Ready-to-Eat Foods: A Potential Vehicle for the Spread of Coagulase-Positive Staphylococci and Antimicrobial-Resistant Staphylococcus aureus in Buea Municipality, South West Cameroon

The consumption of ready-to-eat (RTE) foods contaminated with coagulase-positive staphylococci (CoPS) and especially Staphylococcus aureus puts consumers at a potential risk of food-borne disease or colonization and subsequent infection. This cross-sectional study determined the levels of CoPS and the presence of S. aureus in RTE foods sold in Buea municipality. A total of 420 RTE food samples, comprising 70 each of cake, bread, fruit salad, meat hot-pot, suya, and boiled rice were randomly purchased from February to August 2020. The CoPS counts were determined by culturing on Baird-Parker agar, and S. aureus was identified by amplification of the nuc gene using the polymerase chain reaction. All S. aureus isolates were screened for the presence of classical staphylococcal enterotoxin genes. To determine antimicrobial resistance profiles, each isolate was tested against 11 antimicrobials. Oxacillin-resistant S. aureus strains were analyzed for the presence of the mecA gene. Overall, 161 (38.3%) samples had detectable levels of CoPS ranging from 2.0 to 5.81 log10 CFU/g. Based on CoPS levels, 37 (8.81%) of the 420 RTE food samples–only fruit salad and meat hot-pot, had unsatisfactory microbiological quality. A total of 72 S. aureus isolates, comprising 52.78% from fruit salad, 16.67% from meat hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread, and 4.17% from cake, were recovered. None of the S. aureus isolates possessed any of the classical enterotoxin genes. All the isolates were susceptible to vancomycin and ofloxacin, while 68 (94.44%) and 66 (91.67%) were susceptible to oxacillin and ciprofloxacin, respectively. Resistance to penicillin (93.06%) was highest, followed by amoxicillin (91.67%) and erythromycin (79.17%). Four isolates were identified as methicillin-resistant S. aureus, all of which carried the mecA gene. A total of 24 antibiotypes were identified. Our findings showed that RTE foods sold in the Buea municipality are likely vehicles for the transmission of CoPS and antimicrobial-resistant S. aureus.


Introduction
Ready-to-eat (RTE) foods are perishable food types that are ready for consumption, without any further heat treatment or washing, most often at the point of sale [1].Convenience, ease of production, afordability, palatability, availability, and unique favours are some of the appealing factors that make RTE foods very popular and cherished by people of all age groups and particularly workers in urban areas, students, and roadside dwellers [2][3][4].Te popularity of RTE foods varies from country to country and depends on the food type and the staple diets of the population [5].In low-and middle-income countries like Cameroon, RTE vendors have been associated with low literacy levels, leading to a lack of knowledge about good hygiene and food handling practices [3,6].Furthermore, some RTE foods are prepared, stored, and served under unhygienic conditions, and vending is often done in outdoor environments, thereby exposing the foods to aerosols, insects, and rodents, which may serve as sources of food contaminants [7,8].
Food safety in the community is a major focus area in public health [9].Tree methods, considered gold standards, have been recommended for the determination of the microbiological quality and safety of RTE foods.Tese methods are aerobic colony count, identifcation of hygiene indicator organisms, and identifcation of specifc food-borne pathogens.Although these methods can be used in combination, each of them is sufcient to provide evidence for the microbiological quality and safety of RTE foods [10].Despite having many advantages, RTE foods continue to be associated with a growing number of food-borne illnesses and outbreaks, mainly due to bacterial contamination [11,12].So far, CoPS are considered the main bacterial aetiology of food poisoning [13].
CoPS are opportunistic pathogens that can exist as commensals in humans, companions, and food-producing animals but can cause severe or even lethal diseases [14].Tey are facultative anaerobic gram-positive, nonsporeforming spherical-shaped bacteria.At least nine species of CoPS have been identifed, and they include Staphylococcus aureus, S. hyicus, S. intermedius, S. pseudintermedius, S. lutrae, S. schleiferi subsp.coagulans, S. delphini, S. argenteus, and S. schweitzeri [14,15].Although several of these Staphylococcus species can produce staphylococcal enterotoxins (SEs), the majority of staphylococcal food poisoning is attributed to SE produced by S. aureus [16,17].Te number of CoPS per gram or per millilitre of RTE food sample can be used to determine the microbiological quality and safety of RTE foods [18].
Staphylococcus aureus is one of the major bacterial agents causing food-borne disease in humans worldwide [19].Some pathogenic strains of this bacterium elaborate heat-stable SEs, and so far, 23 SEs have been identifed, comprising fve major classical types (SEs: SEA to SEE) and the nonclassical SE-like toxins (SEl: SEG to SEU) [13,20].Approximately 95% of food poisoning outbreaks are caused by classical enterotoxins [21].Ingestion of food containing SEs can induce severe symptoms, including vomiting, high fever, nausea, and diarrhoea, with rapid onset in typically less than 8 h [22].Consumption of RTE food contaminated with S. aureus has been reported in several developed countries, including the U.S.A., Hong Kong, Germany, Japan, and Italy [1].In China, several RTE food products have been associated with the introduction of microbiological hazards, including Listeria monocytogenes, Cronobacter, Salmonella, and S. aureus [23].Although evidence on food-borne illnesses in developing countries is still limited [24], cases of staphylococcal food poisoning linked to RTE foods have been reported in some parts of Africa [21] and in two major towns in Cameroon [25].It is, therefore, important to monitor and control the presence of bacterial contaminants in RTE foods [12,24].
Over the years, several pathogenic strains of S. aureus isolated from RTE food have developed resistance to one or more antimicrobials commonly used in the treatment of infection.Antimicrobial-resistant bacteria (ARB) are a major threat to global public health.In order to efectively counter the threat of infections with antimicrobial-resistant bacteria, it is critical to identify potential ways that humans can be exposed to these bacteria [26].Although foods have been reported as vehicles for the transmission of antimicrobial-resistant bacteria, the role of RTE foods in the spread of these pathogens is scantily documented [23].Some RTE foods have been reported to carry antimicrobialresistant enterococci [5], antimicrobial-resistant Staphylococcus species [27], and other bacterial species [28,29].Unfortunately, the presence of antimicrobial-resistant bacteria in RTE food is not routinely investigated in low-and middle-income countries, and data are only available from a small number of studies [21].Although the circulation of antimicrobial-resistant and multidrug-resistant bacteria among humans, animals, and the environment has been reported as a real and rising public health threat in Cameroon, there is no integrated surveillance of these superbugs in the context of One Health [30].Although foods, especially of animal origin, have been reported elsewhere as important reservoirs of antimicrobial-resistant bacteria, similar reports are rare in Cameroon.Hence, this study was carried out to investigate the levels of CoPS and determine the prevalence and characteristics of S. aureus isolated from commonly consumed RTE food samples collected in Buea, South West Cameroon.

Study Area.
Tis study was carried out on RTE foods from vendors in Buea municipality, the administrative headquarter of the southwest region of Cameroon.Buea is located on the slope of Mount Cameroon between latitude 4 °14″ north of the equator and longitude 9 °20″ east of the Greenwich Meridian, with an altitude of about 1000 m above sea level [31].Buea is a cosmopolitan and multicultural town and had an estimated population of 300,000 inhabitants in 2013 [32].Tis town is the seat of many educational institutions at the primary, secondary, and tertiary levels and is experiencing a very high rate of population growth and urbanization.At least 7,000 people relocate to Buea each year, according to the municipal council statistics [33].

Study Design and RTE Food
Types.Tis was a crosssectional study carried out from February to August 2020.Buea was purposefully selected because of its large concentration of vendors with intensive food vending activities throughout the week, from Monday through Sunday and from the early hours of the day to late evening.Tese vendors are patronized by people from all walks of life, particularly shoppers, workers, passers-by, and students.
Six types of RTE foods including fruit salad, suya (roasted beef ), cake, boiled rice, bread, and meat hot-pot (cooked meat in tomato sauce) were investigated in this (1)

Molecular Identifcation of Staphylococcus aureus.
All colonies that were used for the confrmation of CoPS were further investigated for the identifcation of S. aureus by PCR targeting the species-specifc thermonuclease (nuc) gene of S. aureus.DNA was extracted from each colony using the simple boiling method.Briefy, 150 µL of phosphatebufered saline (PBS) were pipetted into a sterile 1.5 mL Eppendorf tube, and a loopful of the bacterial colony was suspended in the PBS.Te tubes were vortexed at 14000 rpm for 10 sec.Te bacterial solution was heated in a water bath (Termo Fisher Scientifc, MA, USA) at 100 °C for 15 min after which it was removed and immediately chilled in ice for another 15 min.Te bacterial solution was allowed to thaw at 37 °C in the water bath and then chilled on ice for another 15 min.Te tubes were centrifuged at full speed (14000 rpm) for 5 min, and the supernatant was pipetted into another labelled tube and used as template DNA in the PCR assay.Presumptive S. aureus isolates were confrmed by amplifcation of a 280 bp fragment of the nuc gene of S. aureus using primer sequences previously described (Table 1).
Each PCR reaction in this study comprised 2X BioMix Red master-mix (Meridian Life Science, OH, USA) (10 μL), 1 μL of each primer from a 10 μM working stock (fnal concentration, 0.5 μM), 1 μL DNA template, and nuclease-free water (Termo Fisher Scientifc, MA, USA) to make a fnal volume of 20 µL.Each PCR run had a negative control which was nuclease-free water, and a positive control which was a previously characterized S. aureus isolate stored in the laboratory.
Te PCR cycling conditions were optimized with an initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 94 °C/60 sec, annealing at 54 °C/40sec, and extension at 72 °C/60 sec.Te fnal extension was at 72 °C for 7 min, and the reaction was stopped by holding tubes at 4 °C until removed from the thermal cycler (MyCycler ™ Termal Cycler BIORAD, USA).Te PCR products were electrophoresed in a 1.5% agarose gel stained with SYBR Safe DNA Gel stain (Invitrogen, CA, USA) for 1 h at 90 V in 1X Tris borate EDTA, visualized, and photographed with a Molecular Imager Gel Doc XR system (BIO-RAD, Hercules, CA, USA).

Detection of Classical Enterotoxin Genes in S. aureus.
All confrmed coagulase-positive S. aureus isolates were screened for the presence of S. aureus enterotoxins types A, B, C, and D using multiplex PCR [34] with primers previously described (Table 1).Te multiplex PCR run was optimized at initial denaturation for 5 min at 94 °C, followed by 30  Pure colonies (two to fve) of each S. aureus isolate from the nutrient agar plate were used to prepare the inoculum for AST.Te turbidity of the inoculum was adjusted to that of the 0.5 MacFarland standard and was used to inoculate the Mueller-Hinton agar using the spreading method.Antimicrobial disks were applied frmly on the agar surface, and plates were incubated at 35 °C for 24 h.Te diameter of the zone of inhibition was measured and interpreted as resistant, susceptible, and intermediate according to CLSI [37] guidelines.Staphylococcus aureus isolates that showed resistance to antimicrobials in three or more antimicrobials classes were considered multidrug-resistant.

Data Quality Assurance, Management, and Statistical
Analysis.Te handling and analysis of samples and bacterial isolates were done following standard operating procedures to ensure the quality and reliability of the study fndings.Te sterility of culture media was maintained and confrmed by the incubation of uninoculated culture media as a negative control.Based on CoPS count (colony-forming units/gram), the food samples were classifed as satisfactory (<20 CFU/g), borderline (20-≤ 10 4 CFU/g) or unsatisfactory (potentially injurious to health and/or unft for human consumption, >10 4 CFU/g).Data were entered into Microsoft Excel 2010, exported into SPSS V27.0.1.0,and analyzed using descriptive statistics such as prevalence, percentages, mean, and standard error.Te CoPS counts were transformed to log 10 CFU/ mL prior to statistical analysis.Data on the prevalence of S. aureus and multidrug-resistant isolates were analyzed using a chi-squared test to determine whether there were significant diferences in their prevalence between sample types.Te confdence level was held at 95% and the p value at less than 5% for all analyses.

CoPS Counts.
Te CoPS counts in the RTE foods ranged from 2.0 to 5.81 log 10 CFU/g.Overall, 161 (38.3%) of the RTE food samples had detectable levels of CoPS contamination.Based on CoPS counts, few (37, 8.81%) of the 420 RTE food samples investigated, comprising only fruit salad and meat hot-pot had unsatisfactory or unacceptable quality.More than half (259, 61.67%) of the RTE food samples had satisfactory quality (Table 3).

Prevalence of Staphylococcus aureus in Food Samples.
Molecular confrmation of S. aureus relied on the amplifcation of the nuc gene by singleplex PCR.Te amplifed PCR products showed the desired band of the required size (280 bp) when separated on a 1.5% (W/V) agarose gel (Figure 1).
Out of the 652 CoPS isolates screened, 72 (17.14%) were confrmed to possess the nuc gene and were confrmed to be S. aureus.Te 72 S. aureus isolates comprising 52.78% from fruit salad, 16.67% from meat hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread, and 4.17% from cake were recovered from 46 RTE food samples.Overall, S. aureus was detected in 10.95% of all RTE food samples examined, with some samples having more than one S. aureus isolate.Similarly, the fruit salad was the most contaminated sample with S. aureus, followed by boiled rice and meat hot-pot (Table 4).
Te mecA gene (Figure 1) was detected in 5.56% (4/72) of the S. aureus isolates from three RTE food types, while none of the S. aureus isolates possessed any of the classical enterotoxin genes (Table 4).

Antimicrobial Susceptibility of Staphylococcus aureus
Isolates.Te results of antimicrobial susceptibility testing (Figure 2) were interpreted following the guidelines published by the CLSI [37].All the S. aureus isolates were susceptible to vancomycin (100%) and ofoxacin (100%), while 68 (94.44%) and 66 (91.67%) of the bacterial isolates were susceptible to oxacillin and ciprofoxacin, respectively.On the other hand, the isolates showed high resistance rates to penicillin (93.06%), followed by amoxicillin (91.67%) and erythromycin (79.17%) (Table 5).Four isolates were identifed as methicillin-resistant S. aureus (MRSA) based on their resistance to oxacillin.Each of the MRSA isolates was resistant to at least fve antimicrobials.Of the 72 S. aureus isolates, 45 (62.5%) were multidrugresistant.In this study, multidrug resistance was defned as resistance to one antimicrobial in three or more classes of antimicrobials.

Antibiotypes of S. aureus from the Food Samples.
A total of 24 antibiotypes were identifed in this study (Table 6).Te most prevalent antibiotype was P-AML-E-AZM-C which was detected in 9 (12.5%) of the 72 S. aureus isolates.More antibiotypes were identifed in fruit salad followed by meat hot-pot.

Discussion
Te high rate of consumption of RTE foods in Cameroon and especially in the Buea municipality, coupled with the scarcity of data on the microbiological quality of these foods, is a call for concern.Elsewhere, there are several reports that highlight the role of RTE foods in the emergence or reemergence of microbial threats to health by providing information on the contamination of RTE foods by bacteria or resistant bacterial species to guide public health and food safety interventions [38].Staphylococcal food poisoning, caused by the consumption of one or more of the 23 heat-stable enterotoxins and many variants elaborated by CoPS, particularly Staphylococcus aureus, is a global public health concern [13,39].Most food types, including RTE foods, support the growth of staphylococci and are ideal for enterotoxin production [21].Staphylococcal food poisoning is usually self-limiting and resolves within 24 to 48 h after onset.
Occasionally, it can be severe and require hospitalization [1].Staphylococcus aureus is ubiquitous in the environment, a commensal in the human body, and a notorious zoonotic and major food-borne pathogen that quickly acquires resistance to antimicrobials [40,41].Terefore, this study, that investigated RTE foods for contamination with CoPS with particular focus on S. aureus and its antimicrobial-resistant profles, has both public health and food safety relevance.
Of the 420 RTE food samples analyzed in this study, 161 (38.3%) had detectable levels of CoPS contamination, with counts ranging from 2.0 to 5.81 log 10 CFU/g.Based on the CoPS counts, 37 (8.81%) of the 420 RTE food samples investigated, comprising only fruit salad and meat hot-pot had unsatisfactory or unacceptable quality (Table 3).Our fndings corroborate a previous study that analyzed 275 RTE food samples and reported that 25 (9.1%) were inappropriate for consumption according to Turkish microbiological guidelines [42].Monitoring the CoPS count in RTE foods is very helpful for a risk assessment.Te number of CoPS per gram or millilitre of RTE food sample is an indication of the microbiological quality of the food sample [18].In an earlier study that analyzed 60 RTE foods served in long-term care facilities in Spain, the highest CoPS count in the samples was 1.9 ± 0.3 log 10 CFU/g; hence, all samples had acceptable quality based on United Kingdom guidelines [43].In a very recent study that investigated 83 RTE food samples, 25% (23/83) of those analyzed were classifed as unsatisfactory or potentially hazardous [44].
In this study, fruit salad samples showed high levels (51/70, 72.9%) of contamination with CoPS (Table 2).High contamination of RTE fruit salads has been reported in previous studies.Brooks [45] reported a higher rate (90%) of staphylococcal contamination in 20 RTE salad samples analyzed in his study carried out in Calabar, Nigeria.In another recent study that analyzed 36 RTE pineapple slices in Port Harcourt, Nigeria, high levels of bacterial contamination were detected in all the samples, and the contamination levels were even higher for samples collected in the evening than for those collected in the morning from the same vendors [46].Te high levels of bacterial contamination of RTE fruits could be multifactorial and include washing the fruits with a poor-quality source of Canadian Journal of Infectious Diseases and Medical Microbiology        water; cross-contamination from other fruits washed in the same water many times [47,48]; use of dirty trays or dirty processing utensils like knives and slicing tables; not washing the hands thoroughly; and contamination from the air [46].A total of 12 (17.14%) of the 70 suya samples analyzed in this study had the lowest detectable levels of CoPS, and they ranged from 2 to 2.91 log 10 CFU/g (Table 2).While these 12 samples had borderline quality, the remaining 58 (82.86%) samples had satisfactory quality (Table 3).Hence, all the 70 suya samples analyzed in this study were suitable for consumption at the time of sample collection.Falegan et al. [49], in a recent study carried out in Ado-Ekiti Metropolis, Ekiti State, Nigeria, reported comparably higher values of 4.99-5.45log 10 CFU/g for total bacterial counts in the 20 suya samples analyzed and stated that all the samples had unsatisfactory quality.In another earlier study by Egbebi and Seidu [50] carried out in Ado and Akure, South West Nigeria, bacterial counts ranged from 4.48 to 4.60 (in Ado) and 4.48-4.93(in Akure), and these values placed the suya samples in an acceptable but not satisfactory range.Te low values of CoPS in the suya samples reported in our study could be linked to the fact that these samples were purchased shortly after preparation (i.e., freshly prepared), and not much postprocessing contamination had occurred.
In this study, the majority of the samples of boiled rice (75.71%), bread (61.43%), and cake (70.0%) (Table 3) had satisfactory quality, while 24.29% (boiled rice), 38.57% (bread), and 30% (cake) samples had CoPS counts on the high threshold but within microbiologically acceptable limits recommended by regulatory bodies for vended meat and meat products [51,52].Our results did not agree with those of Wogu et al. [53], who reported that most of the 48 rice samples from hotels in Benin City, Nigeria, examined in their study had unsatisfactory quality.A very recent study that examined 50 rice samples obtained from cafeterias at Akwa Ibom State University in Nigeria reported that most of the rice samples had unsatisfactory quality [54].Te reasons for the diferences in the results reported in these studies could be multifactorial, ranging from rice species diferences, methods of preparation, holding temperature and time, and methods of sample analysis.
A total of 72 S. aureus isolates, comprising 52.78% from fruit salad, 16.67% from meat hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread, and 4.17% from cake (Table 4) were recovered from the 46 S. aureus-positive samples in this study.Since S. aureus is heat-labile and also readily destroyed by most sanitizing agents, its presence in RTE foods may be an indication of poor handling and sanitation [38].Elsewhere, contamination of RTE foods has been linked to the preparation by small-scale local producers without quality control, the reuse of improperly washed dishes, the open-air distribution environment, and holding temperatures during distribution [60].We investigated the S. aureus isolates for the presence of the classical enterotoxin genes, and none were found to possess any of the genes.In a similar study that investigated 84 S. aureus isolates for the presence of the classical enterotoxin genes, only 2 (2.38%) of the isolates possessed the enterotoxin B gene, while the rest of the 82 isolates were negative for other enterotoxins genes [21].In another study, a high prevalence (58.1%, 18/31) of classical staphylococcal enterotoxin genes was identifed in meat samples in Zanjan, Iran [61].Although a positive PCR confrms the presence of enterotoxins, the negative PCR in our study does not point to the absence of the corresponding operon because there is always a possibility of mutation at the level of the corresponding gene.Furthermore, the differences between our results and those reported elsewhere could also be due to other factors like sample source and geographical origin [21].[60,62,63] and the notorious resistance of this bacterium to several commonly used antimicrobials [62][63][64] warranted antimicrobial susceptibility testing for all 72 S. aureus isolates identifed in this study.All the S. aureus isolates showed 100% susceptibility to vancomycin and ofoxacin, followed by ciprofoxacin (91.67%) (Table 5).Tese results agree with those of Sina et al. [65] in a study carried out in Cotonou Benin and those of Wang et al. [66], who reported that all S. aureus isolates from food in Shaanxi Province, China, were susceptible to vancomycin.Tis high susceptibility of the S. aureus isolates has great therapeutic relevance because these antimicrobials can be used to treat infections caused by this bacterium in our resource-poor community.High S. aureus antimicrobial resistance was observed for penicillin (93.06%), followed by amoxicillin and erythromycin, and our results are comparable to those reported by Tsehayneh et al. [67].Forty-fve (62.5%) S. aureus isolates in this study showed multidrug resistance, and this was lower than 98% (54 out of 55 S. aureus isolates) recently reported by Tsehayneh et al. [67].Tis diference in resistance reported may be due to the extent of usage of antimicrobials in a locality and possibly the type of samples analyzed [60].
In this study, resistance to oxacillin was used to identify methicillin-resistant S. aureus (MRSA) strains.A total of four (5.56%) of the 72 S. aureus investigated were MRSA, and all possessed the mecA gene.Saad et al. [55] reported that two (25%) of the eight S. aureus isolates identifed in their study were MRSA; Yang et al. [23] reported 10.14% (7/69) prevalence of MRSA, out of which six were mecA-positive; Naas et al. [56] reported only one (16.67%) of six S. aureus isolates as MRSA but did not screen for the presence of the mecA gene.All MRSA strains in this study were susceptible to vancomycin and, as expected, resistant to penicillin and other antimicrobials.MRSA is a pathogen of global concern and is placed on the WHO priority pathogen list [68] because infections caused by it are extremely difcult to treat.Tus, understanding the epidemiology of MRSA and other antimicrobial-resistant strains of S. aureus, in the context of the one health approach, is relevant to efective prevention and control [63], especially at this time when the antimicrobial pipeline is nearly empty [68].A total of 24 antibiotypes were identifed among the 72 S. aureus isolates in this study, and they did not show any clustering with the origin of the S. aureus isolates.

Conclusion
Tis study presents very important fndings in terms of public health and food safety because these RTE foods are consumed without further heat treatment.Te potential role of these RTE foods in the spread of CoPS and S. aureus cannot be ignored.Te contamination of these foods with CoPS, S. aureus, and even MRSA poses a potential risk to consumers.To combat the spread of antimicrobial-resistant S. aureus and particularly MRSA, it is necessary and urgent to identify all possible sources and potential pathways by which these pathogens spread.Moreover, our study also provides insight into the antibiotypes of S. aureus circulating in the study area.Tis study did not take into consideration the duration of each RTE food in exposed air at room temperature before sample collection, as this could favour the growth of microorganisms.Our results emphasize an urgent need for policies to enforce hygienic practices within the RTE food outlets for the improvement of food safety.Food vendors and food handlers should undergo training on basic food hygiene and preservation to enhance the strict implementation of the hygienic practices.To ascertain the enterotoxigenic potential of S. aureus isolates from RTE foods, the isolates should be tested for the presence of more staphylococcal enterotoxin genes.More research is also required to understand the epidemiological aspects of MRSA and S. aureus isolates in RTE foods.
Te frst draft of this manuscript was deposited on a preprint server [69].

Figure 2 :
Figure 2: Antimicrobial susceptibility test results of Staphylococcus aureus isolate on a Mueller-Hinton agar culture plate using the Kirby-Bauer disk difusion method.Te diameters of the zones of inhibition were measured and values were interpreted as susceptible, intermediate, or resistant according to the guidelines of CLSI [37].
cycles of denaturation at 94 °C for 60 sec, annealing at Canadian Journal of Infectious Diseases and Medical Microbiology 3 50 °C for 60 sec, and extension at 72 °C for 60 sec.A fnal extension at 72 °C for 5 min was done at the end of the cycles.
A singleplex PCR run was performed for the detection of enterotoxin E using the same cycling conditions except for the annealing temperature that was set at 48 °C.All PCR products were electrophoretically separated in 1.5% agarose gel as described above.2.7.Detection of mecA Gene in Methicillin-Resistant S. aureusIsolates.Staphylococcus aureus isolates that were phenotypically confrmed as methicillin-resistant were further screened by PCR for the presence of the mecA gene.Tis gene encodes a modifed penicillin-binding protein designated as PBP2a with reduced afnity for β-lactams.Te amplifcation of the 533 bp fragment of the mecA gene was optimized under the following conditions: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1 min with a fnal extension at 72 °C for 5 min, and cooling to 4 °C.Similarly, PCR products were separated electrophoretically, as described above.ciprofoxacin (CIP-5 µg), penicillin (P-10 I.U), ofoxacin (OFX-5 µg), amoxicillin (AML-30 µg), azithromycin (AZM-30 µg) and oxacillin (OX-1 µg).Antimicrobials were selected to represent diferent antimicrobial classes and also comprised commonly used and most available antimicrobials for the treatment of staphylococcal-related infections in human and veterinary medicine.

Table 1 :
Primer sequences of S. aureus used for the PCR assay in this study.Canadian Journal of Infectious Diseases and Medical Microbiology Te fruit salad samples were the most contaminated (51, 72.9%), while the suya samples were the least contaminated (12, 17.1%) (Table2).A total of 652 presumptive staphylococcal isolates were screened in the confrmatory testing for CoPS.

Table 3 :
Microbiological quality of the RTE food types based on CoPS counts.

Table 2 :
CoPS counts obtained from the ready-to-eat foods analyzed.