and buffaloes (p = 0.8654; X 2 = 0.029). Out of them, the prevalence of MRSA was 35.7% (30/84) with 37.7% (20/53) in cattle and 32.2% (10/31) in buffaloes and no significant difference between cattle and buffaloes (p = 0.6203; X 2 = 0.245). Detailed results of CMT screening in the collected milk samples are shown in Table 4.  Percentages were calculated in comparison to the total number of examined samples of each grade.

Antimicrobial Susceptibility Phenotypic Profiles of the S. aureus Isolates
The antimicrobials susceptibility test for S. aureus strains showed that 64.3% (54/84) of the samples were resistant to penicillin, 59.5% (50/84) were resistant to tetracycline and 35.7% (30/84) of the strains were resistant to cefoxitin (defined as MRSA). In addition, 58.3% (49/84) of the tested strains exhibited intermediate sensitivity to cefotaxime, whereas 64.3% (54/84) of the tested strains were sensitive to cefoxitin. Our results also showed that 78.6% (66/84) of the strains were sensitive to amoxicillin-clavulanic acid, 72.6% (61/84) to ampicillin-sulbactam, and 63.1% (53/84) were sensitive to erythromycin. Details about the antimicrobial susceptibility phenotypic profiles of the S. aureus strains are shown in Table 5.

Virulence Determinant Genes of MRSA Strains
Thirty isolates of MRSA strains were subjected to PCR for the detection of coa, spa, pvl and mecA genes. All the 30 tested strains (100%) were positive for coa gene that showed no gene polymorphism, while 26 out of 30 strains (86.6%) were positive for spa gene and showed a remarkable gene polymorphism with different amplicons size (140 bp, 270 bp and 290 bp). Only 3 out of 30 strains (10%) were positive for pvl gene. Furthermore, all the examined strains (100%) were positive for mecA gene. There is a significant difference in the prevalence of virulence determinant genes (p < 0.0001) among the examined MRSA strains (Table 6). No of the examined MRSA strains= 30. There were significant differences between spa and pvl at X 2 = 34.659, between coa and pvl at X 2 = 48.273; and between mecA and pvl virulence genes at X 2 = 48.273 (p < 0.0001). There were significant differences between sea and seb/sed enterotoxins genes at X 2 = 9.051 and p = 0.0026. * Significant difference in the prevalence between different virulence genes and between different enterotoxins genes.

Prevalence of Enterotoxins Genes among MRSA Strains
The prevalence of enterotoxins genes (sea, seb, sec and sed) among MRSA strains were detected using multiplex PCR. Our results showed that eight out of 30 strains (26.6%) were positive for sea gene, two out of 30 strains were positive for sec gene, one out of 30 strains (3.3%) was positive for both sea and sec genes, and none of the tested strains harbored seb and sed genes. Hence, 11 out of 30 strains (36.6%) were enterotoxignic ( Table 6). The prevalence of the enterotoxins genes showed a significant difference (p < 0.0001) among the examined MRSA strains.

Discussion
Subclinical mastitis is a significantly important disease, causing economic losses in the livestock industry, not only in Egypt, but also worldwide. Antibiotic resistance has increased among various bacterial pathogens, which is considered an emerging problem with a major public health concern due to the risk of resistance transmission to human as well as its influence on the effectiveness of the current antibiotic therapy [26][27][28][29]. Further, MRSA strains can cause nosocomial infections and high mortality in humans [3]. In this study, the detected prevalence of subclinical mastitis was 44% and 52.1% in buffaloes and cattle, respectively, with a total prevalence of 48.6% and no significant difference in the prevalence between both animal species (Table 3). This high prevalence of bovine subclinical mastitis was similar to the results obtained in other previous studies [16,30]. The increased incidence of subclinical mastitis in dairy livestock is attributed to multiple predisposing factors including: (1) contaminated milking machines, (2) improper housing, (3) bad sanitation, and (4) bad handling of animals. Furthermore, the failure in the treatment always occurred due to: (1) chronic infection accompanied with fibrosis, (2) inadequate dose of antibiotics, and (3) emergence of multidrug-resistant bacterial pathogens [31].
The prevalence of S. aureus recovered from subclinical mastitis infected animals was 36% (p < 0.0001). This result was similar to previous studies [16,32] and lower than the prevalence (6.5%) reported by Haltia, et al. [33]. S. aureus transmission between animals is due to using of contaminated milk utensils and is also due to contaminated milker's hands [34]. Additionally, cefoxitin resistance (MRSA) was used determine the methicillin-resistant S. aureus isolates, and antimicrobial susceptibility testing showed that 35.7% of the recovered S. aureus strains were resistant to cefoxitin (Table 5). Further, the identified MRSA strains were confirmed using PCR for detection of mecA gene, where all the tested strains (100%) harbored this gene (Table 6) conferred marked resistance to various antimicrobial agents such as cefoxitin, penicillins, cephalosporins, macrolides, aminoglycosides and tetracyclines. Since the 1990s, most MRSA isolates possessed a multidrug-resistant phenotype and carried many resistant determinants in chromosome and plasmids. Resistance to methicillin is attributed to the existence of mecA gene on the S. aureus chromosome, which is encoded for the synthesis of PBP 2a . Methicillin is stable in the presence of β-lactamase enzymes and is effective in the treatment of S. aureus infection, but not against MRSA that resist methicillin [35][36][37].
In this study, 64.3% S. aureus strains were resistant to penicillin which was in agreement with other studies (more than 50%) [38] and lower than resistance rate (7.1%) determined by Bengtsson, et al. [39]. The failure of treatment of subclinical mastitis with penicillin was mainly due to the release of β-lactamase by S. aureus, which causes hydrolysis of β-lactam rings. The majority of S. aureus strains release penicillinases which consequently make S. aureus resistant to the β-lactam group of antibiotics [40]. Furthermore, 78.57% S. aureus strains were sensitive to amoxicillin-clavulanic acid, while 72.62% was sensitive to ampicillin-sulbactam. The isolation of a naturally occurring β-lactamase inhibitor (clavulanic acid) from Streptomyces clavuligerus was a major step in the formulation of new antibiotic combinations. Sulbactam and clavulanic acid (β-lactam antibiotics) have very poor bactericidal ability, however, they are a very strong β -lactamase inhibitors [41].
We observed that 59.5% of the strains were resistant to tetracyclin, a broad-spectrum antibiotic groups that, due to its extensive use, resulted in the development of resistant strains. Resistance to tetracyclines resulted from the production of ribosomal protection protein by S. aureus that makes a competitive binding to tetracycline. The tet gene that is carried on conjugative plasmids of S. aureus is responsible for their production [42].
In addition, 58.3% of the S. aureus strains were moderately sensitive to cefotaxime, while 63.1% were sensitive to erythromycin. In general, third-generation cephalosporins have strong activity against Gram-negative bacteria and moderate activity against Gram-positives, such as S. aureus and Streptococci. Unlike penicillin, third-generation cephalosporins have stable activity in presence of β-lactamase enzyme. Erythromycin belongs to the macrolide class and showed a potent antibacterial activity against both Gram-positive and Gram-negative bacteria, including Staphylococci, Streptococci and E. coli [41,43].
The coa gene was 100% prevalent in the tested MRSA strains. These results are similar to those reported by Akineden, Annemüller, Hassan, Lämmler, Wolter and Zschöck [24]. This gene exhibited no size polymorphisms [44]. However, in other studies, coagulase gene amplification resulted in different amplicons, indicating coagulase gene size polymorphism [34]. The spa gene displayed remarkable gene polymorphisms where different sized amplicons were found (140, 270, and 290 bp). The X region of spa gene usually undergoes variable repetitions (up to 24 repeats) which might be different in different strains [10]. Number of repeats is associated with the dissemination potential of S. aureus, where strains that have more than seven repeats in the X region were considered as epidemic, whereas the presence of seven or less repeats were considered as non-epidemic MRSA [14]. Further, the pvl gene was detected in 10% of the tested strains. The presence of pvl gene in S. aureus was similar to that obtained previously [45] and disagrees with the results obtained by Ikawaty, et al. [46]. The pvl gene is considered as the most powerful staphylococcal leukotoxin that could resist bovine neutrophils [47]. Therefore, pvl may contribute to resistance by attacking the bovine polymorph-nuclear cells and increase pathogenicity against the host [46]. Interestingly, the sea gene was detected in 26.6% of the tested strains followed by sec gene (6.6%) then mixed (sea and sec) genes in 3.3%, while none of MRSA strains harbored seb and sed genes ( Table 6). The high prevalence of sea and sec gene was previously reported by Rall, et al. [48]. The sea gene was the most predominant enterotoxins gene isolated in other studies conducted on S. aureus [49]. This gene is very resistant to pasteurization heat and maintain some biological activity after 28 min at 121 • C [50]. It's also considered as the most frequently detected gene in the US food poisoning outbreaks followed by sed and seb and 95% of these outbreaks have sea and see enterotoxins [51].

Conclusions
In conclusion, subclinical mastitis caused by S. aureus is considered as one of the major economically important diseases with public health significance. The most predominant virulence genes associated with MRSA strains in bovine milk were coa, mecA and spa, whereas, the most predominant enterotoxin gene is sea that causes food poisoning in human consumers after ingestion of contaminated milk. Based on our knowledge, this is the first study that emphasizes the occurrence of pvl in MRSA strains originating from bovine species milk in Egypt. The continuous application of the antimicrobial susceptibility testing of S. aureus in the future will be necessary to determine the drug choice for disease control.