Toxin Production and Resistance of Staphylococcus Species Isolated from Fermented Artisanal Dairy Products in Benin

Staphylococcus species are considered as one of the major pathogens causing outbreaks of food poisoning. The aim of this work was to assess the toxinogenic and antibiotic susceptibility profiles of the strains of Staphylococcus spp isolated from three types of fermented dairy products (yoghourt, millet dêguê, and couscous dêguê). The isolation of the Staphylococcus strains was performed on selective media, and their identification was done using biochemical and molecular methods. The susceptibility at 15 antibiotics tested was assessed using the disc diffusion method. The immunodiffusion method was used to evaluate the toxin (luk-E/D, luk-S/F, ETA, and ETB) production. Biofilm formation was qualitatively researched on microplates. Less than half (42.77%) of the collected samples were contaminated with Staphylococcus spp. The yoghourt and millet dêguê samples collected in the afternoon were more contaminated than those collected in the morning. The S. aureus, S. capitis, and S. xylosus strains, respectively, were the most present. S. aureus was the only coagulase-positive species identified in our samples. The highest resistance to antibiotics was observed with penicillin (100%) irrespective of the nature of the sample. S. aureus strains were highly (71.4%) resistant to methicillin. The S. aureus strains were the most biofilm-forming (27.6%), followed by S. capitis strains. Panton and Valentine's leukocidin (luk-S/F) was produced by only S. aureus strains at a rate of 8.33%. Only coagulase-negative Staphylococcus (CNS) produced Luk-E/D. The high rates of Staphylococci contamination indicate bad hygiene quality during the production and distribution of dairy products. It is, therefore, necessary to improve the quality of fermented milk products.


Introduction
Milk and its derivative products are very important in children's diets. Fluid milk is a single food providing calcium, potassium, phosphorus, lactose, casein phosphopeptide, and vitamin D in children [1]. Thus, dietary calcium comes mainly from dairy products such as milk, yoghourt, and cheese [2,3] as well as other dairy products [4]. In West African countries, the most consumed are dèguè (millet and couscous), yoghourt, gappal, and tchobal [5]. Over the last few years, food poisoning and food safety have become very topical subjects, drawing much public concern [6]. Foodborne infections are very common and are related to the consumption of many foods including drinks sold in catering and contaminated with certain bacteria or their toxins. Milk products derived from dairy cows milk can harbor a variety of microorganisms [7] and can be important sources of foodborne pathogen [8]. Various bacteria strains (Clostridium botulinum, Clostridium perfringens, Campylobacter, Escherichia coli, Salmonella, and Staphylococcus aureus) can cause food poisoning [9]. Among the mentioned bacteria, Staphylococcus aureus is a major bacterial pathogen responsible for a broad and divergent range of human and animal infections, including toxin-mediated foodborne diseases [10,11]. S. aureus is reported to be one of the important causes of bovine mastitis and one of the most cost-intensive diseases in the dairy industry [12][13][14].
To control bacterial infections, the uncontrolled use of antibiotics by self-medication has select resistance bacteria. Unfortunately, most of the antibiotics known to date face bacterial resistance [15]. It was thus reported that methicillin-resistant S. aureus (MRSA) can form a powerful biofilm and easily colonize the mucous membranes, which is one of the reasons causing chronic, recurrent, or invasive infections [16]. Their capacity to adhere and form biofilms on the surface of milk processing equipment could contribute to be a source of S. aureus contamination of dairy production [17,18]. Recently, MRSA strains have been detected in various types of food products, including meat products, raw products, milk, and dairy products all over the world [19][20][21].
Resistance makes the treatment of bacterial infections through conventional antibiotics difficult. In addition, S. aureus is often associated with a wide variety of virulence factors such as the production of Panton and Valentine's leukocidin (PVL) and heat-resistant staphylococcal enterotoxins which, when ingested, can cause gastrointestinal disorders [22]. Dairy products generally harbor enterotoxigenic S. aureus strains that can induce foodborne intoxications in humans [23,24]. The produced enterotoxins are reported to cause abdominal cramps, nausea, emesis, and eventually diarrhea [25,26].
They are increasingly diagnosed in Benin where collective catering, which has become a phenomenon of modern societies due to its nutritional and socioeconomic importance, has considerably increased in recent years [27]. In view of the many cases of food poisoning caused by S. aureus, the increased level of methicillin-resistant and toxin producer Staphylococcus spp strains from food is to be considered. It is necessary that research be carried out in order to prevent the children health risks linked to the consumption of fermented dairy products. The aim of this study was to assess the toxinogenic and antibiotic susceptibility profiles of Staphylococci strains isolated from three types of fermented dairy products (yoghourt, couscous dêguê, and millet dêguê) sold in secondary schools in Cotonou and Abomey-Calavi in Benin.

Sampling and Sample Collection.
In this study, 15 schools in Cotonou and Abomey-Calavi were selected using the "purposive" sampling technique for the sample collections [28]. Using this sampling method, 180 samples of the three selected fermented milk products (yoghourt, millet dêguê, and couscous dêguê) were collected from vendors located inside the schools or outside within a radius of 20 meters. The collected samples include 60 yoghourt samples, 60 millet dêguê samples, and 60 couscous dêguê samples. For each type of fermented dairy products, two samples were taken twice a day (morning and evening) and twice per week. Once collected, the samples were transported into an icebox (about 4°C) to the laboratory for microbial analysis.

Isolation and Identification of Staphylococcus spp.
Staphylococcus strains were isolated on Baird-Parker agar (OXOID CM0275) enriched with egg yolk and potassium tellurite [29]. Briefly, 10 g of each collected sample was homogenized into a sterile bottle, a stomacher with 90 ml of sterile tryptone salt water. After incubation at 37°C for 24 hours, the isolated colonies were characterized by biochemical tests (catalase test, coagulase test, DNase test, and gallery API ® STAPH) [30].

Molecular Confirmation of Staphylococcus spp.
The isolated Staphylococcus strains were confirmed molecularly through the PCR. The total DNA was manually extracted using the boiling method [31]. The PCR was performed in 30 µl containing 15 µl of 2x Master Mix (Biolabs), 1.5 µl of forward primer (G1: 5′-GAAGTCGTAACAAGG-3′), 1.5 µl of reverse primer (L1: 5′-CAAGGCATCCACCGT-3′), and 3 µl of DNA. 25 cycles (94°C for 1 minute, 50°C for 30 s, and of 72°C for 1 min) was performed in a thermocycler (MultiGene, Labnet International, Inc.). The initial denaturation was done at 94°C for 5 min, the final elongation was done at 72°C for 7 min, and the amplified product was stored at 4°C until electrophoresis migration. The electrophoresis was performed at 150 V for 30 min on a 1.5% agarose gel containing ethidium bromide. A 100 bp standard molecular ladder was used. The bands of 16 S-23 S gene of Staphylococcus spp were visualized at approximately 437 bp [32] on the UV transillumination.

Phenotypic Detection of Toxins.
The production of Panton and Valentine's leucocidin (luk-S/F), leukotoxin Luk-E/D, and the epidermolysins (ETA and ETB) was investigated on isolated strains by the radial immunoprecipitation method [33]. Briefly, fresh Staphylococcus colonies were cultured in 500 µl of yeast casamino acid-pyruvate (YCP) broth at 37°C for 18-24 h with stirring (200 rpm). The supernatants of each sample of bacterial culture are collected after centrifugation (5,000 rpm for 5 min). On a 0.6% agarose gel prepared with PBS, seven wells spaced 8 mm were dug. About 30 µl of each supernatant was deposited in the corresponding external well. The appropriate purified rabbit antibodies (OD � 3) are deposited in the central rosette well and control antigens (OD � 0.2) in the top and bottom wells. The experiment was incubated at room temperature for about 16 hours. After incubation time, the precipitation arcs were observed directly or after staining with Coomassie blue [33].

Biofilm Training Research.
From an 18 h culture in brain heart infusion, 48-well microplates (polystyrene) were inoculated with 10 µl of dilute bacteria suspension to which 150 µl of BCC was added. The microplates were incubated for 24 h at 37°C. After incubation, the wells were washed three times with sterile physiological water (about 0.2 ml) to remove free bacteria (plankton). The biofilms formed by the adhesion of sessile organisms to the polystyrene support in each of the wells were stained with crystal violet (0.1%) for 10 min. The excess dye was thoroughly removed with sterile distilled water, and the plates were left at room temperature for drying. The results were compared to the positive and negative controls after incubation [34].

Antibiotics Susceptibility of Isolated Strains of Staphylococcus spp.
The susceptibility of each Staphylococcus strain to antibiotics was determined by the diffusion method [35]. The tested antibiotics (Bio-Rad) were as follows: penicillin G (P 2.6. Data Analysis. Microsoft Office Excel 2010 spreadsheet was used for data processing. The statistical analysis and graphs were made using the R 3.6.1. The test is considered statistically significant if p < 0.05.  Figure 1). Figure 2 shows the distribution of the 13 identified Staphylococcus strains according to the kind of fermented milk products. The distribution of species according to the different types of fermented milk products is not statistically significant (p > 0.05). However, it appears that S. aureus was the most isolated irrespective of the milk products. Ten different species were identified in the couscous dêguê, seven species in yoghourt, and six species in the millet dêguê. S. aureus, S. xylosus, and S. capitis were present in three kinds of products. Figure 3 shows that couscous dêguê samples were more contaminated in the morning (16.29%) than in the afternoon (14.29%). The yoghourt samples were more contaminated in the afternoon (23.38%) than in the morning (18.18%).  (Figure 4). None of S. cohnii spp cohnii, S. cohnii ssp urealyticus, and S. schleiferi produced biofilm. The Staphylococcus strains isolated from yoghourt (48.28%) made more biofilm while those isolated from couscous dêguê (17.24%) made less ( Figure 5). Biofilm production by the different isolated species in the function of sample types is statistically significant (p � 0.042).

Distribution of Toxin Production by Isolated Staphylococcus spp Strains.
The isolated Staphylococcus spp were predominantly producing epidermolysin B (ETB) at a rate of 37.5% for coagulase-negative Staphylococcus strains and 25% for coagulase-positive Staphylococcus. Luk-S/F was     produced by only S. aureus strains at a rate of 8.33%. Only coagulase-negative Staphylococcus have produced Luk-E/D ( Figure 6).
In the samples collected in the morning, only ETA and ETB toxins were produced by the Staphylococcus strains. ETB-producer strains were present in the afternoon samples. Luk-E/D and Luk-S/F toxins were produced only by Staphylococcus strains isolated in the afternoon (Figure 7).

Susceptibility of Isolated Staphylococcus spp Strains to
Antibiotics. All the Staphylococcus strains were resistant to penicillin, and 93.51% were resistant to lincomycin. About 70.13% were resistant to cefoxitin, and 60% were resistant to the antibiotics of the β-lactam family. The lowest resistance rate was observed with ciprofloxacin (22.08%) (Figure 8).

Discussion
The evaluation of the microbiological quality of fermented milk (yoghourt, millet dêguê, and couscous dêguê) products reveals the presence of total coliforms and Staphylococcus germs. This observation is consistent with the work of Kukhtyn et al. [36], who find that the greatest danger in the process of making milk and milk products is the contamination of pathogens. Dairy products are highly moisture and offer suitable growth conditions for many pathogens including S. aureus that has frequently been the cause of foodborne diseases [37]. It is thus important to implement 14  microbiological regulations for each dairy product to reduce food poisoning by fermented dairy products. The presence of S. aureus illustrates a failure in hygiene and in the implementation of good manufacturing practice. The search for these germs at the industrial level constitutes a test of overall hygienic quality. The microbiological quality of dairy products is important in the prevention of food poisoning. Thus, in this study, we observed a very high presence of total coliforms and Staphylococci. It emerged from our investigations that 36.36% of the strains of Staphylococci isolated were coagulase-positive (Staphylococcus aureus) and 63.64% were coagulase-negative (Figure 1). The presence of S. aureus in dairy products could alter the microbiological quality of these products and cause food poisoning [38]. Worldwide, S. aureus has been isolated from several ready-to-eat products. The presence of those microorganisms indicates potentially cross-contamination that may be due to improper staff hygiene and/or poor surface sanitation. Indeed, S. aureus strains were isolated from food handlers, foods, and patient specimens [39]. This microorganism is capable of surviving on dry stainless steel, and it can easily be transferred from sponges to stainless steel surfaces and subsequently to food products [40]. In milk transformation, S. aureus can contaminate the production at almost every step. Therefore, S. aureus can be shed directly into the milk and produce enterotoxins that represents one of the most common food safety concerns from raw milk products [41][42][43].
As in our study, some authors observed in the United States [44], Egypt [45], and Morocco [46] diversity in the presence of coagulase-negative Staphylococcus strains. The ones commonly detected in raw milk are S. epidermidis, S. simulans, S. hominis, S. caprae, S. warneri, and S. xylosus [44,47,48]. In our study, the most dominant coagulasenegative strain was S. xylosus. But Organji et al. [48] observed in their study on milk in Egypt that S. saprophyticus was the most dominant coagulase-negative strain. This difference in proportion can be explained by the fact that our samples were not fermented enough because S. saprophyticus plays a role in the fermentation process [49]. In this respect, probably no health risks are associated with all Staphylococci detected in fermented dairy products samples. Considering the collection period, the yogurt samples are most contaminated by Staphylococci in the 27  afternoon (23.38%) than in the morning (18.18%). This result is lower than the results found by Tondo et al. [50] who found 90.4% contamination of S. aureus in raw milk taken in the morning. This difference could be explained by the fermentation and pasteurization processes that yogurt undergoes.
Concerning the formation of biofilm capability, S. aureus displays the highest rate (27.6%). In addition, the yogurt samples were those that contained more strains (48.28%) of biofilm-forming Staphylococci. Indeed, S. aureus can produce a multilayer biofilm incorporated into a glycocalyx with the expression of heterogeneous proteins overall, forming at least two types of biofilms: ica-dependent and ica-independent [51]. The formation of biofilm by foodborne staphylococcal strains (especially yogurt) is very serious for human prognosis, especially for children who are the biggest consumers of it. This biofilm formation is more observed in clinical strains [52]. Biofilm formation in dairy equipment as well as insufficient acidification during fermentation opens S. aureus niches for multiplication and efficient contamination of the dairy processing lines [17].
The pathogenicity of Staphylococcus strains is attributable to toxin production and their antibiotic-resistant profile [53]. Toxin research revealed that Panton and Valentine's leucocidin was produced (8.33%) only by S. aureus. The most produced toxin is epidermolysin B (62.5%) by all strains of Staphylococcus. This result is higher than the 27.59% founded by Ahouandjinou et al. [54] among the Staphylococci strains isolated from bovine carcasses in Benin. The PVL production obtained in our study is lower than the 15% obtained in Benin by Baba-Moussa et al. [55] for direct debits of all origins. Baba-Moussa et al. [56] showed that 21.50% of the S. aureus strains isolated from CHU infections produced PVL. This toxin, in clinical practice, is associated with skin diseases such as boils and abscesses [57]. Thus, the production of PVL by food strains must also challenge us especially in terms of its virulence. This presence may be due to the carrying of these types of ailments by sellers that would facilitate the transmission from humans to food. S. aureus is able to survive on dry stainless steel and it can easily be transferred from any surfaces to food products [40]. Therefore, in the milk transformation process, S. aureus can contaminate the production at almost every step. S. aureus can be shed directly into the milk and produce exotoxins that represents one of the most common food safety concerns from raw milk products [41][42][43]58]. In addition, toxins can be produced in the product during storage of the fermented dairy product if conditions allow the growth of S. aureus.
The study of the sensitivity to antibiotics of isolated Staphylococcus strains showed the existence of resistance, 48  with variable proportions according to the families of antibiotics. In fact, the Staphylococcus spp show a high resistance rate to penicillin (100%), lincomycin (93.51%), and cefotaxime (90.91%). These results are consistent with those reported on staphylococcal strains isolated from street foods in Benin [10] and Ethiopia [59]. This high resistance level to antibiotics observed can be due to self-medication and excessive and uncontrolled use of antibiotics. The lowest resistance of S. aureus observed was with ciprofloxacin (28.6%). This result is close to the 31.3% obtained in studies carried out on milk by Wang et al. [60]. About 71% of the S. aureus were resistant to methicillin (cefoxitin). These results are higher than the 15.18% found by Sina et al. [10]. The coagulase-negative staphylococcal strains displayed methicillin resistance rates of 69.4%. Our results concerning this antibiotic are slightly lower than those reported for clinical strains in Turkey on coagulase-negative Staphylococci [61]. This difference may be caused by the fact that clinical strains are more in contact with the antimicrobial molecule than food strains. The Staphylococcus spp. methicillin resistance observed in this study suggests that it involves the resistance of most of the β-lactams currently available [62]. However, the proportion is frightening for food since it is reported that methicillin-resistant Staphylococcus strains began to develop resistance to many antibiotics (quinolones, macrolides, aminoglycosides, tetracyclines, trimethoprim-sulfamethoxazole, clindamycin, and chloramphenicol) widely used to control staphylococcal infection [63,64], such as food poisoning. The sellers who do not protect their hands and head and do not observe hygiene rules during the manufacturing of dairy products quickly find themselves contaminated by potentially pathogenic Staphylococcus strains that are antibiotics-resistant, 8  producing toxins, and forming biofilms. This situation is a real problem for food safety.

Conclusion
Staphylococcal food poisoning is of major concern in public health programs worldwide. Results clearly indicated that the fermented milk products analyzed were contaminated with 13 species of Staphylococcus, namely, S. aureus. Human, animal, and environmental sources could be incriminated in the contamination of dairy products. Resistant and toxinproducing strains were also isolated from the collected fermented milk products. There is a high risk of food poisoning associated with the consumption of those dairy products. We can say that good hygienic and manufacturing practices along each step of the selected dairy fermented production chain are of great importance and necessary to eliminate the dissemination of potentially pathogenic Staphylococci in the community. Thus, more measures focusing on hygienic prevention are required to reduce contamination by Staphylococci.

Data Availability
The data are available from the corresponding author upon request.

Conflicts of Interest
The authors declare there are no conflicts of interest in the publication of this manuscript.  Resistance rate (%) Tested antibiotics CNS CPS Figure 9: Antibiotic resistance rate of strains of negative coagulase Staphylococcus spp (NCS) and S. aureus (CPS) isolated.