Prevalence, Enterotoxigenic Potential and Antimicrobial Resistance of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Algerian Ready to Eat Foods

Staphylococcus aureus causes a foodborne intoxication due to the production of enterotoxins and shows antimicrobial resistance, as in the case of methicillin-resistant strains (MRSA). Herein, we analyzed 207 ready-to-eat foods collected in Algeria, reporting a S. aureus prevalence of 23.2% (48/207) and respective loads of coagulase positive staphylococci (CPS) ranging from 1.00 ± 0.5 to 5.11 ± 0.24 Log CFU/g. The 48 S. aureus isolates were widely characterized by staphylococcal enterotoxin gene (SEg)-typing and 16S-23S rDNA intergenic spacer region (ISR)-PCR, as well as by detecting tst and mecA genes, genetic determinants of toxic shock syndrome toxin-1 and methicillin resistance, respectively. We found that the S. aureus isolates belonged to seven different SEg-types harboring the following combinations of genes: (1) selW, selX; (2) egc (seG, seI, seM, seN, seO), selW, selX; (3) seA, seH, seK, seQ, selW, selX; (4) seB, selW, selX; (5) seD, selJ, seR, selW, selX; (6) seH, selW, selX, selY; and (7) seA, egc, selW, selX, while among these, 2.1% and 4.2% were tst- and mecA- (staphylococcal chromosomal cassette mec-type IV) positive, respectively. Selected strains belonging to the 12 detected ISR-types were resistant towards antimicrobials including benzylpenicillin, ofloxacin, erythromycin, lincomycin, tetracyclin, kanamycin, oxacillin, and cefoxitin; 8.3% (1/12) were confirmed as MRSA and 16.7% (2/12) were multidrug resistant. The present study shows the heterogeneity of the S. aureus population in Algerian ready-to-eat foods as for their toxigenic potential and antimicrobial resistance, shedding the light on the quality and safety related to the consume of ready-to-eat foods in Algeria.


Introduction
Food-associated health problems, caused by food contamination or malnutrition, have a worldwide impact on public health and economy [1]. Particularly, foodborne diseases (FBDs) represent a major issue for public health, and there is a growing number of outbreaks  94 1.00 ± 0.5-5.11 ± 0.24 a Coagulase positive staphylococci count expressed as Log CFU/g ± standard deviation in S. aureus positive samples. The count is expressed as median ± standard deviation for the categories "meat/fish-based foods", "vegetable-based foods", "pastries", and "cereals" where more than one S. aureus positive sample was found, and "total". b Minimum and maximum counts of coagulase positive staphylococci found in S. aureus positive samples, expressed as Log CFU/g ± standard deviation.

16S-23S rDNA Intergenic Spacer Region (ISR)-PCR, Staphylococcal Enterotoxin Gene (SEg)-Typing and Presence of tst and mecA Genes
The 48 S. aureus isolated from the ready-to-eat foods sampled in Algeria are listed in Table 2. Minced meat c I 1 --SA54 Beet salad b I 1 -- Vegetable and meat sauce c Sautéed beef with potato c XII 2 The 16S-23S rDNA intergenic spacer region (ISR)-PCR allowed us to identify 12 different ISR-types, as shown in Figure 1.
It is noteworthy that S. aureus isolates belonging to the same ISR-type harbored the same genes as detected by PCRs (Table 2).
Lastly, the distribution of the S. aureus in relation to their genotypes and the corresponding sources of isolation is displayed in Table 3.

Characterization of the Staphylococcal Chromosomal Cassette mec (SCCmec)
The multiplex PCR used for the characterization of the staphylococcal chromosomal cassette mec (SCCmec) [30] showed that the two mecA positive isolates (Table 2)

Characterization of the Staphylococcal Chromosomal Cassette mec (SCCmec)
The multiplex PCR used for the characterization of the staphylococcal chromosomal cassette mec (SCCmec) [30] showed that the two mecA positive isolates (Table 2)

Discussion
In the present study, we found S. aureus in Algerian ready-to-eat foods with a prevalence of 23.2%. Other studies analyzed the presence of this microorganism in ready-to-eat foods in African countries, but only fragmented and incomplete information about the enterotoxigenic S. aureus and MRSA isolated from African ready-to-eat foods is available so far. Similar to our findings, Chaalal et al. [31] reported an overall S. aureus prevalence of 23.8% in pastries and cooked dishes sampled from supermarkets and university cities in Western Algeria, and Titouche et al. [32], in the same country of the aforementioned study, found a prevalence of 14.46% in pastries collected from several market points (in Tizi Ouzou area), while no S. aureus contamination was found by these authors in the sampled sandwiches [32]. A prevalence of 33.26% was reported in ready-to-eat meat products collected in all the provinces of South Africa [33], while in Egypt (in Benha city), a prevalence of 50.8% was reported for ready-to-eat meat products sampled from restaurants and street vendors [34], and in Nigeria (in Port Harcourt Metropolis), 100% of the analyzed street ready-to-eat meals were reported to be contaminated by S. aureus [35].
As shown by these studies, the prevalence of S. aureus contamination in ready-to-eat foods may greatly vary and, as highlighted by some authors [32,36], such differences are related to several factors that include the source (e.g., street-vendors or shops) and type of samples (e.g., animal-or non-animal-derived foods), the sample size, the accuracy of the identification method (based on cultivation characteristics, biochemical tests, or molecular biology techniques), the manufacturing procedures (e.g., involving bactericidal temperatures or not), and the overall hygienic measures that are implemented during the preparation and handling of the foods. In particular, S. aureus, being a commensal bacterium present on the skin, the nose, and mucous membranes of animals and humans [28], may contaminate food especially when poor hygienic practices and conditions exist. It is noteworthy that, beyond the contamination that may originate from animals at the primary production stage, food handlers are recognized as the main source of food contamination with S. aureus [31,32,37], and Sezer et al. [38] found that the 79% of the food handlers employed in a catering establishment were carriers of S. aureus. Nevertheless, it should be taken into account that contamination may also happen and spread by cross-contamination among foods and/or surfaces in the environment surrounding the manufacturing and storage of the ready-to-eat foods, since S. aureus may survive on inanimate surfaces for prolonged times [39], especially in cases of improper or ineffective sanitizing procedures [40].
When contamination occurs, S. aureus may replicate, and its load may increase in food. S. aureus is mostly a coagulase positive bacterium, and according to the Interministerial Decree of the Algerian Republic Official Journal No 39 (published on 2 July 2017) establishing the microbiological criteria for food products, the limits of CPS in ready-to-eat foods are set to values equal to 10 2 CFU/g ("m" value), below which the product quality is considered satisfactory, and 10 3 CFU/g ("M" value), above which the product quality is considered unacceptable. In the ready-to-eat products sampled in this study, we found CPS loads, above the "M" value, in four of the five analyzed food categories (meat/fish-based foods, vegetable-based foods, pastries, and cereals), in which the highest CPS counts reached values higher than 4 and 5 Log CFU/g. Additionally, according to the Decree, samples in which CPS counts are superior or equal to 10 5 CFU/g are considered as toxic. Therefore, these findings shed light on the quality and safety of the Algerian retail ready-to-eat foods.
Some other studies assessed CPS loads, even reported as S. aureus counts, in readyto-eat foods in the African continent. Although Mahami et al. [41] found low contamination of cooked and smoked sausages, reporting S. aureus loads ranging from 1.85 to 2.15 Log CFU/g in samples purchased from a factory in Ghana (in Accra city) and less than 1 Log CFU/g in samples from a shopping mall, Oguttu et al. [42], analyzing readyto-eat chicken sold in informal markets in South Africa (in Tshwane Metropolitan City), reported the presence of unsatisfactory quality samples with loads of S. aureus greater than 3 Log CFU/g, and an overall mean of 3.6 Log CFU/g. Similarly, Shiningeni et al. [43] found ready-to-eat beef and chicken meats of unsatisfactory quality, with loads of S. aureus equal or greater than 3 Log CFU/g, that were purchased from street vendors in Namibia (Windhoek city). These authors reported that, in relation to the vending sites, the mean counts in the ready-to-eat meat samples ranged from 0 to 3.46 Log CFU/g, and the highest load was 5.12 Log CFU/g [43].
Since the issue regarding S. aureus and corresponding loads in foods is related to the possible contamination of food by staphylococcal enterotoxins, that in very round figures may occur at hazardous levels if S. aureus load reaches around 5 Log CFU/g [40], a great importance was given in this study to the investigation of the enterotoxigenic potential of the S. aureus we isolated, and to the best of our knowledge this is the first study in Algeria in which the presence of a total of 27 se and sel genes was sought in foodborne S. aureus.
In our study, we found that 100% (48/48) of the analyzed S. aureus harbored selW and selX. These are located in the chromosome and are reported as highly prevalent genes; indeed, they were detected in more than 92.0% and in more than 79.0% of the S. aureus analyzed in previous studies, respectively [46,47]. The recent identification of this and other new se and sel genes indicates that the pathogenic potential of S. aureus may be greater than previously thought [48]. Moreover, based on the findings of Aung et al. [46], it was suggested that selW, and to a greater extent selX, may play a universal role in the virulence of S. aureus [46].
The enterotoxin gene cluster (egc) was found in the 18.8% (9/48) of the S. aureus herein analyzed. This cluster, located in a variable genomic island (vSaβ) inserted in the chromosome [37], has been similarly reported with a prevalence of 16.2% [49] and of 26.4% [13] in S. aureus from various sources, including food. In our study, the presence of the egc was confirmed by the detection, in the same strains, of the egc encoded genes seG, seI, seM, seN, and seO. Various types of egc exist in S. aureus (egc1 to egc5) and, considering our findings, the strains herein analyzed may harbor egc1 [50] or the newly described egc5 [13]. It should be specified that these two egc types, beyond harboring the enterotoxin genes we detected (seG, seI, seM, seN, seO), might also include two pseudogenes (ψent1-ψent2) (egc1) or selU2 (egc5) [13,50].
Since selU2 results from a single adenine deletion in the ψent1-ψent2 region (being just a 1 nucleotide frameshift of the ψent1-ψent2 sequence that deletes the stop codon of pseudogene ψent1) [51], the design of primers able to specifically detect ψent1-ψent2 or selU2 is not achievable, making impracticable their easy and affordable detection by a PCR screening such as the one we performed.
The seH gene was detected in the S. aureus herein analyzed with a prevalence of 16.7% (8/48). This gene, located on a presumptive transposon [52], was reported with lower prevalence in some previous studies, being in the range of 4.05-4.8% in S. aureus from food and other sources [32,45], while a higher prevalence, more similar to our findings, and ranging between 24.6 and 28.0%, was reported by other authors analyzing S. aureus from raw milk, raw meat, and ready-to-eat foods [13,44,53].
The egc-encoded enterotoxins are reported with increasingly probability to have a role in staphylococcal foodborne poisoning (SFP) [54,55], and the seH encoded enterotoxin (SEH) is the first non-classical SE that has been reported to have caused SFP outbreaks [56,57], highlighting therefore the relevance of the detection of these genes in foodborne S. aureus.
In our investigation, we also detected selJ, seK, seQ, seR, and selY. In general, scarce knowledge is available on the prevalence of these new se and sel genes in foodborne S. aureus. This study helps to improve this paucity of data, representing one of the few African reports available to date. Interestingly, we observed that some of the abovementioned genes were found in association in the S. aureus herein analyzed, in particular, selJ and seR along with seD, and seK and seQ along with seA. The first association is carried on plasmids (pIB485-like), and the second is carried on prophages [37], and both of these associations are being found in S. aureus isolates from SFP outbreaks [61][62][63][64]. Considering that the emetic properties of the encoded SEs (SEK, SEQ, and SER) have been demonstrated [65,66], these new se genes may also have a role in the genesis of staphylococcal intoxication that to date is still not fully understood.
tst and mecA genes were found at low prevalence in the S. aureus analyzed in our study (2.1% (1/48) and 4.2% (2/48), respectively), and they were not in association in the same strains. These genes, being the genetic determinants of toxic shock syndrome toxin-1 (tst), which causes a range of systemic diseases, and methicillin resistance (mecA), which confers a general resistance to β-lactam antibiotics in MRSA, represent important virulence and antimicrobial resistance attributes that can worsen clinical conditions when S. aureus infections occur.
In general, a low prevalence of tst gene in foodborne S. aureus has also been reported in previous studies conducted in various parts of the world including African countries [31,[67][68][69]. Although the association of tst and mecA genes has been found in S. aureus isolated especially from clinical samples [70,71], our findings resemble those of Chaalal et al. [31] that recently analyzed S. aureus from food products in Algeria detecting a low prevalence of tst gene (3.2%) that was found only in mecA-negative isolates.
Nevertheless, the presence of mecA has been reported in foodborne S. aureus isolates in Algeria, with a prevalence of 3.03 [72], 4.81 [32], and 16.9% [31], and, additionally, in the aforementioned studies, an overall low prevalence of MRSA in the analyzed foods has been reported, which is consistent with our findings [31,32,72]. The massive and inappropriate use of antimicrobials in veterinary and human medicine is considered the main cause for the emergence of antimicrobial-resistant strains [73]; however, the prevalence of MRSA in food is described to still be low and reported in the range of 1.6-6.4% in investigations also conducted in other countries [74].
Transmission of MRSA occurs between animals and humans but, notably, food contamination may serve as a vehicle to increase the dissemination of MRSA [73]. The communityacquired MRSA (CA-MRSA) but also the livestock-acquired MRSA (LA-MRSA) frequently carry the SCCmec-type IV [75,76], and they principally spread in human community settings and livestock populations. The detection of the SCCmec-type IV in our S. aureus isolates tentatively suggests their origin and, consistently with our findings, it was also the prevalent SCCmec-type found in the MRSA isolated from food in previous studies [31,[77][78][79].
Interestingly, the ability of MRSA strains to act as a foodborne pathogens has been reported [80], and the first foodborne gastrointestinal illness outbreak caused by an enterotoxigenic MRSA strain has been described in 2002 [81]. However, to date, the actual MRSA involvement in SFP (cases or outbreaks) is not precisely elucidated, and a general underestimation is suggested [80]. Our mecA-SCCmec-type IV positive isolates harbored seH, selW, selX, and selY genes (SEg-type 6) suggesting their potential role as SFP agents, especially considering that the seH encoded enterotoxin (SEH) has been already reported to have caused SFP outbreaks [56,57], as mentioned above. Additionally, the association of seH and mecA gene is known and has been previously described [52]. Moreover, our results also corroborate those of previous studies that detected se and sel genes in MRSA isolated from food products [32,77,79,82].
To further characterize our S. aureus isolates, we employed ISR-PCR, previously used also by other authors as a practical tool for S. aureus genotyping [13,83]. Interestingly, unlike results previously reported by Chieffi et al. [13], we observed that ISR-PCR had a discriminatory power higher than SEg-typing since ISR-PCR enabled us to detect 12 ISRtypes despite the seven SEg-types. On the other hand, we observed that S. aureus belonging to the same ISR-type harbored the same genes as detected by PCRs, corroborating the aforementioned study that reported that strains belonging to the same ISR-type showed the same SEg-type.
Therefore, ISR-PCR allowed us to select 12 representative genetically diverse S. aureus strains that, when tested for antimicrobial resistance, showed four patterns of resistance.
In particular, the antimicrobial susceptibility test allowed us to confirm one selected strain (1/12, 8.3%) as MRSA that is in agreement with the detection in the same strain of the mecA gene. Moreover, two selected strains (2/12, 16.7%) were classified as multidrugresistant, being resistant to three (i.e., benzylpenicillin, kanamycin, and tetracycline) and four (i.e., ofloxacin, erythromycin, lincomycin, and tetracycline) antimicrobials belonging to different classes.
Multidrug-resistant S. aureus isolated from food products were detected also in previous studies conducted in Africa and in other parts of the world, whose reported prevalence ranged between 15.38-33.3% [31,32,84] and 10.4-57.5% [85,86], respectively. Such findings represent a threat to public health since the assortment of effective antimicrobials to treat S. aureus infections is reduced. Moreover, as for MRSA, these multidrug strains also harbored se and sel genes belonging to SEg-type 2 [egc (seG, seI, seM, seN, seO), selW, selX] and SEg-type 4 (seB, selW, selX), being therefore enterotoxigenic strains with the potential to cause SFP. Interestingly, the presence in food products of multidrug-resistant S. aureus strains carrying se and sel genes has also been reported by other authors [87].
The resistance to penicillin is reported with high prevalence; indeed, from 60% to more than 90% of foodborne S. aureus isolates showed penicillin resistance in many studies [31,32,85,86,88], which is consistent with the high prevalence of resistance observed also in our investigation (10/12, 83.3%).

Conclusions
The information on foodborne S. aureus in Africa is, to date, fragmented and still incomplete, calling for studies that can cover this lack of data from most of its countries. Herein, the detailed molecular characterization, in particular aimed to investigate the actual enterotoxigenic potential of the analyzed S. aureus isolates, along with the findings of MRSA and multidrug-resistant strains, highlight the pathogenicity as well as the heterogeneity of S. aureus population in Algerian ready-to-eat foods. Additionally, the finding of generally high CPS loads in the analyzed ready-to-eat foods, with respect to the microbiological criteria established by the Algerian legislation, draws attention to the quality and safety of such foods in Algeria.
Therefore, S. aureus contamination represents a current risk for consumers' health, and ready-to-eat foods seem to still be far from the relevant Algerian standard, but we should point out that better hygienic practices during the manufacture of ready-to-eat foods may be an affordable strategy to successfully address these current issues.

Sampling
A total of 207 samples of ready-to-eat foods were randomly collected from hotels, restaurants, fast foods, and pizzerias in Algiers, capital of Algeria, during 2018 and 2019. Those samples included 55 meat-based foods, 72 vegetable-based foods, 43 pastries, 17 cereals, and 20 various foods (milk-and egg-based foods). The samples were transported on ice to the laboratory and analyzed immediately.

Isolation of Presumptive Staphylococcus aureus
The analysis was carried out using 25 g of homogenized food in 225 mL of preenrichment diluent tryptone-salt broth (Oxoid, Dardilly, France) using a Stomacher-type homogenizer. Further decimal dilutions were carried up to 10 −5 . Thereafter, the corresponding dilutions were plate-counted in accordance with the standard reference culture method recommended by the International Organization for Standardization [91] for the enumeration of coagulase-positive staphylococci, using Baird Parker with egg yolk emulsion (BPEY) incubated at 37 • C for 24-48 h. From each food sample processed, one presumptive Staphylococcus aureus colony was subcultured on BPEY (Oxoid, Dardilly, France) and purified by repeated streaking. The pure cultures were stored at −80 • C in brain heart infusion broth (BHI; Conda Pronadisa, Madrid, Spain), amended with 0.6% yeast extract (Biolife Italiana, Milano, Italy) added with 20% glycerol.

DNA Extraction
The presumptive S. aureus pure cultures were cultivated in BHI broth (Oxoid, Dardilly, France) amended with 0.6% yeast extract (Biolife Italiana, Milano, Italy) and incubated at 37 • C for 24 h. Five hundred microliters of each broth culture were centrifuged at 12,000 rpm for 90 s, and the resulting pellets were washed with 1 mL of sterilized distilled water. DNA was extracted with InstaGene Matrix (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions. Three to five microliters of the resulting DNA solutions were used for the conventional polymerase chain reaction (PCR) protocols, while two microliters were used for the real time PCR protocols, as described below.

Identification of Staphylococcus aureus
The identification of Staphylococcus aureus isolates was carried out by a species-specific simplex polymerase chain reaction (PCR) targeting the nuc gene [92]. Briefly, each reaction Conventional and real time PCR assays targeting the classical (seA to seE) and the newly described (seG to selZ, sel27 and sel28) se and sel genes were carried out following the protocols described by Chieffi et al. [13], while the presence of the egc was assessed using the SYBR Green real-time PCR protocol described by Fusco et al. [11]. Amplicons obtained by conventional PCR were separated on agarose gel by electrophoresis in TAE buffer at 100V. S. aureus strains used as controls in the PCR assays are reported in Table S1 [13,50,[93][94][95][96][97].

16S-23S rDNA Intergenic Spacer Region PCR (ISR-PCR)
16S-23S rDNA intergenic spacer region PCR (ISR-PCR) was carried out using 5 µL of each S. aureus DNA following the protocol described by Chieffi et al. [13]. DNA of S. aureus DSM20231 T was also included in the PCR reaction as control reference strain. The resulting patterns were visualized as described by Fusco et al. [98], on agarose gel (1.7% w/v) electrophoresized in TAE buffer at 60 V for 6.5 h. Two patterns were considered different ISR-types if one or more DNA bands differed in size. The relevant analysis was carried out to construct an UPGMA (unweighted pair group method with arithmetic mean) dendrogram using the Bionumerics software version 5.1 (AppliedMaths, Sint Martens Latem, Belgium) [99], setting the Pearson correlation as a fingerprint similarity coefficient and choosing 1.0% position tolerance and 0.0% optimization as position tolerance settings.

Detection of tst and mecA Genes and Characterization of the Staphylococcal Chromosomal Cassette mec (SCCmec)
Five µL of S. aureus DNA were employed in the multiplex PCR protocol described by Oliveira and de Lencastre [30] for the detection of the mecA gene and the characterization of the SCCmec as well. The resulting amplicons were separated, loading 2 µL of the reaction mixture on agarose gel (2% w/v) and performing electrophoresis in TAE buffer at 100 V for 1 h 50 min. The tst gene was detected as described by Johnson et al. [100] with minor modifications in the reaction mixture that was prepared as follows: 3 µL of the extracted DNA, 0.4 µM of each primer, 2.5 mM of MgCl 2 (Promega, Madison, WI, USA), 0.25 mM of each deoxynucleotide triphosphate (Promega, Madison, WI, USA), 1 U of GoTaq G2 hot start polymerase (Promega, Madison, WI, USA), 1X reaction buffer (Promega, Madison, WI, USA), and nucelase-free water to a final volume of 25 µL. Amplicons were separated by electrophoresis in TAE buffer at 100 V on agarose gel (1.3% w/v). S. aureus strains used as controls in the PCR assays are reported in Table S1 [13,50,[93][94][95][96][97].

Antimicrobial Susceptibility Testing
Twelve S. aureus strains belonging to the different ISR-types were selected for testing ( Table 4).
The pure cultures of each strain were cultured in Brain Heart Infusion (BHI) agar (Conda Pronadisa, Madrid, Spain) and incubated at 37 • C for 24 h. Suspensions of 0.5 McFarland were prepared from these cultures.
Antimicrobial susceptibility was performed by Vitek 2 (bioMérieux Inc., Durham, NC, USA) using AST-P631 cards. The cards were inoculated with the prepared suspensions and loaded into the Vitek 2 automated reader. Results were interpreted by the Vitek 2 Advanced Expert System software according to the reference criteria of CLSI and EU-CAST [101,102]. S. aureus DSM 20231 T was used as control. Strains resistant to cefoxitin and oxacillin were considered MRSA, and strains resistant to ≥3 antimicrobials of different classes were considered multidrug-resistant. The following antimicrobial agents were included: benzylpenicillin, oxacillin, cefoxitin, gentamicin, kanamycin, tobramycin, ofloxacin, erythromycin, lincomycin, clindamycin, pristinamycin, linezolid, teicoplanin, vancomycin, tetracycline, fosfomycin, nitrofurantoin, fusidic acid, rifampicin, and co-trimoxazole.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/toxins13120835/s1, Table S1: S. aureus strains used as controls in conventional and real time PCR assays in the present study.