Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat

Contamination of meat with foodborne pathogens represents a major public health threat. The increasing volume of trade and travel is considered as a potential risk factor facilitating the global transport and dissemination of pathogenic bacteria in food. Imported animal products are considered as a clear example, and risk analyses have been previously applied to characterize these products [1]. This risk analyses strategy have been implemented following the World Trade Organization (WTO) creation. The application of sanitary and phytosanitary measures in response to the sanitary and phytosanitary measures agreement (SPS Agreement) [2] requires the WTO members to remove barriers on the trade of agricultural products, except in situations where such trade can potentially create risk to the animal, human or plant health in the importing country.

Staphylococcus aureus is one of the most common foodborne pathogens causing food poisoning outbreaks worldwide [3]. Other than S. aureus, the clinical and veterinary importance of coagulase-positive staphylococci (CPS) and coagulase-negative staphylococci (CNS) have often been neglected. In recent years, the risk of CPS and CNS has been highlighted by recent reports [4–6] with special reference to the CNS that have been commonly found in food [7, 8]. CNS have been recorded as conveying vector for virulence genes and have been implicated in some cases of food poisoning [9]. Furthermore, food-related staphylococci could act as dissemination vectors for antibiotic resistance genes to other potentially pathogenic microorganisms causing immediate threat to the public health. The mecA-harboring CNS (MRCNS) have been reported to have a reservoir in animal farm facilities and in meat products, with the ability to be conveyed to S. aureus [10–12].

The remarkable global concern of antibiotic- resistant pathogens in the food chain and the potential for these resistant pathogens to spread through the food chain prompted the Codex Alimentarius Commission to establish an ad hoc Intergovernmental Task Force on antimicrobial resistance. The main task of this commission is to apply a complete risk assessment strategy on the use of antimicrobials belonging to both clinical and veterinary classes. While domestic control over antimicrobial usage policies and monitoring is achievable, negligible information is available for imported food [13], with special reference to beef meat that has been incriminated to contribute to the emergence of multidrug resistance among humans through the dispersion of resistance genes carried by resistant pathogens transmitted by contaminated meat [14, 15].

Livestock-associated methicillin-resistant S. aureus (LA-MRSA) have acquired a number of novel and unusual antimicrobial resistance genes including multi-resistance genes such as the cfr gene, that confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A [16]. Oxazolidinones are last resort antimicrobial agents for the control of serious infections caused by MRSA and vancomycin-resistant enterococci in humans. MRSA is also notoriously difficult to treat due to resistance to β-lactams (including penicillin, oxacillin, and methicillin) represent a class of antibiotics generally prescribed as the first line of defense against clinical infections caused by staphylococci, which include drugs like penicillin, oxacillin, and methicillin. This resistance was attributed to the carriage of mecA. The mecA gene encodes a different form of penicillin-binding protein, PBP2a, which β-lactam drugs cannot inactivate.

Therefore, the objectives of this study were to characterize the diversity of Staphylococcus strains recovered from imported meat sold in the Egyptian market and to assess recovered isolates as potential dispersion vectors for the spread of antimicrobial resistance. To achieve that aim, Staphylococcus isolates were tested for the presence of antimicrobial resistance phenotypes and genetic determinants (mecA, cfr, gyrA, gyrB, and grlA) and for their biofilm formation.

Globally, an increasing recognizable concern exists on the status of antimicrobial resistant microbial contaminants in the food chain and their capacity to be widely dispersed through the international trade of food. This prompted the Codex Alimentarius Commission to establish an ad hoc Intergovernmental Task Force on Antimicrobial Resistance in 2007, bearing in mind the occurrence of national and regional diversity in antimicrobial misuse, human subjection to resistant microorganisms and determinants and their prevalence in foodborne pathogens. Egypt imports different meat types to fill the gaps in animal protein supply. According to the U.S. Meat Export Federation (USMEF) that examines key statistics and trends in beef and pork trade from 2008 to 2017, Egypt’s projection for beef-importing in 2017 would reach about 332,000 metric tons of meat imports and this is supposed to grow to 2025 by 52.28% [25]. While the importation of beef meat in Egypt is crucial to close the gap in animal protein requirements, monitoring the frequency of antimicrobial resistance in imported meat must assure that quality and safety standards are met.

The spread of MRSA is a serious public health concern for both human and veterinary medicine. Due to the occurrence of MRSA, methicillin and other β-lactamic antibiotics have become useless for clinical therapy, leaving the term MRSA to be used to describe S. aureus strains resistant to effectively all β-lactamase-resistant penicillins and harboring the mecA gene [23]. Although the development and spread of multiple antibiotic-resistant MRSA have gained much attention over the past years, yet the role of imported meat has not been given much attention. In a Danish study, Agersø et al. reported that MRSA was found in 18% of the imported broiler meat and 7.5% of the imported pork [26]. MRSA are often resistant to other antimicrobials different to methicillin, highlighting the necessity for new and effective antimicrobials. Attributed to their extended antimicrobial spectra, fluoroquinolone compounds such as ciprofloxacin and norfloxacin were recommended as useful candidates for eradicating MRSA [27]. Nonetheless, due to the misuse of these compounds in the clinical practice, resistance of MRSA to these compounds has been observed [28]. For this reason ciprofloxacin is not recommended to be used in empirical therapy against MRSA infections. Furthermore, the use of other fluoroquinolones is only allowed following accurate antimicrobial susceptibility testing. Nonetheless, vancomycin remains the drug of choice to treat MRSA infections [29].

Identification of methicillin resistant staphylococci in the laboratory is often problematic due to difficulty in detecting heterogeneous methicillin-oxacillin resistance in staphylococci [30, 31]. Moreover, Standard interpretive breakpoints for oxacillin susceptibility reporting published by the CLSI (formerly the National Committee for Clinical and Laboratory Standards) were changed in 2004, and NSA isolates of veterinary origin are now more likely than in previous years to be deemed resistant by testing laboratories that use those guidelines [32].

In this study, the susceptibility of 23 Staphylococcus spp. strains isolated from imported meat to 12 antibiotics from different classes was evaluated. An extreme resistance was found against penicillin and sulfamethoxazole/trimethoprim as compared to other tested antibiotics, which could be attributed to the extensive use of these antibiotics in treating mastitis cases and/or as growth promoters in animal feed in the countries from which the beef meat was imported. Resistance to other clinically important antibiotics, including β-lactamic antibiotics (such as ampicillin-sulbactam, methicillin, oxacillin), fluoroquinolones (ciprofloxacin), macrolides (erythromycin), aminoglycosides (gentamicin), glycopeptides (vancomycin), lincosamides (clindamycin) and tetracycline also occurred and, in some cases, multidrug resistance phenotypes were identified. Indeed, correlation analyses showed co-occurrence of resistance to series of antibiotics. Extended-spectrum beta-lactamases (ESBL) -producing pathogens are often resistant to fluoroquinolones and aminoglycosides since resistance mechanisms for these classes of antibiotics are often carried on the same large plasmids that contain the genetic elements for ESBL production [33]. Our results corroborate this observation. Thus, for instance, resistance to methicillin was significantly associated with resistance to ciprofloxacin and gentamycin, among others (clindamycin, oxacillin, tetracycline and vancomycin). The colonization ability in conjunction with the occurrence of MDR highlights the hazardous nature of the Staphylococcus spp. isolated from imported beef meat.

The antimicrobial resistance determinants mecA, gyrA, gyrB, grlA and cfr, frequently reported in S. aureus strains were also screened in recovered isolates. The emergence of the multiple drug resistance gene cfr in staphylococci is of global clinical and veterinary importance that has been previously investigated in staphylococci [34] and has been identified as a phenicol and lincosamide resistance gene [35]. In this study, the absence of the cfr gene among imported beef meat isolates suggests a decreased potential risk as an outcome of harboring the gene in the food chain through imported meat. Furthermore, the absence of the gene cfr poses a significant and interdisciplinary public health positive factor to the Egyptian consumers. One isolate showed chloramphenicol resistance but was negative for the cfr gene. This observation might be attributed to a possible heterogeneous expression of the cfr gene [36] or to different potential chloramphenicol resistance mechanisms.

The mecA gene confers methicillin resistance. It encodes the penicillin binding protein 2a, an enzyme that has low affinity for beta-lactams, and has been reported to lead to resistance to ciprofloxacin, gentamycin, methicillin, oxacillin and vancomycin [37]. In our study, the mecA gene was carried by 5 out of 23 Staphylococcus spp. isolates, and, as expected, presence of mecA was directly correlated with resistance to ciprofloxacin, gentamycin, methicillin, oxacillin, and vancomycin. Nevertheless, not all methicillin or oxacillin resistant isolates were mecA positive. It was previously indicated that some isolates missing the mecA gene could be identified as phenotypically resistant to oxacillin (MRSA) [38]. These results suggests that other potential resistance mechanisms might exist. Molecular investigations of a S. aureus isolate, which was found to be phenotypically resistant to methicillin but negative for the mecA gene, were able to identify the presence of a novel mecA homologue, which was found to be associated with cattle [39], suggesting the existence of a zoonotic MRSA reservoir [40]. In 2012, the International Working Group on the Classification of Staphylococcal Cassette Chromosome elements (IWCC) renamed the mecA variant, mecC [41]. Methicillin-resistant S. aureus strains carrying the mecC gene have been shown to cause a range of infections in humans and appear to be predominantly community associated [40]. The prevalence of mecC in CNS has been recently reported for 13 European countries, where it has been isolated from 14 different host species (Holmes et al. 42), and an allotype of the mecC gene has been detected in a S. xylosus strain [42]. It is also worth mentioning the fact that one S. lugdunensis isolate was observed to harbor the mecA resistance gene but was phenotypically characterized as MSNSA. The phenotypic susceptibility to oxacillin, methicillin, penicillin and ampicillin (β-lactam antibiotic of the penicillin class) regardless of the mecA presence may be attributed to the heterogeneous expression of mecA [43], which is more common in CNS than that in S. aureus [44]. Nevertheless, it should be noted that in 2014, Bhargava and Zhang emphasized the importance of MRCNSs as important reservoirs of mecA that could act as precursors of MRSA [11]. In addition, temperature abuse during storage and transportation to the importing country may result in multiplication of MRS [45]. An additional important finding was introduced by the identification and characterization of another gene on the SCC element called mecR2 that regulates and increases mecA expression when MRSA bacteria encounter β-lactam drugs [46].

Besides resistance genes, mutation-mediated resistance is especially common among resistances to synthetic antibacterial agents, such as fluoroquinolones and oxazolidinones in S. aureus and other bacterial species [47]. The primers used in this study amplified only the copies of gyrA, gyrB, and grlA that contained mutations in the quinolone resistance-determining regions (QRDRs). In this study, the amplification of a grlA and gyrA product represented the presence of a mutation conferring resistance. Mutations in the gyrB gene does not play an important role in quinolone resistance [48]. Although the presence of resistance genes are a primary reason for antibiotic resistance, the resistance phenotype and gene presence are not exclusively linked in this study. Similarly, this inconsistency was common in other classes of antibiotics, demonstrating that the presence of a certain resistance gene was not necessary an indicator of antibiotic resistance [49]. A potential explanation for this inconsistency could be the lack of expression of some of the resistance genes and the presence of other genes encoding resistance. In addition, chromosomally encoded multidrug resistance pumps have been shown to have other primary functional or structural roles [50].

The connection between microbial biofilms and antibiotic resistance is of considerable interest to biomedical researchers. Previous reports have suggested that a correlation between antibiotic resistance and biofilm formation ability exists, a phenomenon that may be responsible for a decrease in the efficaciousness of antimicrobial agents against S. aureus infections [51]. As a consequence of biofilm development, the capability of horizontal gene transfer is increased and may facilitate the dispersion of antibiotic resistance [52]. In the present study, MRS and MDR isolates showed a good potential to form biofilms. In fact, correlation analyses showed that biofilm formation, as assessed by the CRA tests, was directly correlated with mecA presence, methicillin resistance and vancomycin resistance, and biofilm producing MRNSA showed high resistance to almost all antibiotic classes compared to non-producers, which corroborates previous observations [53].

Despite the alert generated by the outbreaks of foot and mouth disease and swine fever, which have been attributed to international trade of meat, little information or actionable risk management information is available yet on the dissemination of microbial hazards through retail imported meat. This study reveals that imported meat can act as a transmission vector for MRSA or MRNSA harboring the mecA gene, which may represent a risk for both human and veterinary medicine. The data obtained on the resistance of Staphylococcus spp. to antimicrobials, may be used for implementing an antimicrobial resistance spread monitoring and prevention program.