Graduate Student Literature Review: Enterotoxigenic potential and antimicrobial resistance of staphylococci from Brazilian artisanal raw milk cheeses*

More than 30 types of artisanal cheeses are known in Brazil; however, microorganisms, such as Staphylococcus spp., can contaminate raw milk cheeses through different sources, from milking to processing. Staphylococcal food poisoning results from the consumption of food in which coagulase-positive staphylococci, mostly Staphylococcus aureus , have developed and produced enterotoxins. In addition, an emerging public health concern is the increasing antimicrobial resistance of some Staphylococcus strains. Furthermore, the ability of Staphylococcus spp. in sharing antibiotic resistance-related genes with other bacteria increases this problem. In light of these observations, this review aims to discuss the presence of, enterotoxins of, and antibiotic-resistant of Staphylococcus spp. in Brazilian artisanal cheese produced with raw milk.


INTRODUCTION
Brazil stands out for being the fourth largest cheese producer in the world, producing approximately 804 thousand tonnes of cheese per year, with this number in continuous growth (ABIQ, 2021).More than 30 types of artisanal cheeses are known in Brazil.Brazilian artisanal cheeses are those made by traditional methods that have territorial, regional, or cultural attachment and value and that respect the elaboration protocols established for each variety, generally produced from raw milk (Brazil, 2019a).
The current trend toward the consumption of natural, less processed, and clean label products is related to the potential consumption of raw milk in countries where this regulatory possibility exists (e.g., United States, Italy).Although, in Italy, for example, it is recommended to boil raw milk before consumption.This is supported by a biased public perception that raw milk is associated with nutritional benefits which are supposedly lost after thermal processing.In the same way, cheeses produced with raw milk gain relevant importance in this context.Due to the need to regulate the production of artisanal cheese made from raw milk, the Brazilian Ministry of Agriculture, Livestock, and Food Supply (MAPA) published the Normative Instruction No. 30 dated August 7, 2013 (Brazil, 2013).This regulation allows the production of artisanal cheese made from raw milk with less than 60 d of ripening if it meets some requirements, such as technical-scientific studies proving that the reduction in ripening time does not affect product quality and safety (Brazil, 2013).In addition, Decree No. 9,013 dated March 29, 2017, which provides regulation for the industrial and sanitary inspection of animal-based products, excludes the obligation to pasteurize milk meant for production of cheese when exposed to a ripening process at a temperature above 5°C during a period of 60 d or less, if safety is proven through analytical tests (Brazil, 2017).
The concern of the regulatory agencies with the minimum ripening time derives from the potential for the development of pathogenic bacteria in raw milk products.Pathogens can contaminate raw milk cheese through different sources, such as the raw milk itself, but also the dairy environment (from milking to processing) and people having close contact with milk and cheeses during production and distribution (Rola et al., 2015).Among the microorganisms considered as risks for the safety of this food, the foodborne pathogens Salmonella spp., Campylobacter spp., Escherichia coli pathotypes, Listeria monocytogenes, and Staphylococcus aureus can be mentioned.These pathogens are responsible for some of the morbidity and mortality in the world, resulting in significant damage to socioeconomic development worldwide (WHO, 2015).
According to data from the Foodborne Diseases Active Surveillance Network (FoodNet), which monitors the incidence of foodborne diseases (FBD) in the United States, a total of 18,462 cases of FBD were recorded in 2020, with 4,788 hospitalizations and 118 deaths.In Brazil, the Health Surveillance Secretariat reported 2,504 FBD outbreaks between 2016 and 2019, with 37,247 affected patients and 38 deaths.Among the total outbreaks, 541 (21.6%) were reported with the identified etiological agents.In 11.5% (62 outbreaks), the etiological agent was identified as S. aureus.Among the total outbreaks, in 894 (35.7%), information was present regarding the type of food involved, with milk and dairy products (9.06%; 81 outbreaks) being responsible (Brazil, 2020).It is important to note that even with some Brazilian data published, these numbers may be underestimated, due to no obligation for notification of foodborne outbreaks in Brazil (de Medeiros Carvalho et al., 2019).To the best of our knowledge, information concerning how frequently Brazilian artisanal raw milk cheeses have been implicated in staphylococcal food poisoning does not exist in official epidemiological surveillance data in Brazil.Staphylococci have a particular relevance in raw milk cheeses, once they can contaminate the milk due to poor hygienic milking conditions, conservation, transport, and mastitis occurrence.Staphylococci are naturally present in skin and mucosae from humans and animals, leading to direct contamination from improper handling.In addition to the hygiene indicator role, staphylococcal food poisoning results from the consumption of food in which coagulase-positive staphylococci (CPS), mostly S. aureus, have grown and produced staphylococcal enterotoxins (SEs; Silva et al., 2021).Similarly, an emerging public health concern is the growth of antimicrobial resistance in this bacterial group.Studies demonstrate the relationship between the abusive use of antimicrobials in humans, as well as in agriculture and the treatment of animals, with the increased incidence of resistant bacteria (Rola et al., 2015).Furthermore, the bacterial capacity of Staphylococcus spp.sharing resistance genes increases this problem.The presence of Staphylococcus spp. with resistance to antimicrobials isolated from food has been reported worldwide.In light of these observations, this review aims to discuss the presence, SEs, and antibiotic-resistant of Staphylococcus spp. in Brazilian artisanal cheeses produced with raw milk.

STAPHYLOCOCCUS SPP.
The Staphylococcus genus combines more than 70 species and subspecies characterized so far (https: / / lpsn .dsmz.de/genus/ staphylococcus).Among the variety of diseases caused by these pathogens (e.g., respiratory infections, skin abscesses, soft tissue infections, bacteremia, infective endocarditis; El-Far et al., 2021), poisoning caused by the consumption of food contaminated with heat-resistant SEs is a worldwide public health concern (Chieffi et al., 2020).Staphylococcus spp.also are organized into 2 groups according to their ability to produce coagulase, an enzyme enrolled in the plasma coagulation cascade (Podkowik et al., 2013).Usually, CNS have a low virulence potential compared with CPS.However, due to the higher incidence of nosocomial infections, CNS species such as S. epidermidis and S. haemolyticus are associated with patients who have common risk factors for hospital-acquired infection (Chajęcka-Wierzchowska et al., 2019).
Staphylococcus aureus is considered 1 of the 3 most important foodborne pathogens that can cause diseases in humans (Al-Nabulsi et al., 2020).These bacteria are ubiquitous, and specifically, S. aureus colonizes the skin, the nasopharyngeal mucosa, and to a lesser extent, the gastrointestinal tract of humans and animals (Leroy et al., 2020).This makes this species an important indicator of hygiene quality because contaminated handlers can contaminate the product.However, periodic and intensive training in good manufacturing practices (GMP), hygiene, and health of the handlers can minimize the problem.
Another worrying factor regarding contamination by S. aureus associated with food handlers is related to the transmission of methicillin-resistant strains, known by the acronym MRSA (methicillin-resistant S. aureus).
A study carried out with food handlers in hospitals located in northeastern Brazil pointed out that of 140 food handlers, 50% were colonized with CPS on their hands or nostrils, and 93% and 28.6% were colonized, respectively, by penicillin-resistant strains and MRSA (Ferreira et al., 2014).
Staphylococcus aureus cells are not able to survive pasteurization and sterilization temperatures during food processing, however, preformed SEs show resistance to high temperatures in milk, which is represented by D-values at 121°C and 100°C ranging from 9.9 to 11.4 to 70 min, respectively (Medvedová and Valík, 2012) survive, the preformed SEs on food can resist the usual thermal control strategies of the food industry (Ziuzina et al., 2018).
Foodborne diseases caused by SEs are characterized by the International Commission on Microbiological Specifications for Foods as belonging to risk group III, which includes moderately dangerous diseases, usually with a short duration and without the threat of death or sequelae, with self-limiting symptoms but causing severe discomfort.In general, the incubation period and severity of symptoms depend on the number of ingested SEs and the individual's susceptibility.According to Rajkovic (2016), symptoms are quick and may occur between 30 min up to 8 h after eating the contaminated food.In moderate to severe clinical conditions, the symptoms found include abdominal pain, emesis, diarrhea, dizziness, lassitude, myalgia, headache, moderate fever, and low blood pressure.In most cases, recovery is uneventful and takes 24 to 48 h to occur.Mortality rates are low (0.03% of confirmed cases) with the most incidences found in children (<5 yr), the elderly, and immunocompromised patients (Rajkovic, 2016;Stewart, 2017).Although the clinical manifestations are well described, the pathophysiology of symptoms is only partially understood (Rosengren et al., 2013;Hu and Nakane, 2014).

STAPHYLOCOCCUS SPP. IN BRAZILIAN ARTISANAL CHEESES
Raw milk and raw milk cheeses are often contaminated with enterotoxigenic Staphylococcus spp., and considered potentially dangerous foods for SEs transmission (Titouche et al., 2019;Leroy et al., 2020).Feitosa et al. (2003) (Brazil).The results revealed a high incidence (91.3%) of CPS in artisanal cheese samples compared with industrialized samples (8.7%).Ferreira et al. (2016) reported the presence of S. aureus in 56 samples of artisanal and in 10 samples of industrialized Minas Frescal cheese commercialized in the State of Goiás (Brazil).Among the samples analyzed, 20% presented CPS counts above the limit currently established in Brazil (3 log 10 cfu/g; Brazil, 2019a).The results also showed that 13 (44.8%) of the S. aureus isolates from raw milk cheeses had genes encoding enterotoxins.Tigre and Borelly (2011) analyzed 51 samples of Coalho cheese sold in the city of Salvador (Brazil) and detected CPS counts above 35% of what is permitted by Brazilian legislation.Borges et al. (2003) reported the occurrence of CPS in 93% of the samples of Coalho cheese analyzed in the State of Ceará (Brazil), with 91% of these samples falling outside the current microbiological standards.Sousa et al. (2014) (Brazil).
Nunes and Caldas (2017) performed a quantitative microbial risk assessment, obtaining CPS data (from 350 samples of Minas Frescal cheese) from Brazilian monitoring platforms.The data were used as a procurator for S. aureus contamination.The work of Arcuri et al. (2010) was used to set (73%) the variable prevalence of toxigenic strains.The study preliminary assessed the risk associated with poisoning by SEs due to the consumption by the Brazilian population.In the evaluation, the SE toxigenic dose of 100 ng or more was used to calculate the cumulative probability of ingestion events.Although the study identified a lack of data that are needed in order to obtain a better quantitative microbial risk assessment, the evaluation indicated that consumption poisoning is low risk.Silva et al. (2021) isolated 10 S. aureus strains from Minas Frescal cheese, but none presented classical enterotoxin-related genes.Alves et al. (2018) collected 64 cheese samples from 3 Minas Frescal cheese dairies.The presence of Staphylococcus spp. was investigated in processing environments, raw materials, and the final products.Thirty-three cheese samples were contaminated with S. aureus.Among 66 S. aureus isolates, only one was identified as MRSA (recovered from brine).The authors observed that S. aureus was most frequently isolated from cheese (82%), followed by raw milk, curd, whey, and brine (50%), non-food contact surfaces (40%), and food contact surfaces (34.6%).High prevalence rates of S. aureus contamination were reported in the 3 cheese processing lines evaluated, ranging from 21 to 68.2%.André et al. (2008) studied a dairy processing plant in the State of Goiás (Brazil) and obtained 73 S. aureus isolates from samples of food handlers (75%), raw bovine milk (66.7%), and Minas Frescal cheese (70.8%).The results showed that raw milk was the most likely source of contamination in cheeses.However, Arcuri et al. (2010) investigated 291 S. aureus strains isolated from mastitis milk (125), process tank milk ( 96), and Minas Frescal cheese (70) for the presence of tst-1 gene encoding staphylococcal toxic shock syndrome toxin 1 and SE genes (sea, seb, sec, sed, see, seg, seh, sei, selj, and sell).For at least 1 of the 11 genes, 109 (37.5%) S. aureus were positive.Of these, 72.9% came from cheese, 41.7% from milk from the process tank, and 13.6% from milk from cows with mastitis.Twenty-three distinct genotypes were observed for SEs genes.The high genotype diversity of potential toxigenic S. aureus strains found in the study suggests that the source of contamination can be multifactorial including, in addition to raw milk, the processing environment and the food handlers.Borges et al. (2008) detected the presence of high levels of Staphylococcus ssp.and SEs in a Coalho cheese production line in the city of Fortaleza, CE, Brazil.The population of Staphylococcus spp.ranged from <1 cfu/mL in pasteurized to 3.2 log 10 cfu/mL in raw milk, whereas that of CPS ranged between <1 cfu/mL in pasteurized to 6.8 log 10 cfu/mL in raw milk.The CPS population was detected in 100% (25) of the raw milk samples and in 8% (2) of the cheese samples (2).The presence of staphylococcal enterotoxin was found in 20% of raw milk samples and, consequently, was also detected in pasteurized milk, curd, and cheese.Campos et al. (2021) reported the presence of CPS above Brazilian legal limits in samples of 38 (42%) Canastra cheeses (artisanal cheese from the Serra da Canastra region, MG, Brazil).Grecelle et al. ( 2020) isolated Staphylococcus spp. of products and surfaces in the main stages of production of Colonial cheese (artisanal cheese from southern Brazil).Of the 72 isolates, 43% (31) were typified as CPS with some isolates having one or more genes for enterotoxin production.These studies are examples that reinforce the need to improve GMP in the search for strategies to reduce contamination levels.The same concern has been highlighted with artisanal raw milk cheeses produced in other countries (Johler et al., 2015a(Johler et al., ,b, 2018)).

STAPHYLOCOCCAL ENTEROTOXIN CHARACTERISTICS AND ENTEROTOXIGENIC POTENTIAL OF STAPHYLOCOCCI ISOLATED FROM BRAZILIAN ARTISANAL CHEESES
The nomenclature for SEs used in this review follows the rules proposed by the International Nomenclature Committee for Staphylococcal Superantigens Nomenclature (INCSSN).The INCSSN proposed that only toxins exhibiting emetic activity after oral administration in a primate model should be designated as SEs.The other related toxins without emetic activity or not yet tested for this activity have been recommended to be designated as staphylococcal enterotoxin-like (SEl; Lina et al., 2004).Therefore, as it may cause emesis, SEs (Table 1) can be separated into 2 main groups, classical SEs and newer SE-like enterotoxigenic-type proteins (Abolghait et al., 2020).The SEls are considered enterotoxins, due to their molecular structure, but do not demonstrate emetic potential in primate models as recommended by the INCSSN (Umeda et al., 2017).Zhang et al. (2018) reported 2 new enterotoxins from Staphylococcus argenteus and Staphylococcus schweitzeri species were recently recognized as new species closely related to S. aureus.The related proteins, designated as SE-like toxin 26 (SEl26) and 27 (SEl27), were identified and characterized.Suzuki et al. (2020) described a new enterotoxin (SE02) produced by 2 S. aureus strains (Tokyo12480 and Tokyo12482) isolated from an outbreak that occurred in Tokyo in 2004.The new SE02 has superantigenic and emetic bioactivity, showing resistance to heating (boiling at 100°C) and to digestion with pepsin.
Staphylococcal enterotoxins are considered the most important factor linked to food poisoning by the Staphylococcus genus, and these toxins are generally found in milk and dairy products.Staphylococcal enterotoxins consist of a superfamily of 24 structurally related proteins based on sequence homology that combines superantigenic and emetic activities, their molecular weights range from 22 to 28 kDa (Argudín et al., 2010).They are low-molecular-weight pyrogenic exotoxins sharing functional similarities (Fox et al., 2020).Also, SEs are soluble in water and saline solutions, highly stable, and resistant to environmental conditions that would easily destroy the producing vegetative cell, being resistant to proteolytic (e.g., trypsin, pepsin) and coagulating enzymes (e.g., papain, renin, chymotrypsin), and low pH values, persisting during cheese-making process and in gastrointestinal tract acidity after ingestion (Le Loir and Hennekinne, 2014;Abolghait et al., 2020).
Staphylococcal enterotoxin thermal denaturation generates a loss of biological activity.The potency of these toxins gradually decreases as the temperature increases (Hu and Nakane, 2014).Some outbreaks resulted from ingestion of foods that were heat-treated after SE production (Le Loir and Hennekinne, 2014).In this context, to study the influence of heat treatment used in industrial food production on the viability of S. aureus and its SEs, Necidova et al. (2016) inoculated SE producing S. aureus (A, B, C) in milk samples and then applied heat treatment at temperatures of 72, 85, and 92°C for 15 s; after thermal processing, SEs detection rates varied from 45 to 87%.Although it has been observed that pasteurization temperatures can partially reduce SEs, their inactivation also depends on the amount present in the food.Poisoning typically occurs due to ingestion of only high nanogram to low microgram quantities of SE, a level usually reached when populations of enterotoxigenic Staphylococcus spp.exceed 5 log 10 cfu/g of food (Fisher et al., 2018).However, the symptoms vary with the individual's degree of susceptibility and weight, the concentration of enterotoxins in the food, and the amount of food consumed.In more susceptible persons, an intake of 0.1 µg may be sufficient to cause symptoms (Nunes and Caldas, 2017).The multiplication of S. aureus and the production of enterotoxins are associate; however, a controversy exists regarding the number of viable S. aureus cells per gram needed to initiate the production of enterotoxins.Values of viable cells related to quantities sufficient to cause intoxication differ between substrates and types of enterotoxins.Studies usually show amounts above 5 log 10 cfu/g associated with the release of enterotoxins in an amount capable of affecting humans, as long as the strain involved is capable of producing it (Rajkovic et al., 2020).
On the other hand, it is worth noting that the potential to cause staphylococcal poisoning cannot be predicted only when a high number of S. aureus cells are found in the food.This is because the only determination of the population to establish the presence or absence of enterotoxins has limited value, as the toxins are thermostable and once produced could persist in heated or fermented foods, whereas viable cells decline in number, reaching undetectable levels (Rajkovic, 2016).In this context, Cardoso et al. (2013) studied microbiological parameters of Minas Serro cheese during the ripening, considering the dry and rainy seasons in Brazil.In total, 100 samples obtained from 5 dairies were analyzed, and the presence of SEB and SEC was detected in 1 and 4% of the cheeses, respectively, after 30 d of ripening.The results suggest that the fermentation and ripening processes are not effective in mitigating the presence of SEs.Omwenga et al. (2019) consider that enterotoxins are one of the most important virulence factors in S. aureus species.According to Necidova et al. (2016), 50 to 70% of S. aureus strains are capable of producing SEs under suitable conditions.The superantigens cause lymphocyte activation preliminarily, but then result in clonal deletion and anergy, leading to immunological tolerance; that is, exposure to high concentration levels of SEs can inhibit antibody production.Such exposure to superantigens causes memory, which can make cells anergic and refractory to bacterial antigen stimulation.Hence, the superantigenicity of SEs and the immune  result to interruption of the response are probably more significant than the enterotoxic properties (Stewart, 2017).Food poisoning, in the case of dairy products, can occur from the ingestion of raw milk or dairy products contaminated with the preformed SEs.The increased frequency of enterotoxigenic S. aureus strains in dairy products may pose a risk to consumers' health (Abdeen et al., 2020).
Staphylococcal enterotoxin A is recognized as the major causative enterotoxin of food poisoning in humans worldwide (Argudín et al., 2010;Hu and Nakane, 2014).In a study carried out by Rosengren et al. (2013), the presence of SEA was detected when S. aureus levels in food were above 6.8 log 10 cfu/mL.Abdeen et al. (2020) detected a high prevalence of SEA, SEB, and SED in raw milk samples in Egypt.De Freitas Guimarães et al. ( 2013) compared the prevalence of genes coding for the production of SEs and found a higher prevalence in CNS (66%) when compared with CPS strains (34%) in Brazilian raw milk samples.Rodrigues et al. (2017) detected enterotoxin genes in samples of several kinds of Brazilian cheeses, where the seh gene (coding for SEH) was the most found (62%), followed by the coding genes for enterotoxin SElX and SER (42%).In the study conducted by da Silva Cândido et al. (2020), on the production chain of Minas Frescal conventional and organic cheeses, the presence of Staphylococcus spp.carrying enterotoxin encoding genes was detected, with the seg gene (coding for SEG) standing out in 35.8% of the isolates, followed by the sei gene (coding for SEI; 21.1%), with no differences between organic and conventional samples being verified.The main critical points of transmission of S. aureus in the cheese production chain are considered to be the brining process, the utensils used, and the type of milk (raw, pasteurized, or both; da Silva Cândido et al., 2020).Additionally, Mehli et al. (2017) analyzed several dairy products and identified in 72 S. aureus isolates that 87.5% (63) harbored genes encoding SEC, SEG, and SEH.
The high diversity of isolates carrying enterotoxin genes demonstrates real reasons for concern with the safety in the production of artisanal cheeses.Nevertheless, the presence of enterotoxigenic genes alone does not necessarily indicate the microorganisms ability to produce enough biologically active SEs to cause clinical manifestations.In this context, during the microbial evaluation of the safety of Brazilian artisanal Serro cheese, using genotypic and phenotypic approaches, Andretta et al. (2019) did not detect the presence of classical SEs (SEA, SEB, SEC, SED, and SEE) or genes encoding these enterotoxins, suggesting that proper milking practices and adequate processing conditions of artisanal products can reduce the risk of contamination in the production chain of dairy products.
The results found by Chieffi et al. (2020) suggest that new SEs are potential sources of outbreaks.Therefore, the detection of SEs in food, especially those involved in cases of poisoning, should relate not only to classic SEs, but also to all types of new enterotoxins (SEls) identified.In order for this to happen, it is necessary to use methods capable of detecting the production of all types of SEs and SEls that have been known up to now.

STAPHYLOCOCCAL ENTEROTOXIN DETECTION ASSAYS
Several methods are used to detect bacterial enterotoxins in food, for example, bioassays, immunoassays, and molecular methods (Hennekinne et al., 2010;Cretenet et al., 2011).Bioassays are based on the ability of the food sample suspected of contamination to induce the characteristic symptoms of intoxication, such as emesis, gastrointestinal symptoms, or superantigenic action in cell cultures (Le Loir and Hennekinne, 2014).Historically, SEs were detected through intraperitoneal tests based on their emetic activity in primates, cats, and animal models such as the Asian house shrew (Suncus murinus).A disadvantage of this technique is that the symptoms appear when the dose of SEs administered to animals is above 2.3 mg, an amount considered greater than those involved in human food poisoning, and, therefore not being a technique to accurately characterize outbreaks (Le Loir and Hennekinne, 2014).
Staphylococcal enterotoxin A are produced in small amounts in cheese (usually <0.1 ng/g); however, highprotein products can interfere with detection tests (Cretenet et al., 2011).Thus, different methods have been developed for the detection and identification of in dairy products (Table 2), and among the main rapid and sensitive ones, some are based on ELISA.Immunological technology has high specificity and efficiency and does not require complex equipment.The limitation of the ELISA tests, in this case, is the lack of enterotoxin-specific antibodies that allow the identification of all existing SEs and has the disadvantage of probable false-positive results during detection (Le Loir and Hennekinne, 2014).
Quantitative MS methods have been developed and have demonstrated the potential to overcome the limitations of ELISA methods to detect and quantify SEs.However, the MS method cost per analysis disfavors them.According to Le Loir and Hennekinne (2014), MS methods have not been consolidated as a choice in routine analysis, but only for confirmation of outbreaks related to SEs.
Molecular techniques are based on PCR.These techniques detect regions of genes encoding SEs in strains isolated from contaminated food, reporting the pres-Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL ence or absence of genes.However, they do not provide any information about the expression of target genes in food, and cannot be used as the only method to confirm the production of the enterotoxin or S. aureus strain potentially responsible for the food outbreak (Le Loir and Hennekinne, 2014).In general, molecular methods are the most used for the detection of genes encoding SEs and genes for identifying the species in dairy products (Table 2).Singleplex or multiplex PCR methods were designed to screen for genes that encode each enterotoxin, requiring sets of specific primers targeting each gene (Rodríguez et al., 2016).
The European Screening Method (ESM) based on extraction, concentration by dialysis, and qualitative immunochemical detection has as main target of 5 SEs (SEA, SEB, SEC, SED, and SEE).However, ESM is not able to distinguish among the SEs, making it necessary to analyze the sample with a confirmatory method, such as the ELISA.As mentioned before, the ELISA can generate false-positive results, due to interference arising from the food matrix or caused by endogenous enzymes, such as alkaline phosphatase and raw milk lactoperoxidase (Zeleny et al., 2015).
Upadhyay and Nara (2018) reported a method of growing interest for the rapid detection of SEs called lateral flow immunoassay that could be used in Brazilian artisanal cheese in the future.The assay is based on the sandwich format and uses 2 specific monoclonal antibodies against SEs, one of which is labeled with gold nanoparticles and the other is immobilized on the control line.The method can be suitable for testing the presence of SEA in milk samples with easy execution, reproducibility, and stability at 4°C, being also fast and effective without the need for pre-incubation with toxin control samples.
In developing methods for detecting SEs, Zeleny et al. (2015) highlighted the importance of studying reference materials (RM) for accurate measurements of S. aureus SEA in food.Reference materials consist of toxins and matrix materials that contain one or more toxins at different levels in food matrices, such as cheese, milk, or ham.Within this approach, the authors carried out a study that made it possible to predict the RM characteristic and the assignment of SEA value at levels below ng/g in cheese matrices, constituting an important step toward the development of a certified RM for SEA.
Especially in the cheese matrix, the detection and quantification of SEs in dairy products are required by various regulatory bodies, such as the ANVISA through Normative Instruction No. 60 dated December 23, 2019 (Brazil, 2019a).The detection of enterotoxin in foods is the most conclusive way to associate the presence of the toxin with the consumption of a certain food that causes intoxication.Thus, several commercial immunological methods for detecting enterotoxins in food have been developed (Table 3).
Among the immunological methods, sandwich ELISA is the most used due to the commercial availability of the reagents in monovalent and polyvalent formats, which means that it can be used both for screening enterotoxins and for identifying a specific SE.Commercial ELISA kits for detecting SEA and SEE are affordable and inexpensive.Kits based on monoclonal antibodies eliminate the cross-reactivity that can be observed between similar SEs (e.g., SEA, SEE).The tests show a detection limit of an average of 0.1 to 1.0 ng of enterotoxin per gram of food.Reverse passive agglutination tests with a sensitivity of 0.5 ng/mL are also commercially available.

ANTIBIOTIC-RESISTANT STAPHYLOCOCCUS SPP. AND METHODS TO DETERMINE ANTIMICROBIAL RESISTANCE
The World Health Organization describes S. aureus as one of the most relevant pathogens because it represents a threat to human health in terms of multiresistance to antimicrobial agents (WHO, 2017).The emergence of antibiotic-resistant S. aureus in the agricultural environment may be mainly associated with the selective pressure put into place by the consolidated use of antimicrobials to treat infections such as bovine mastitis.In this context, the risk of emergence and transmission of antimicrobial-resistant pathogens may increase as a result of the excessive use of drugs in animals, either as a promoter of zootechnical development or to prevent or treat infectious diseases (Zeinhom and Abed, 2021).Some examples are tetracyclines, aminoglycosides, macrolides, and sulfamethoxazole, which are the most common drugs for therapeutic use in food-producing animals (Rola et al., 2015).In addition, several strains have resistance to the β-lactam group.Resistance to penicillin, also called resistance to methicillin or oxacillin, manifests as resistance to most antimicrobial agents of the β-lactam group, including cephalosporins and carbapenems (Oniciuc et al., 2017).The fifth-generation cephalosporins, such as ceftaroline, are the unique β-lactam that remain active against MRSA (Oniciuc et al., 2017).
Methicillin-resistant Staphylococcus aureus resistance is related to the acquisition of a DNA fragment called Staphylococcal Cassette Chromosome mec (SC-Cmec), which carries the mecA gene responsible for encoding a protein with low binding affinity to the β-lactam ring, named Penicillin Binding Protein 2A (PBP2A).The synthesis of PBP2a allows bacteria that carry SCCmec to synthesize the bacterial cell wall even in the presence of β-lactams, providing resistance to most antimicrobials in this class (Otto and Chatterjee, 2013).Methicillin-resistant Staphylococcus aureus causes important nosocomial and community infections and currently represents about 60% of S. aureus isolated from hospitalized patients in countries such as the United States and Brazil (Reis et al., 2020).Rodrigues et al. (2017) found dairy-related staphylococcal strains resistant to penicillin, cefoxitin, oxacillin, clindamycin, erythromycin, tetracycline, tobramycin, and gentamicin, ciprofloxacin, and chloramphenicol and reported the presence of community-acquired-MRSA isolates in raw milk and cheese samples from Brazil.These are considered a threat to public health because they are easily disseminated by carriers that are considered healthy and because they have an increased potential for virulence when compared with nosocomial strains (Herrera et al., 2016).
Casaes Nunes et al. (2016) investigated the presence of CNS in Minas Frescal cheeses marketed in Southwest Brazil.The results showed the presence of multiresistant strains to β-lactam antimicrobial agents such as oxacillin, penicillin, and cefoxitin (100%); vancomycin (80%); linezolid (30%).The isolates (10 strains from 6 Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL dairies) also showed high resistance to other classes of antimicrobials, such as sulfamethoprim (60%), the aminoglycosides ciprofloxacin, neomycin, and gentamicin (50%), chloramphenicol (60%), and clindamycin (50%).Concerning the presence of enterotoxin-related genes, all strains carried multiple SEs genes: sea (90%); seb (70%); sec/see (60%); seh/sei (50%); sed (40%); sec/ selk/selq/selr (20%); selu (10%).The authors confirmed the enterotoxigenic character of the strains that were able to express and produce enterotoxins in vitro.As a result, the presence of multiantimicrobial-resistant CNS strains found in the Brazilian dairy processing environment is also a concern for this food production chain (Rodrigues et al., 2017).In general, the implementation of GMP is essential to reduce the risk of dissemination of antimicrobial-resistant bacteria throughout the production chain, associated with studies that aim to assess and to monitor the resistance and virulence of staphylococcal isolates.
Several methods have been described to determine antimicrobial resistance in Staphylococcus spp.Among phenotypic methods, the agar disk-diffusion (solid culture media) and broth microdilution (liquid culture media) are the most used to perform antimicrobial susceptibility testing for strains of food origin.Additionally, the MALDI-TOF-MS method can be used in specific protocols to detect antimicrobial resistance, for example, for predicting whether the bacterium produces enzymes that hydrolyze antibiotics, such as carbapenemases and extended-spectrum β-lactamases (March-Rosselló, 2017).
Among the genotypic methods, those based on the detection of specific resistance genes stand out (Kadlec et al., 2015).Genotypic tests provide information about which resistance genes are present in the genome of a bacterial isolate.To detect resistance genes, present in the genome, 2 main methods are used: (1) amplification of DNA segments encoding resistance genes (PCR and its variations), and (2) expression analysis by DNA microarrays.The disadvantage of these methods is that only known nucleotide sequences can be detected.Polymerase chain reaction-based methods must be associated with phenotypic assays, as they do not provide information on whether the detected gene is functionally expressed (Kadlec et al., 2015).In staphylococci, it is known that different resistance genes may be as-sociated with one or more resistance phenotypes, such as the ermA, ermB, ermC, and ermT resistance genes.These genes confer resistance to macrolide, lincosamide, and streptogramin B, and may occur alone or in different combinations per isolate (Wendlandt et al., 2013).Different studies performed the identification of antibiotic resistance genes of MRSA isolates in samples of raw milk and Brazilian artisanal cheeses (Table 4).
As described by Wang et al. (2014), real-time multiplex PCR was used to simultaneously detect 16SrRNA sequence for S. aureus and the nuc and mecA genes for methicillin resistance, incorporating 3 target-specific TaqMan probes labeled with different fluorophores (Cy5, HEX, and FAM).Detection of resistance genes can be coupled with the detection of species.The DNA microarray method can be used to assess gene expression, being able to detect many resistance genes simultaneously; however, the test is costly and laborious to perform (Kadlec et al., 2015).These approaches have not yet been tested on samples of Brazilian artisanal cheeses.

CONTROL OF STAPHYLOCOCCI GROWTH AND ENTEROTOXIN PRODUCTION IN ARTISANAL CHEESES
Brazilian artisanal cheeses are characterized by the use of raw milk and, occasionally, the use of natural whey starter (known in Brazil as pingo).The manufacturing process is based on traditional techniques developed over the centuries by cheesemakers, with environmental and sociocultural influences.Artisanal cheeses have a diverse and complex microbiota, and the microbial consortium plays an important role during ripening regarding biosafety through its action in  Ferreira et al. (2016) modulating the development of pathogens (de Almeida et al., 2020;Pineda et al., 2020).Brazil, Resolution No. 331, dated December 23, 2019, covers the entire food production chain and deals with the microbiological standards for foods and their application (Brazil, 2019b).This resolution includes the production, storage, transport, distribution, commercialization, or any other stage that is part of the food chain.The Normative Instruction No. 60, dated December 23, 2019, which complements the Resolution No. 331, presents the lists with the microbiological standards for foods presented to the consumer (Brazil, 2019a).According to these issues, food cannot contain pathogenic microorganisms, toxins, or metabolites in amounts that harm the consumers' health.Normative Instruction No. 60 states that for cheeses, 2 out of every 5 samples of the batch may have positive CPS per gram counts between 2 and 3 log 10 cfu/g.Meanwhile, SEs should be absent in all samples (n = 5) from each lot.The methods limit of quantification for SEs must be less than or equal to 1 ng/g.Currently, artisanal cheeses must meet the microbiological standards established in Normative Instruction No. 60 or must comply with the Technical Regulation of Identity and Quality, exclusive to each variety of artisanal cheese, to guarantee the minimum service characteristics, both from a microbiological and process point of view.In Brazil, these criteria for each artisanal cheese are established by MAPA.However, there is the same effort from MAPA for artisanal cheeses to meet the same criteria established for industrial cheeses concerning the microbiological quality of the products, including CPS and SEs criteria in the products.
In addition to microbiological control, antimicrobial resistance is one of the greatest challenges for public health.Due to its importance and complexity, this topic is treated in a global context based on a One Health approach, working together with human, animal, and environmental health.In this sense, MAPA implemented the Surveillance and Monitoring Program for Antimicrobial Resistance in the Scope of Agriculture (2019Agriculture ( -2022) ) to assess risks, trends, and patterns in the occurrence and dissemination of resistance to antimicrobials through the food of animal origin produced in Brazil, as well as providing essential data for risk analysis (Brazil, 2021).The program is part of the activities established in the National Action Plan for the Prevention and Control of Antimicrobial Resistance in Agriculture (PAN-BR AGRO), which describes the specific actions to be developed by the sector related to the topic.For now, this program does not cover dairy cattle farming or artisanal dairy products yet.However, the initiative considers this important topic, and the dairy production chain will probably be included in the national surveillance program.
Apart from official surveillance programs, several strategies are being tested and verified to minimize or to prevent the SEs production in cheese, as the use of protective cultures.The use of protective bacterial cultures represents a potential intervention to increase the safety of dairy products, with most of these cultures belonging to the group of the lactic acid bacteria (LAB) (Aljasir and D'Amico, 2020).Lactic acid bacteria are important microbiota components that actively participate in the processes that define the physicochemical, sensory, and microbiological characteristics of cheese.Among the various microbiological interactions present in cheeses, the inhibitory activity of LAB on S. aureus is one of the most relevant (Silva et al., 2020).
Some LAB strains are capable of producing antimicrobial substances, such as bacteriocins.Perin et al. (2015) investigated the potential of Lactococcus lactis ssp.lactis GLc05 as a protective culture added to raw milk to control pathogens' growth.As a result, the study demonstrated that CPS counts were lower when LAB was used and after 60 d of ripening, CPS counts were not registered in the cheese (<10 cfu/mL).This probably occurs due to the ability of Lc. lactis ssp.lactis GLc05 to produce nisin.The addition of protective cultures to fermented products does not require additional labeling and can meet the current consumer demand for clean label food.Biocontrol by LAB can be considered a viable alternative to the use of chemical agents in food; however, the mechanisms of this antagonism have not been fully elucidated.It is crucial to understand how pathogens are affected at molecular and phenotypic levels for a bio preservation strategy's success (Nogueira Viçosa et al., 2019).In this sense, some predictive microbiology approaches can be used to model microbial interactions in real matrices, such as in the production of artisanal cheeses made with raw milk.
Other approaches are being tested to prevent cheese contamination with S. aureus and SEs around the world.These approaches could be used or tested in Brazilian artisanal cheeses in the future, such as the use of natural antimicrobials.Botanical essential oils with antimicrobial properties have aroused interest for their use in foods as a natural antibacterial alternative, as a source to control contamination by multiresistant bacteria, and to meet consumer demand for safer foods (Zhu et al., 2021).In this context, de Melo et al. (2020) reported the effectiveness of microencapsulated lemongrass essential oil against CNS during Brazilian artisanal Coalho cheese ripening.The velame essential oil was proposed as an alternative for the control of MRSA  (de Oliveira et al., 2020), while rosemary pepper essential oil was effective against S. aureus from Minas Frescal cheese (Castro et al., 2011).On the other hand, when thyme oil and oregano were used in Coalho cheese, the inhibition of S. aureus was observed but also the starter cultures Lc. lactis ssp.lactis and Lc.lactis ssp.cremoris were highly affected by the antimicrobial agent (de Carvalho et al., 2015;de Souza et al., 2016).Thus, the choice of the essential oil must consider the autochthonous or starter LAB.
Regarding the use of antimicrobial peptides produced by Staphylococcus ssp.(called staphylococcins), Miceli de Farias et al. ( 2019) investigated its potential use to prevent or reduce contamination by S. aureus in Brazilian Minas Frescal cheese with reduced sodium content.The antistaphylococcal activity tested in cheeses demonstrated that 3 types of staphylococcins (aureocin A53, Pep5, and lysostaphin) showed lytic activity against enterotoxigenic and resistance to β-lactam antibiotics strains.Lysostaphin revealed the best inhibitory activity and significantly reduced the number of viable cells.The results of the study suggested that staphylococcins could be a viable alternative for application in the cheese industry regarding bio preservation aspects; however, the industrial application requires extensive investigation regarding the safe use of these substances in food.
Quorum sensing blockers are also suggested as promising new agents for antimicrobial therapy, especially for the treatment of infections with multidrug-resistant strains of S. aureus.However, it is noteworthy that the inhibition of quorum sensing signaling is not aimed at eliminating the microorganism, but at blocking the expression of the target gene, reducing cells' virulence, and making them more vulnerable to the hosts' immune response (Saeki et al., 2020).According to Xie et al. (2019), interactions between the autoinducing peptide and AgrC (histidine protein kinase) activate virulence in Staphylococcus spp., whereas noncognate pairs generally have an inhibitory effect.Nonetheless, exploring agr for the development of new therapies depends on understanding all the underlying mechanisms and the agonism and antagonism of the 2 components signaling at all stages of the signaling process.The understanding of chemical communication among bacteria in food is still being understood, and despite representing a research field with many challenges, it will contribute to the development of new strategies against food poisoning.Although bio preservation is a promising tool, as mentioned in the paragraphs above, the effective reduction of the level of contamination through improved sanitation and hygienic procedure, in addition to the application of GMP, are still the main control measures.

FINAL CONSIDERATIONS AND PERSPECTIVES
The production of artisanal cheese in Brazil represents economic and cultural relevance, which explains the increasing efforts to guarantee its safety.Despite the advances that have been taking place in the organization of the production chain, artisanal cheeses are usually produced by small farmers with little access to technical support.Knowing that Brazilian artisanal raw milk cheeses are a potential source of enterotoxigenic and antibiotic-resistant staphylococci, the development of the dairy production chain is allowing its adequate organization as well as the adoption of hygienic production procedures.One of the main contributions, in this regard, comes from the more effective control of mastitis in the dairy herd.On the other hand, this system has some weaknesses; for example, there is no systematic knowledge of how much of the artisanal cheese produced in Brazil has enterotoxin contamination.In addition, no consistent FBD data are available in Brazil, mainly associated with artisanal cheeses.Therefore, an increasing number of studies with artisanal cheeses by several research groups in the country are allowing knowledge of the microbiological profiles of each artisanal cheese to define its identity to identify natural bio controllers, which could be a tool to control Staphylococcus spp.Finally, considering the public health emergency that antimicrobial resistance represents, the One Health approach is recommended as a strategy for the study and prevention of antimicrobial-resistant S. aureus in the production chain of artisanal raw milk cheese.
. Although S. aureus cells are not able to Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL

Table 2 .
Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL Nonexhaustive methods for detecting staphylococcal enterotoxins (SEs and SEls) in raw milk and Brazilian artisanal cheeses 1

Table 3 .
Commercial immunoassay kits for detection of staphylococcal enterotoxins in food

Table 4 .
Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL Detection methods of antibiotic resistance genes of Staphylococcus aureus isolates from raw milk and Brazilian artisanal cheeses 1 Carneiro Aguiar et al.: LITERATURE REVIEW: ENTEROTOXIGENIC POTENTIAL in artisanal goat cheese in the northeastern semi-arid Brazilian region