Staphylococcus aureus is an opportunistic human and animal pathogen causing food intoxication and a variety of infections ranging from skin and soft tissue infections to serious diseases including endocarditis, septicemia, osteomyelitis, pneumonia and toxic shock syndrome (Chen and Huang, 2014; Otto, 2014). The drug resistance of S. aureus has gradually increased in the recent decades due to the misuse of antibiotics (e.g., using antibiotics without prescription, uncontrolled doses, and useless application of drugs), which resulted in bacterial evolution (Gharieb et al., 2020; Guo et al., 2020). Consequently, multidrug-resistant S. aureus especially methicillin-resistant (MRSA) strains are a major human health concern causing severe morbidity and mortality, in particular in hospitals (hospital-associated MRSA; HA-MRSA), and in healthy persons (community-associated MRSA; CA-MRSA) (Weber, 2005). MRSA, one of twelve priority pathogens that threaten human health as classified by the World Health Organization (WHO), has been increasingly detected in food products (Craft et al., 2019; Li et al., 2019). Animal- derived foods serve as vehicles for the transmission of livestock- associated MRSA (LA-MRSA) (Mossong et al., 2015; Anjum et al., 2019). Further, human-mediated contamination of carcasses, meat product or ready-to-eat (RTE) foods at abattoirs, meat processing plants or during handling may be a source of MRSA and could represent potential risk for consumers (Hadjirin et al., 2015; Li et al., 2019).

Apart from the studies focused on LA-MRSA, the prevalence of staphylococci in RTE foods is receiving widespread attention due to the increasing number of food poisoning cases (Chajecka-Wierzchowska et al., 2015; Yang et al., 2017). MRSA strains exhibit resistance to various antimicrobials such as penicillins, cephalosporins, and carbapenem through the acquisition of the mobile staphylococcal cassette chromosome mec (SCCmec) that carries mecA gene, which encodes for an altered penicillin binding proteins (PBP2a or PBP2′), thereby making their treatment difficult (Hiramatsu et al., 2014). Vancomycin has long been considered the last-resort treatment for MRSA infections (Holmes et al., 2015). However, its excessive use resulted in the emergence of vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), and heterogeneous vancomycin-intermediate S. aureus (hVISA) strains (Amberpet et al., 2019). In addition to antimicrobial resistance, MRSA strains are able to form biofilm as a fitness and survival mechanism that is mediated by the strong adhesion, increase in drug resistance and reduction in the effectiveness of sanitizers (Craft et al., 2019). In the presence of biofilm, resistance of S. aureus to antimicrobials was reported to increase to 1,000 times that of planktonic cells (Guo et al., 2020). Therefore, MRSA biofilm formation is an important persistence and dissemination mechanism of food contamination (Vergara et al., 2017; Avila-Novoa et al., 2021). Biofilm development is thought to be a two-step process in which bacteria adhere to a surface via a capsular antigen, capsular polysaccharide/adhesin (PS/A), and then multiply to form a multilayered biofilm, which is triggered by the synthesis of polysaccharide intercellular adhesin (PIA) from β-1, 6- linked N-acetyld-glucosamines (Periasamy et al., 2012). The intercellular adhesion (ica) locus is comprised of icaA, icaD, icaB, and icaC genes that mediate the production of PIA and PS/A (McKenney et al., 1998). The icaA gene encodes N-acetylglucosaminyltransferase enzyme. Furthermore, icaD has been shown to play a key role in the maximum expression of this enzyme, which leads to capsular polysaccharide phenotypic expression (Arciola et al., 2001). The icaC functions as a polysaccharide receptor (McKenney et al., 1998). Furthermore, the biofilm-associated protein (Bap) is required for S. aureus initial attachment and biofilm development (Cucarella et al., 2004). Vaccine candidates consisting of several antigens related to multiple stages of biofilm formation and biofilm-related macromolecules have been proposed as a strategy for prevention and treatment of staphylococcal infection (Mirzaei et al., 2016, 2019, 2021; Bahonar et al., 2019; Gholami et al., 2019).

Indeed, data on the prevalence and biofilm formation by MRSA and VRSA in RTE food of animal origin remain scarce. In Egypt, most of the studies on the prevalence of MRSA isolates in RTE meat has focused on their antimicrobial resistance profile (Saad et al., 2019; Mahros et al., 2021), yet these studies did not analyze biofilm formation potential by the isolates, did not analyze the clustering pattern of such isolates in this important source and no studies exist that included isolates from humans. Here, we provide a characterization of a set of MRSA and VRSA isolates from humans (patients and food handlers) and RTE meat (shawarma and burger) focusing on their frequency, antimicrobial resistance phenotype, biofilm formation ability and how antimicrobial resistance and biofilm formation, as fitness traits, could be linked.

Nowadays, there is an increased trend of RTE food afforded by infinite number of restaurants and street vendors all over the world, notably in Egypt. While this provides the advantage of fast food, it comes with the challenge and risk of bacterial infection especially these RTE products are not further exposed to heating. Raw meat contaminated with S. aureus is considered to be a major cause of food poisoning worldwide (de Boer et al., 2009; Wang et al., 2014; Raji et al., 2016). The risk of infection is being increased when the contaminated meat is not cooked sufficiently or RTE food is contaminated with this bacterium by cross-contamination (Wang et al., 2017). Recently, RTE food products have been reported as reservoirs for methicillin-resistant coagulase-negative staphylococci (MRCONS) (Yang et al., 2017), but the occurrence, resistance profile, and biofilm formation ability of methicillin-resistant coagulase-positive staphylococci (MRCOPS) and VRSA have received much less attention and require further in-depth investigation. Indeed, the available studies in Egypt have provided detailed characterization of MRSA strains in raw beef meat (Osman et al., 2015), and the available studies on RTE meat have described the occurrence of antimicrobial resistance without analyzing the biofilm formation ability, although being an important survival feature and none of these studies have analyzed the clustering and association of isolates coming from humans and RTE meat (Saad et al., 2019; Mahros et al., 2021). Here we tried to fill in these gaps by characterizing a set of MRCOPS and VRSA isolates from human and RTE meat with respect to their occurrence, antibiotic resistance profile, and biofilm formation ability.

In this study, 23.4% of RTE meat samples tested positive for S. aureus. These findings are higher than that documented in other studies on RTE meat products associated with foodborne illness where isolation rates of S. aureus were 9.5% in southern Taiwan (Wei et al., 2006), 11.8% in China (Yang et al., 2016), and 15.4% in Taiwan (Fang et al., 2003), but lower than the rates reported in chicken (40.9%) and pork meat (34.4%) from Shaanxi Province, China (Wang et al., 2014), and Qalubiya Governorate, Egypt (50.8%) (Saad et al., 2019). Our findings also revealed non-significant difference (P > 0.05) in the occurrence of S. aureus in shawarma (36.8%) and burger sandwiches (34.6%), which were lower than that reported in burger (43.3%) and nearly similar to that observed in shawarma (36.6%) in another Egyptian study (Saad et al., 2019). The differences in the microbiological quality of raw ingredient, the utensils and equipment used for food preparation, cooking, the presence of seasoning ingredients, isolation methods may contribute to the observed differences in the bacterial detection rate (Daelman et al., 2013; Wang et al., 2014). Our study reported a higher frequency of MRSA isolates from the examined COPS isolates recovered from shawarma and burger sandwiches (100%) than that obtained previously in beef burger and hot dog sandwiches (22.2%) (Mahros et al., 2021) and in other RTE meat products (25%) (Saad et al., 2019). This suggests the increased occurrence of MRSA in this kind of RTE meat. The current study showed that high number of MRSA strains were also VRSA (17/60, 28.3%). This is already higher than that obtained previously in Egypt by Mahros et al. (2021). The prevalence of MRSA (23.4%, 15 isolates/64 sample) and VRSA (6.25%, 4/64) in RTE meat samples in our study was higher than that observed (6.4% MRSA and 0% VRSA) in RTE meat (cooked pork, chicken, and duck) from China (Yang et al., 2016). In addition, a lower prevalence of MRSA (2.3% in chicken meat and 0.6% in pork meat) and VRSA (0%) were detected by Wang et al. (2014). However, Al-Amery et al. (2019) found that 4% (8/200) of camel meat samples from Egypt were positive for VRSA.

Food can become contaminated with S. aureus or MRSA from the food-producing animals or from infected food handlers during various processing stages (de Boer et al., 2009; Crago et al., 2012; Al-Amery et al., 2019). Since S. aureus is vulnerable to destruction by heating and sanitizing agents, its presence in RTE foods might be related to human contamination rather that contamination from animal origin and is indicative of poor sanitation during processing (U.S. Food and Drug Administration, 1998; Fontes et al., 2013). Generally, infected food handlers are reported to be responsible for causing 20% of bacterial foodborne illnesses. Here, the carriage rate of S. aureus in food handlers was 30% (12 isolates/40 samples), all isolates were MRSA and 25% of which was VRSA, indicating that food handlers, which are clinically healthy could pose a serious risk to public health and food safety. Other researches from different countries reported various rates of MRSA carriage on hands and noses of food handlers ranging from 1.6% in Bosnia and Herzegovina (Uzunoviæ et al., 2013), 28.6% in Brazil (Ferreira et al., 2014), and to 79% in Turkey (Sezer et al., 2015). Moreover, Ferreira et al. (2014) reported that 72.9 and 82.5% of the isolates from the handlers’ hand and nose were VRSA, respectively. However, VRSA isolates were not identified in the study done in Bosnia and Herzegovina (Uzunoviæ et al., 2013). This variation could be based on different factors including health conditions of food handlers, cleaning habits, level of education, the state of development of the country, and the regulations on inquest (Sezer et al., 2015). This suggests the importance of the continuous survey and proper hygiene of the food handlers, which could decrease contamination by this pathogen and minimize the risk of disease.

Because MRSA and VRSA have been identified in RTE food worldwide, it is supposed that it could be transmitted to humans, causing illness. This study showed that 45.8% of patients’ samples were positive for S. aureus, all isolates were of MRSA type and 30.3% of patients’ isolates were VRSA. In support of our findings, a prospective surveillance study in Egypt has reported an upsurge in the prevalence of HA-MRSA and VRSA as the rate of MRSA has doubled from 48.6 to 86.8% in a 8-year period (from 2005 to 2013) and the proportion of VRSA has increased from 4.2 to 25.8% during the same period (Abdel-Maksoud et al., 2016). Conversely, comparatively lower proportions have been reported in other countries: S. aureus isolation rate was 14.3%, MRSA was 17.5%, and VRSA was 5.1% in Ethiopia (Dilnessa and Bitew, 2016) and 63.1, 72, and 15.9%, respectively, in Eritrea (Garoy et al., 2019). The observed variation is possibly attributed to the study population and duration, types of the specimen, and the used laboratory and control procedures.

MRSA isolates pose more risk if they resist treatment by antimicrobials. Alarmingly, all MRSA isolates characterized in the current study from all sources were MDR showing resistance to clinically important antimicrobials including cefepime, penicillin, and ampicillin-sulbactam, ciprofloxacin, nitrofurontoin, gentamicin, vancomycin, erythromycin, sulfamethoxazole - trimethoprim, and clindamycin. Other studies done in Egypt also reported higher resistance rates of HA-MRSA to clindamycin (56%), gentamicin (80.7%), and erythromycin (64.4%) (Abdel-Maksoud et al., 2016). Nearly similar results were observed for penicillin and vancomycin among MRSA isolates from Ethiopian patients but higher resistance was noted for erythromycin and trimethoprim-sulfamethoxazole (Dilnessa and Bitew, 2016). Other authors have reported considerably lower frequency of vancomycin and gentamicin resistance in Eritrea (Garoy et al., 2019).

The higher resistance rates of MRSA isolates cultured from RTE meat for cefuroxime, ciprofloxacin, gentamicin, and erythromycin than those isolated from humans is consonance with results from previous studies in retail foods (Wang et al., 2014) and RTE foods (Yang et al., 2016) in China and food animals in Tanzania (Mdegela et al., 2021). Our findings are even more serious in terms of public health hazard because shawarma and burger sandwiches are consumed without further cooking, which if happened would eliminate or reduce the load of pathogen. Overall, the high frequency of MDR isolates in the study area has been related to prevail, excessive use and imprudent use of antibiotics, including in livestock husbandry, self-medication, and substandard infection control and prevention practices (Garoy et al., 2019). Accordingly, governments and individuals should pay attention to prevent further spread of MRSA and VRSA.

In addition to exhibiting antimicrobial resistance, MRSA isolates are able to produce biofilm as a defense mechanism enabling further survival in an infection niche (Archer et al., 2011). It is plausible to assume that a successful isolate would be able to form biofilm and resist antimicrobials. Our study supports this idea, where MRSA isolates with strong biofilm producers were resistant to 91.6% of the studied antimicrobials. There was also significant association between phenotypic resistance to cefuroxime, nitrofurantoin, gentamicin, vancomycin and biofilm formation. In agreement, Weigel and coauthors reported that vancomycin resistance is associated with and enhanced by the microenvironment of a biofilm (Weigel et al., 2007). These observations are similar to those reported in a study in Italy, in which food isolates of MRSA (milk and pork) have the ability to produce biofilms, as has been found previously for clinical MRSA (Vergara et al., 2017).

In S. aureus, the ica genes play a key role in biofilm formation. Strains with the icaADBC cluster especially icaA and icaD have previously been identified as potential biofilm producers (Arciola et al., 2001). These genes were detected in 92% of the biofilm-producing isolates with icaA and icaD being the most prevalent (76 and 74%, respectively). Our findings are consistent with the results of prior studies that found the ica genes in all isolates (Chen et al., 2020) especially both icaA and icaD genes (Szweda et al., 2012; Dai et al., 2019). However, Bissong and Ateba (2020) detected the ica genes in 75.3% of isolates with a lower proportion of icaA and icaD combination. These findings imply that S. aureus strains may differ in their ability to produce biofilms depending on their source (human or animal) and geographical origin. The results of this study, like those of previous research, indicated that the bap gene was not detected in biofilm-producing isolates (Szweda et al., 2012; Dai et al., 2019; Chen et al., 2020). Although not all genes involved in biofilm formation were examined, this might imply that the ica-dependent pathway is predominantly responsible for adhesion and biofilm formation in these isolates. Other biofilm-associated genes may be responsible for biofilm formation in isolates that tested negative for any of the genes investigated.

Based on the biofilm-forming capability in chronic S. aureus-related infection, some chemoprophylaxis procedures such as vaccine candidates were recently documented (Mirzaei et al., 2016, 2019, 2021; Bahonar et al., 2019; Gholami et al., 2019). Gholami et al. (2019) declared that a vaccine containing a mixture of PIA and glycerol teichoic acid (Gly-TA), biofilm related macromolecules, could be effective in preventing S. aureus biofilm formation and protect against infection. Furthermore, PIA-rSesC conjugate vaccine has been developed to raise antibodies against PIA in order to eliminate biofilm-forming S. aureus and S. epidermidis (Bahonar et al., 2019; Mirzaei et al., 2019).

In the absence of advanced sequencing technologies, a comprehensive and deep characterization of bacteria, and thus similarity among isolates from various hosts, is difficult to attain. However, studying targeted antimicrobial resistance and biofilm formation ability in a set of isolates could unravel important isolate’s characteristics, especially looking at homogenicity among isolates within and among hosts as shown in our previous investigations in Campylobacter jejuni (Abd El-Hamid et al., 2019), Aeromonas hydrophila (Tartor et al., 2021), and Escherichia coli (Ramadan et al., 2020). This could initiate hypothesizing some zoonotic aspects and transmission scenarios across hosts. Here we showed that considering both antimicrobial resistance and biofilm formation potential, all isolates within most of the S. aureus clusters (i.e., the 5 clusters) were from human and none of the RTE meat isolates have identical profiles and thus formed no clusters. In addition, only two clusters were formed that contain mixed RTE meat and human isolates. In support of this, our correlation analyses revealed certain degree of disparity between human and RTE meat isolates, where gene correlation differed between both isolates in the two sources (Figure 3). While this should be interpreted using more isolates, this initially reflects the differential use of antimicrobials in each source (i.e., humans vs. RTE meat) (Pokharel et al., 2020). It also suggests the uniqueness of within-hosts isolates and that isolates within a single host (e.g., humans in the current study) could have developed similar fitness strategies such as antimicrobial resistance or biofilm formation ability. There are some clues from previous comprehensive genomic analyses that the majority of the animal-associated S. aureus clustered into animal-specific lineages a part from human lineages (Sung et al., 2008). Moreover, human lineages of S. aureus were rarely found in animals (Sakwinska et al., 2011). This was also supported by our observation of the host-dependent correlation between presence of antimicrobial resistance and biofilm formation ability (Figure 3). These observations motivated us to perform further analyses to determine the extent of similarities and dissimilarities among isolates from human and RTE meat using estimates of average binary distance among all isolates as done previously (Abd El-Hamid et al., 2019). Our observation of high binary distances between human isolates and either burger or shawarma isolates supports the uniqueness of within-host bacterial population.

A shortcoming of this study is the small number of examined samples, which was reflected on the small number of obtained isolates ready for characterization and analyses. However, it was convenient at least as an initial study on these two important sources, to balance the sample number, the multi-sample type nature included in the study and the needed analyses. We acknowledge that the traditional characterization methods applied here are not by any way considered as alternative for advance genomics tools (e.g., whole genome sequencing), and it is becoming clear that introducing modern genomics is vital and inevitable if it comes to comprehensive characterization of bacterial population, which we are planning for in future investigation.

The high prevalence of MRSA and VRSA isolates in shawarma and burger sandwiches in the present study suggests that RTE meat could be a potential source of MRSA and VRSA isolates with significant clinical relevance. The ability of MRSA and VRSA isolates to produce biofilms and the presence of high rates of antimicrobial resistance amongst the isolates highlight the danger posed by these potentially virulent microorganisms persisting in RTE meat, food handlers, and patients. Taken together, good hygiene practices and antimicrobial surveillance plans should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA and VRSA biofilm-producing strains.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

RE-M, EA, SE-S, GE, AA, AM, and SA performed investigation, practical work, and supervision. MS performed the statistical and bioinformatics analyses, prepared the figures, and participated with YT in formulating the tables. MS and YT investigated the data and designed the research manuscript. MS, with inputs from YT, took the lead in directing the research manuscript. YT wrote the initial draft of the manuscript. All authors revised the manuscript and agreed to the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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