Abstract: Background and Objective: Staphylococcus aureus is commonly associated with mastitis in dairy herds with potential public health implications. This study was conducted to investigate the existence of S. aureus in mastitic milk and to determine the antimicrobial resistance profiles of the isolated strains as well as the resistance and virulence associated genes. Materials and Methods: Two hundred quarter milk samples were collected from 3 dairy farms at Dakahliya (n = 2) and Damietta (n = 1) Governorates, Egypt from September to December 2016. Conventional culturing and Polymerase Chain Reaction (PCR) assays targeting nuc (thermonuclease) and coa (coagulase) genes were performed. Isolates were tested for its susceptibility against 14 antimicrobial agents using disk diffusion method. All the isolates were screened for the presence of β-lactamases (blaZ, mecA) and virulence associated (pvl and tst) genes by PCR. Results: The S. aureus was detected in 42% (84/200) of the total examined milk samples. Regarding the antibiogram results, S. aureus revealed a high resistance against ampicillin (95.2%) and penicillin (83.3%) and a lower resistance was observed against gentamicin (23.8%), amikacin (16.7%) and ciprofloxacin (14.3%). Multidrug resistances were detected in 83.3% of the isolated S. aureus. Of the 70 penicillin-resistant S. aureus isolates, blaZ gene was identified in 67 (95.7%) isolates. Fifty percent of S. aureus isolates harbored the specific amplicon of mecA gene. Markedly, all mecA positive strains displayed multidrug resistance and were also positive for blaZ gene. The virulence determinants pvl and tst were detected in 7.1 and 11.9% of the isolated S. aureus, respectively. Conclusion: Presence of multidrug resistant and toxin producing S. aureus in dairy farms pose a major risk to public health. Therefore, this study highlighted the importance of developing an efficient control program to inhibit the transmission of S. aureus, particularly multidrug resistant strains to humans.
INTRODUCTION
Staphylococcus aureus, a Gram-positive bacterium, constitutes part of the commensal microbiota in the skin, nose and respiratory tracts of both humans and animals. Other strains of S. aureus are highly pathogenic to human causing variety of diseases that includes skin infections, osteomyelitis, septic arthritis, septicemia and food poisoning1. Food intoxication in human is mainly caused by S. aureus thermostable enterotoxins that can resist the pasteurization temperature2.
Mastitis is one of the most common infectious diseases affecting dairy herds, which lead to sever financial losses and attributed to decrease in milk yield and milk quality. The S. aureus is considered the most significant pathogen associated with mastitis in dairy herds3,4. Zoonotic potential of clinical and sub-clinical mastitis arises from the possibility of shedding the pathogens and their toxins into milk5.
The emergence of antimicrobial resistance among S. aureus has been suggested to cause delay in antibiotic treatment of bovine mastitis. This resistance against wide range of antimicrobial classes may be attributed to the indiscriminate use of these agents in the treatment of bovine mastitis6. In Egypt, the over-prescription and misuse of antimicrobials used for the treatment of bacterial infections in humans and animals, have been associated with the exacerbation of antimicrobial resistance among these pathogenic bacteria7,8.
The β-lactams have been frequently used for treatment of bovine mastitis but their efficiency is decreased due to development of β-lactamase encoded blaZ that hydrolyze penicillins9. Methicillin/oxacillin resistance is another β-lactam resistance mechanism, results from the production of low-affinity penicillin-binding protein (PBP2a) encoded by mecA gene10. Methicillin-resistant Staphylococcus aureus (MRSA) attracted a great concern by mid-1990s because they displayed multiple resistances to a wide range of antimicrobial classes other than β-lactams and disseminated continually through new communities11.
There are wide arrays of virulence determinants associated with the pathogenicity of S. aureus12. These determinants consist of enzymes and cytotoxins that include hemolysins (α, β, γ and δ), nucleases, lipases, proteases, collagenases and hyaluronidases. Additionally, some S. aureus strains produce exoproteins, such as Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin-1 (TSST-1), exfoliative toxins (ETA and ETB) and staphylococcal enterotoxins (SEA-E, G-I)13.
This study aimed to investigate the occurrence of S. aureus in mastitic milk from three dairy herds at Dakahliya and Damietta Governorates, Egypt. Molecular characterization of the isolated strains was performed using PCR targeting thermonuclease (nuc) and coagulase (coa) genes, β-lactamases (mecA and blaZ) and virulence associated genes (TSST-1 gene, tst and Panton-Valentine leukocidin gene, pvl). The antimicrobial resistance profiles were also determined.
MATERIALS AND METHODS
Samples collection and bacteriological culturing: Cattle from three dairy farms located at Dakahliya (n = 2) (Latitude 31.04° N and longitude 31.37° E) and Damietta (n = 1) (Latitude 31.36° N and longitude 31.67° E) Governorates, Egypt were included in this study. A total of 200 individual quarter milk samples were taken from 120 cows during the period from September to December 2016. All the cattle investigated had a median age of 5-7 years. Physical examination of the udder was done before collection of each sample to estimate the existence of any signs of inflammation. All the milk samples were collected aseptically by hand milking in sterile tubes after discarding the first few streams of milk. The collected milk samples were transported aseptically in ice coolers to the Laboratory of Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Mansoura University for bacteriological evaluation. Informed consents were taken from the owners of the farms prior sampling.
Milk samples were subjected to S. aureus isolation procedures as previously described by Wang et al.14. Presumptive S. aureus colonies were picked up and purified by sub-culturing on the surface of tryptone soya agar (TSA; Oxoid, UK) plates. Then, the presumptive colonies were subjected to gram staining and standard biochemical tests15.
Antimicrobial susceptibility testing: Antimicrobial susceptibility profiling was done by disc diffusion method for the isolated S. aureus against 14 antimicrobial agents (Oxoid, UK) and according to Clinical and Laboratory Standards Institute (CLSI) guidelines16. The S. aureus isolates were tested against penicillin (P; 10 IU), oxacillin (OX; 15 μg), amoxicillin (AX; 25 μg), tetracycline (TE; 30 μg), streptomycin (S; 10 μg), amikacin (AK; 30 μg), sulfamethoxazole-trimethoprim (SXT; 23.75/1.25 μg), rifampin (RA; 5 μg), erythromycin (E; 15 μg), ampicillin (AM; 15 μg), chloramphenicol (C; 30 μg), vancomycin (VA; 30 μg), gentamicin (CN; 10 μg) and ciprofloxacin (CIP; 5 μg). Interpretation of the results was done following CLSI guidelines. A single strain was considered multidrug resistant if it exhibited resistance to 3 or more different antimicrobial classes.
Genotypic characterization of S. aureus: The DNA extraction from S. aureus isolates was performed using PureLink Genomic DNA extraction Kit (Invitrogen, Carlsbad, CA) according to the manufacturer guidelines. The PCR was performed on the isolated S. aureus to determine nuc, mecA and blaZ genes as previously reported by Oliveira et al.17. The coa gene was also investigated as previously outlined18. The virulence determinants pvl and tst genes encoding PVL and TSST-1 were determined according to Lina et al.19 and Sallam et al.20, respectively. The primers sequences and their PCR products are summarized in Table 117-20. The PCR for each specific gene was performed using 96 well Bio-Rad (Munich, Germany) thermal cycler. Each PCR reaction mixture was done in a final volume of 25 μL containing 12.5 μL of 2X PCR master mix (Promega, Madison, USA), 1 μL of 20 pmol of each primer (Metabion, Germany), 5 μL DNA template and the volume of the reaction mixture was completed to 25 μL using DNase/RNase-free water. Thermocyclic condition for each PCR reaction was done as summarized in Table 2. The amplified products for each gene were separated by subjecting 3 μL aliquots to agarose (1.2%) gel for 30 min at 100 V followed by a 20 min staining in ethidium bromide solution. Gels were then visualized under UV light and photographed.
RESULTS AND DISCUSSION
In this study, of the 200 mastitic milk samples subjected to bacterial culturing, eighty four (42%) S. aureus isolates were identified based on the morphological and biochemical characters. A wide variety in the prevalence rate of S. aureus from bovine mastitis was detected in many previous studies5,21-27. Lower occurrence of S. aureus was reported in a study conducted by Anderson et al.21, who determined a 13.6% prevalence rate of S. aureus from the lactating cows in three different areas in USA. In another study from Germany, Schlotter et al.22 identified S. aureus in 15.5% of the total examined milk samples. However, higher recovery rates of S. aureus were determined from several countries; Zimbabwe (49.3%)23, South Ethiopia (51.2%)24 and Brazil (53%)5. In Egypt, variable isolation rates of S. aureus from mastitic milk were previously recorded by Elhaig and Selim25 (38.3%), Elsayed et al.26 (11.2%) and El-Ashker et al.27 (5.6%). Since pasteurization process could eliminate S. aureus in milk, yet, the possibility of human infection with S. aureus might be attributed to the consumption of raw milk and dairy products particularly homemade cheese2,28.
| Table 1: | Sequences and amplified products size of primers used in polymerase chain reaction assay |
| Table 2: | Cyclic polymerase chain reaction conditions of the different primer sets |
| Fig. 1: | Representative agarose gel electrophoresis of the expected amplified PCR products for nuc (Lane 1, 270 bp), mecA (Lane 2, 162 bp), blaZ (Lane 3, 861 bp), pvl (Lane 4, 433 bp), coa (Lane 5 and 7, variable products 500-1000 bp) and tst (Lane 6, 543 bp) genes, Lane -ve: Negative control and Lane M: 100 bp DNA ladder |
Moreover, the incorporation of milk from mastitic cows into the bulk milk especially in developing countries implies a serious hazard to humans.
The nuc gene, which is considered a genetic marker used for the rapid and direct identification of S. aureus, was detected using PCR in all the biochemically identified isolates from mastitic milk29,30. Coagulase gene encoded by coa was also used for the confirmation of the isolated S. aureus. In this study, all the recovered S. aureus possessed the specific amplified products of coa gene (Fig. 1). Coagulase is a significant virulence determinant of S. aureus causes coagulation of plasma and its expression is assumed to resist phagocytosis, making the S. aureus more virulent31.
Antimicrobials are considered the best choice for the treatment of mastitis; however, the improper usage of antimicrobial drugs poses great hazards to both human and animal health due to the emergence of antimicrobial resistance32. In this study, about 83% of the isolated S. aureus showed multidrug resistances to 3 or more antimicrobial agents. The isolated S. aureus displayed higher resistance to ampicillin (95.2%) followed by penicillin (83.3%). At the same time, the isolates exhibited lower antimicrobial resistance with gentamicin (23.8%), amikacin (16.7%) and ciprofloxacin (14.3%) (Table 3). Nearly similar results were previously determined by Al-Ashmawy et al.28, Akindolire et al.32 and Prashanth et al.33, who found that the majority of S. aureus isolates from milk and dairy products showed higher antimicrobial resistance against penicillin.
The penicillin resistance in S. aureus is a well-recognized phenomenon worldwide due to the production of β-lactamases. Of the 70 penicillin-resistant S. aureus isolates, genotypic characterization revealed that 67 (95.7%) isolates had blaZ gene (Table 4). Similarly, Da Costa Krewer et al.34 reported the higher occurrence of blaZ gene among β-lactam resistant S. aureus isolated from bovine mastitis in the Northeast of Brazil.
| Table 3: | Antimicrobial resistance of S. aureus isolates from mastitic milk |
| Table 4: | Antimicrobial resistance phenotypes and genotypes of S. aureus isolates from mastitic milk |
| ND: Not determined | |
This could explain that other mechanisms have a role in the resistance of staphylococci to β-lactams than blaZ gene35.
All the isolated S. aureus were screened for the presence of mecA gene using PCR where 50% of the isolates harbored the specific amplicon of mecA gene at 162 bp (Fig. 1). The mecA gene was detected in all the phenotypically identified isolates that resist oxacillin. The existence of mecA-positive MRSA in bovine milk has been reported worldwide in many previous studies36-38. However, mecA-negative MRSA has been also recovered from bovine milk; the resistance revealed by mecA-negative MRSA isolates might be attributed to the presence of mecA homologues as mecC or other β-lactam resistance mechanisms15-39,40.
The results of disk diffusion test for antibiotic resistance revealed that all MRSA isolates were resistant to 9 or more antimicrobial agents. Likewise, several studies reported that MRSA strains isolated from milk and some dairy products exhibited a multidrug resistance28,41,42. The ability of MRSA strains to resist a wide range of antimicrobial agents might be due to production of β-lactamases and PBP2a. Interestingly, all mecA-carrying strains were also positive for the blaZ gene. The MRSA is commonly known to be originated from humans and transmitted to animals under poor hygienic measures; however the existence of MRSA in milk and animal environment pose a major threat to the consumers and occupational contacts37,43.
All the isolates were further assessed for the presence of two additional virulence genes, including tst and pvl. Toxic shock syndrome toxin-1, encoded by tst gene is a superantigen secreted by S. aureus in susceptible hosts and is responsible for toxic shock syndrome in humans44. In this study, tst was detected in 11.9% (10/84) of the tested S. aureus isolates. Similarly, in a pan-European survey that involved 12 European countries and examined 456 S. aureus isolates from cow milk, 12.9% of the isolates harbored tst gene45. However, several previous studies revealed the absence of tst in milk and other dairy products28,46.
The specific amplicon of pvl gene was detected only in 7.1% of the isolated S. aureus. This was opposite to that determined previously in many studies that reported the absence of pvl gene in S. aureus isolates from animal origin46-49. The pvl gene, which is responsible for the severe necrotic inflammation in soft tissues and skin, is commonly associated with the community-acquired MRSA (CA-MRSA) strains from humans50. Nonetheless, the presence of pvl gene in this study from mastitic cows might be attributed to the close contact of animals with its rearing community33.
CONCLUSION
In conclusion, the existence of S. aureus carrying pvl gene indicates personnel sources of contamination to dairy farms. Special concern should be considered to prevent this source of contamination through prevention of unauthorized persons from entering dairy farms as well as the adoption of restrict hygienic measures. In addition, high resistance against β-lactams which are widely used in veterinary practice and the presence of MRSA constitutes another burden to the public health. Hence, monitoring the emergence of multidrug resistant strains of S. aureus in dairy farms is essential to control the spreading of this pathogen and the related zoonotic hazard.
SIGNIFICANCE STATEMENTS
This study determined the prevalence of S. aureus and some of its virulence determinants as well as antimicrobial resistance in mastitic cows from different dairy herds. Monitoring of S. aureus, which indicates the improper hygienic measures, in dairy herds is a prerequisite for the initiation of effective infection control measures. The pvl is one of S. aureus virulence associated genes that many researchers were not able to explore from animal origin. Thus a new principle on the existence of pvl from animal sources may be arrived at.
ACKNOWLEDGMENT
The authors acknowledge the technical assistance of colleagues at Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Mansoura University. There was no grant from any funding agency for this research.