Abstract: Background and Objective: The emergence of methicillin-resistant Staphylococcus aureus in animals has been an outbreak of mastitis in cattle which represents a great economic loss in milk industry and a serious public health concern worldwide. The objective of this study was to identify the antimicrobial resistance patterns and the prevalence of methicillin resistant and susceptible Staphylococcus aureus harboring the classical staphylococcal enterotoxin genes in bovine mastitis cases. Materials and Methods: A total of 112 Staphylococcus aureus isolates were investigated for antibiotic resistance patterns. Some of the isolates were subjected to genotypic analysis for methicillin-resistant genes (mecA) and classical staphylococcal enterotoxin A through E genes. Results: Methicillin-resistant Staphylococcus aureus was phenotypically detected in 77 (68.7%) of the isolates, while methicillin-susceptible Staphylococcus aureus was identified in 35 (31.2%) of isolates, most of the isolates were showed multidrug-resistance to at least one member of the antimicrobial classes. By genotypic analysis, 54.5% phenotypically methicillin-resistant isolates were mecA gene positive (methicillin-resistant Staphylococcus aureus), while 45.5% of the isolates were mecA gene negative (methicillin-susceptible Staphylococcus aureus). Variation in the staphylococcal-enterotoxin encoding genes was detected. The 66.7% of methicillin-resistant Staphylococcus aureus and 80% of methicillin-susceptible Staphylococcus aureus were encoding staphylococcal enterotoxin genes. The most frequently detected staphylococcal enterotoxin genes were the combination of staphylococcal enterotoxin A, D and E representing 50% of methicillin-resistant Staphylococcus aureus isolates as well as staphylococcal enterotoxin D representing 75% of methicillin-susceptible Staphylococcus aureus isolates. Conclusion: The high prevalence of multidrug-resistant and toxigenic methicillin resistant and susceptible Staphylococcus aureus strains in bovine milk are considered as a potential threat to public health. Hence, the right use of antibiotic of choice in the line of treatment and control of the bovine mastitis caused by Staphylococcus aureus is very crucial in Egypt.
INTRODUCTION
One of the most pathogenic bacteria is Staphylococcus aureus (S. aureus) that is associated with contagious bovine mastitis. Pathogenicity of S. aureus is mainly related to a combination of toxin-mediated virulence, invasive capacity and antibiotic resistance1,2. Virulent S. aureus strains include methicillin-resistant Staphylococcus aureus (MRSA) strains which have become resistant to most antimicrobial agents including β-lactams, aminoglycosides, macrolides and more recently fluoroquinolones3. Therefore, the spread of S. aureus, especially MRSA and its control have become a major challenge in Egypt.
The major challenge of S. aureus related infections has been the difficulty in treatment due to antimicrobial resistance. The levels of antibiotic-resistant infections in the developing countries, especially Egypt, have increased steadily in the last years as a result of the misuse of antimicrobial agents both in therapy and as growth promoters in food animals4.
A variety of toxins is produced by some strains of S. aureus including staphylococcal enterotoxins (SEs) as superantigens which cause a wide range of infections in humans and animals, including staphylococcal food poisoning and bovine mastitis5. The staphylococcal enterotoxins are heat stable exotoxins, include SEA through SEE and SEG through SEJ, being produced sometimes singly and sometimes in combination6.
Little data is available on the epidemiology of MRSA isolates from the bovine origin in Egypt. Therefore, the aim of the current investigation was to study the antimicrobial resistance patterns and the prevalence of methicillin-resistant Staphylococcus aureus harboring the classical SE genes using multiplex PCR as well as the determination of the predominant enterotoxin in each strain. In Egypt, such information could give an indication of the virulence of MRSA present in bovine mastitis.
MATERIALS AND METHODS
Samples: In total, 230 milk samples were collected from cows (n = 115) and buffaloes (n = 115) with clinical or subclinical mastitis from different farms in Dakahlia and Damietta governorates, Egypt in January, 2016. All milk samples were subjected to bacteriological examination.
Microbiological analysis: S. aureus strains were isolated on Baird Parker agar, mannitol salt agar and 5% sheep blood agar (Oxoid). All isolates were identified by conventional tests7 and coagulase tests (both slide and tube)8.
Antimicrobial susceptibility test: The disk diffusion assay9 was performed by using 10 antibacterial disks (Oxoid, England) and interpreted according to NCCLS10.
Oxacillin agar screen: The oxacillin agar screen was performed as described by Felten et al.11. The oxacillin resistant isolates were grown within 48 h.
Molecular identification of mecA gene and staphylococcal enterotoxin A through E genes using Multiplex Polymerase Chain Reaction (mPCR): The DNA extraction and purification from S. aureus isolates (grown in BHI) were performed according to the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) manufacturer’s recommendations with modifications. The DNA was amplified using specific primers supplied from Metabion, Germany with the specific profile (Table 1). Separation of the PCR products was done by electrophoresis on 1.5% agarose gel. The fragment sizes were determined by using gelpilot 100 bp DNA ladder (Qiagen, Germany, GmbH). A gel documentation system (Alpha Innotech, Biometra) was used to photograph the gel, followed by analysis of the data through computer software.
RESULTS
Based on cultural and biochemical properties, a total of 112 (48.7%) S. aureus isolates were recovered from 230 bovines (cows and buffaloes) mastitis milk samples as shown in Table 2. Oxacillin screening test was performed on all 112 S. aureus isolates to detect methicillin-resistant strains. Seventy-seven(77/112, 68.7%) of the tested isolates were positive by the oxacillin screening test and determined to be methicillin-resistant S. aureus (MRSA), while other isolates (35/112, 31.2%) without growth on the oxacillin agar screen plate were identified as methicillin-susceptible S. aureus (MSSA).
In vitro, the antibiotic susceptibility of all 112 S. aureus (77 MRSA and 35 MSSA) isolates against 10 different antimicrobial agents from different antibiotic classes is presented in Table 3.
| Table 1: | Primers sequences, target genes, amplicon sizes and cycling conditions |
| Table 2: | Enzymatic activities of S. aureus isolated from milk |
The resistance of 77 MRSA isolates to oxacillin, penicillin and ampicillin (100%, for each) were the most common finding, followed by tetracycline (91%), clindamycin (77.9%), erythromycin (53.2%) and gentamicin (39%). The highest frequencies of resistance detected by 35 MSSA isolates were those against tetracycline (68.6%), clindamycin (60%), gentamicin (45.7%), ampicillin (40%) and penicillin (34.3%). On the other hand, the resistance to the other antibiotics vancomycin, chloramphenicol and ciprofloxacin were not as high as the other antibiotics either to MRSA or MSSA.
The molecular expression for mecA genes revealed that 42 (54.5%) of 77 isolates possessed the resistant genes (mecA) while the absence of mecA gene was found in 35 (45.5%) of these isolates. Table 4 shows the comparison between the detection of mecA gene by PCR and oxacillin screening test to 77 isolates, 42 strains were genotypically mecA gene positive and phenotypically resistant to oxacillin, whereas other (35) strains were genotypically mecA gene negative and phenotypically oxacillin susceptible.
According to the results of multiplex PCR analysis of 77 isolates, this study revealed about 72.7% (56/77)S. aureus isolates, 66.7% (28/42) of MRSA and 80% (28/35) of MSSA, encoding staphylococcal enterotoxin (SE) genes (Table 4). The most commonly detected genes were the combination of SEA, SED, SEE (14 isolates) representing 50% of MRSA isolates and SED (21 isolates) representing 75% of MSSA isolates. The remaining isolates harbored combination of SEA, SEE genes (7 isolates, 25%) and SED, SEE genes (7 isolates, 25%) of MRSA as well as SEB, SED, SEE genes (7 isolates, 25%) of MSSA. None of the isolates were positive for SEA, SEB, SEC and SEE genes alone.
| Table 3: | Antibiotic susceptibility profiles of MRSA and MSSA isolates |
| R: Resistant, S: Sensitive | |
| Table 4: | Incidence of MRSA isolates harbouring classical SE encoding genes (n = 77) |
DISCUSSION
S. aureus is a major causative bacterium of clinical or subclinical bovine mastitis generates important losses in dairy industries in Egypt12 and is considered as a risk factor for food poisoning in humans13. The high frequency of staphylococcal mastitis (48.7%) in this study confirms the general agreement with other studies supporting the previous postulation that S. aureus is the very important factor in bovine mastitis all over the world4,5,14,15.
MRSA strains have been identified among S. aureus strains isolated from bovine milk16,17, so it is considered an emerging threat to a serious public health hazard. Although the oxacillin disk diffusion test is the least reliable test for detection of resistance to oxacillin in S. aureus18 in comparison to more sensitive and specific oxacillin screening test19,20, there was a similarity between the results of oxacillin screening and oxacillin disk diffusion tests in the identification of S. aureus as MRSA and MSSA. Thus, oxacillin screening test and oxacillin disk diffusion tests have been very useful for the detection of MRSA strains in this study. These results revealed that the MRSA isolates were detected with the high rate (68.7%) in milk samples. Similarly, the high prevalence of MRSA strains was detected by Al-Ashmawy et al.21 (75%) from raw milk in Egypt and Widianingrum et al.4 (50%) from bovine milk in Indonesia. On the contrary, a much lower percentage of MRSA isolation was reported by Intrakamhaeng et al.22 from bovine mastitis cases (19.7%) in Thailand, Kamal et al.12 from raw milk (8.6%) and Ammar et al.23 from examined milk (17.34%) in Egypt. These differences in the MRSA incidence could be contributed to the method of MRSA determination, different national antimicrobial policies and presence of several animals in the same area that facilitate the transfer of genetic material between S. aureus strains. Oxacillin and other β-lactam antibiotics have been used as therapeutic agents for staphylococcal mastitis in Egyptian dairy animals and that may be the reason for the emergence of MRSA in the bovine milk. According to in vitro susceptibility tests, the majority of MRSA strains exhibited resistance to oxacillin, penicillin, ampicillin, tetracycline, clindamycin and erythromycin. A large percentage of the strains were susceptible to the antibiotics vancomycin, chloramphenicol, ciprofloxacin and gentamicin. Furthermore, most MSSA strains were highly resistant to tetracycline, clindamycin, gentamicin, erythromycin, penicillin and ampicillin but were oxacillin, vancomycin, chloramphenicol and ciprofloxacin susceptible. These findings were similar to the previous studies24,25. As well, this coresistance pattern of MRSA strains (β-lactams, tetracyclines, macrolides and aminoglycosides) has been frequently detected by other investigators26,27. On the other hand, the most effective antimicrobials against MRSA and MSSA isolates were vancomycin, chloramphenicol and ciprofloxacin according to this study. Interestingly, the emergence of multidrug-resistant MRSA and MSSA strains are due to the misuse of a large number of antimicrobial agents in Egyptian dairy farms. Consequently, the search for newer and more effective antibiotics against this organism as well as effective measures against the unregulated use of antibiotics is necessary for effective treatment of staphylococcal infections and avoiding the development of multidrug resistant strains.
In the current study, most strains were mecA positive oxacillin-resistant S. aureus. On the other hand, there were some strains mecA negative oxacillin-resistant S. aureus. This indicated the presence of incompatibility between the detection of methicillin resistance phenotypically using oxacillin screening test and the absence of mecA gene in some MRSA strains. This finding is in accordance with Garcia-Alvarez et al.28, who identified phenotypic MRSA isolates without mecA gene. This may be attributed to the presence of PCR inhibitors or other physical factors that may have compromised the sensitivity of PCR in the detection of mecA gene29. Also, methicillin resistance may be occurred by other mechanisms other than the expression of mecA gene28 or instability of the mecA gene in strains with the loss of mecA30. Furthermore, the discovering of phenotypically resistant of some mecA negative isolates to β-lactam antimicrobial agents may be correlated to a less common type of resistance because of overproduction of β-lactamase or the presence of altered penicillin-binding protein (PBP) not related to 2a or 2’ 31. It is also possible that the mecA negative oxacillin-resistant S. aureus may have mecA alleles which could not be detected by the primers used in this study. However, the previous studies showed that mecA gene-positive isolates are not necessary to be associated with its expression to oxacillin resistance by the conventional phenotypic methods because there are some determinants, such as the mecR1-mecI, BlaI and fem genes, that control the expression of oxacillin resistance30. Furthermore, the cost and work load of PCR to detect the mecA gene have prevented their broad use in a clinical microbiology laboratory. Thus, regardless the presence or absence of mecA gene by PCR, the detection of resistance to oxacillin and β-lactams offers an interesting new approach to the rapid characterization of S. aureus as MRSA or MSSA.
Overall, there is a serious health risk of MRSA and MSSA caused by the production of staphylococcal enterotoxins as superantigens, stimulating T-lymphocytes to release cytokines and T-cell proliferation32. Thus, a multiplex PCR-based diagnostic protocol was used in this study to detect the genes for staphylococcal enterotoxins A to E. About 72.7% of S. aureus isolates were enterotoxigenic in this research. Similar results were observed by El-Bagoury33 and Bystron et al.34, who recorded 75 and 66% toxigenic strains of S. aureus isolates, respectively. Moreover, this study detected the high prevalence (66.7%) of MRSA isolates encoding staphylococcal enterotoxin genes. This result is consistent with other researchers who found 82.4% of MRSA isolates harboring SE genes19. Interestingly, the most commonly detected genes were the combination of SEA, SED, SEE genes in MRSA isolates and SED gene in MSSA isolates in this research. MRSA isolates encoded SED as the most predominant enterotoxin genes were observed by other researchers in mastitis isolates35. These classical genes, SEA, SEB, SED and SEE are more frequently detectable genes from MRSA and MSSA isolates from mastitis milk36. The SEC gene could not be detected by this study in comparison to the previous studies36. This finding highlights a potential risk for consumers because MRSA and MSSA of animal origin may contaminate foods and represent a source of potential infection and intoxication in humans.
CONCLUSION
These data indicate the bovine milk represents a potential risk of multidrug-resistant and toxigenic S. aureus either MRSA or MSSA in Egypt. Accordingly, the line of treatment with the prudent use of antibiotics and control of the bovine mastitis caused by S. aureus is very crucial to prevent the emergence and spread of multidrug-resistant S. aureus strains in Egypt. In addition, further detailed analysis of functional genomics is now warranted to get a better understanding of enterotoxin activity and virulence of S. aureus in Egypt.
SIGNIFICANCE STATEMENT
This study discovers the high prevalence of multidrug-resistant and toxigenic methicillin resistant and susceptible Staphylococcus aureus strains in bovine mastitis. Such information can give an indication of the virulence of methicillin resistant and susceptible Staphylococcus aureus present in bovine mastitis as well as a potential threat of bovine milk to public health. This study will help the researchers to uncover the critical areas of the misuse of the antibiotic in the dairy farms on the emergence of multidrug-resistant MRSA and MSSA strains. Thus, the significance of the searching for newer and more effective antibiotics against this pathogen besides effective measures against the unregulated use of antibiotics is essential for effective treatment of staphylococcal infections and avoiding the development of multidrug resistant strains.