Introduction

Foodborne diseases (FBD) occur worldwide and affect a large part of the population, being a cause of international concern among health authorities [1]. FBDs have significant morbidity and mortality, it is estimated that thousands of people are hospitalized and some of them die from this cause. It is very difficult to estimate the actual number of FBD cases because not all of them are registered by the public health system due to misdiagnosis or underreporting [1].

Staphylococcus aureus can be transmitted by contaminated food [2]; and it is one of the pathogens that most cause foodborne outbreaks in Brazil [3]. This transmission is mainly due to the poor handling of food during processing [4, 5]. The consumption of food contaminated with toxins produced by S. aureus can lead to staphylococcal food poisoning, which may cause severe gastroenteritis, nausea, vomiting, diarrhea, and abdominal pain within 1 to 6 h after the consumption of contaminated food [6]. S. aureus can also lead to other diseases [2]; some of them severe, such as sepsis, endocarditis and necrotizing pneumonia [7]. This bacterium is found on human skin and it is commonly identified as a cause of hospital-acquired infections [8]. It is also the leading cause of bacterial infections in humans; around 20% of humans are persistent carriers of S. aureus, 30% are intermittent carriers, 50% of people do not carry this bacterium [7, 9, 10], and a third of people are asymptomatic carriers; the pathogen is commonly found in the nostrils, neck, axillae, groin, and rectum [11,12,13].

S. aureus spp. are non-spore forming Gram-positive bacteria in the form of cocci; they are non-mobile, mesophilic, biofilm-forming, and facultative anaerobes that produce enterotoxins [3]. They were first described by Sir Alexander Ogston in 1881, when the infection caused by this agent was fatal because of the lack of antibiotics [11]. Currently, this microorganism’s ability in developing resistance to antibiotics is notorious. The resistance is usually acquired by horizontal gene transfer, although mutation and selection are also important [14]. Infections caused by resistant strains are common in epidemic waves by one or more clones; methicillin-resistant Staphylococcus aureus (MRSA) is prominent in epidemic waves, being historically associated with hospitals and health units (healthcare-associated MRSA (HA-MRSA). Nowadays, it has emerged as a cause of community-associated infections (CA-MRSA), spreading rapidly among healthy individuals and its presence is a cause of concern due to resistance to various antibiotics, limiting treatment [14].

The incidence of CA-MRSA has been increasing [15,16,17,18]. Furthermore, CA-MRSA strains appear to be especially virulent [14]. It should be noted that CA-MRSA, HA-MRSA, and livestock-associated MRSA (LA-MRSA) have been found in foods intended for human consumption [15]. Researches have been showing the incidence of MRSA isolated from foods [6, 9, 19,20,21]. Studies from different geographical areas have revealed the presence of enterotoxins in MRSA isolates; in addition, the genetic relationship between enterotoxigenic isolates and isolates from human infections has been reported [15]. Therefore, this review aims to explore data that show the importance and incidence of MRSA isolated from foods around the world, and especially in Brazil where S. aureus is one of the main etiological causes of food poisoning outbreaks.

Antibiotic-resistant Staphylococcus spp.

Antibiotics correspond to a group of drugs that are commonly used in hospitals and in the community. However, pharmacological agents do not only affect the patients that use this, but also significantly intervene in the environment through the genetic modification of microorganisms [22].

The use of antibiotics has increased a lot over the years and, consequently, the exposure of these medicines to bacteria has also expanding [23]. S. aureus is a bacterial species known for its ability to become resistant to antibiotics [14]. For Chambers and DeLeo [14], exposure to antibiotics was, without a doubt, the most concentrated selective pressure exerted on the co-evolutive history of S. aureus with humanity.

The indiscriminate use of antibiotics stimulates the development of antibiotic resistance. The most useful antibiotics in the treatment of infections caused by S. aureus are β-lactams, including penicillin, methicillin, flucloxacillin, dicloxacillin, nafcillin, oxacillin, and cloxacillin [24].

Methicillin-resistant S. aureus (MRSA) are those who carry the mecA gene and are resistant to all penicillins, cephalosporins,s and carbapenem [24]. In MRSA cases, the antibiotic of choice has for a long time been vancomycin; however, other options have emerged such oxazolidinones, glycylcyclines, and lipopeptides [25]. Nevertheless, it is relevant to describe how antibiotic-resistant S. aureus arose, especially MRSA.

In 1928, Alexander Fleming discovered penicillin, thus making it possible to treat infections caused by S. aureus, starting the “Antibiotic Era” [14]. However, the use of penicillin to treat infections did not last long, as penicillin-resistant strains started to emerge [11]. In 1940, S. aureus became resistant to sulfonamide, and in 1944, it started becoming resistant to penicillin [11]. Thus, epidemic waves of antibiotic-resistant S. aureus began; in the 40s, the first wave was observed with resistance to penicillin, which still occurring today [14]. Methicillin and oxacillin were used in the 1960s to treat infections caused by S. aureus; however, some years later, resistant strains emerged, which were collectively known as MRSA [25]. In the following years, cases of resistance of S. aureus to different classes of antibiotics such as macrolides, fluoroquinolones, glycopeptides, aminoglycosides, and tetracyclines started being reported [26]. The second wave occurred almost immediately after the introduction of methicillin with the isolation of the first MRSA isolate, type I SCCmec (Staphylococcal Cassette Chromosome mec) [14]. The third wave happened in the mid-1970s with new MRSA strains that had new SCCmec types, type II and III, signaling a MRSA pandemic around the world; and the fourth and latest wave of antibiotic resistance arose in the late 1990s, with the emergence of MRSA strains in the community [14]. The discovery of CA-MRSA happened in the USA [27] when the strains were already resistant to several antibiotics, in addition to those of the beta-lactam type, and were not related to the hospital strains, which contained a new SCCmec, type IV, and a variety of virulence factors [14]. With the increase in MRSA, the use of vancomycin also increased in the treatment of infections caused by these bacteria; in this way, strains with intermediate resistance to vancomycin (Vancomycin-intermediate Staphylococcus aureus (VISA)) emerged, and in 2002, the first strains resistant to vancomycin (Vancomycin-Resistant Staphylococcus aureus (VRSA)) were identified [14].

Given the above, the concern about the presence, distribution, and incidence of resistant strains in any environment becomes evident; especially in health units where weakened individuals may be exposed to them, and in food production, where people from different age groups and with different health states may be contaminated during production and/or consumption.

Methicillin-resistant Staphylococcus aureus

The MRSA is one of the leading causes of infections [28]; 50% of the strains isolated in the USA and in European countries are Methicillin-resistant [25, 29]. Infections caused by MRSA generate higher expenses in the area of public health, and higher morbidity and mortality rate compared with non-resistant strains [25, 30]. Additionally, infectious diseases caused by MRSA are among the leading causes of death caused by infectious agents [11].

More people die each year from infections caused by MRSA than by HIV (human immunodeficiency virus) in the USA [11]. Thereby, methicillin resistance is a very serious health problem when implicated in human infections or in animals [31]. It is worth noting that the rapid detection of the infection may contribute to the effectiveness of treatment and reduction in the mortality rate [32, 33].

Currently, MRSA is spread around the world and its ability to acquire antibiotic resistance mechanisms raises concern; MRSA is often or can easily become resistant to multiple antibiotics, limiting treatment options [14]. At first, the transmission of MRSA occurred in clinical environments, but in recent decades, its presence has been reported in the community, outside the hospital environment [7]. However, investigating the origins of bacteria is complicated. Evidence indicates that resistant S. aureus can be spread in livestock operations and in hospitals, where antibiotics are widely used; thus, it could be disseminated within communities and the environment. It is worth mentioning that more researches are essential to determine how the transfers in fact occur [34].

As mentioned above, MRSA is classified as HA-MRSA, healthcare-associated methicillin-resistant S. aureus, CA-MRSA, community-associated methicillin-resistant S. aureus [35], or LA-MRSA, livestock-associated methicillin-resistant S. aureus. In an even more worrisome scenario, MRSA strains may become resistant to multiple antibiotics (multidrug-resistant MDR) [25]. The biggest problem is the ability of these bacteria to be transferred from animals to humans, causing infections [36]. Reports have been mentioned MRSA in animals, especially in pigs, but it can also affect calves [37], horses [38], and dogs [39]. LA-MRSA can be transmitted to humans who live in close contact with animals [40]. S. aureus can also be transmitted to humans through meat products, for example, due to their improper handling or cross-contamination during processing [34]. A recent study by Caggiano et al. [40] assessed healthy individuals who worked in the food industry, and the presence of S. aureus and MRSA among the individuals represented a risk to public health. LA-MRSA strains have been found in pork and chicken products in the USA, as well in raw turkey meat [34]. Caggiano et al. [40] conclude that the spread of S. aureus and MRSA in non-hospital environments, such as communities and in livestock, demands careful and continuous monitoring.

There are several methods to determine whether a S. aureus strain is methicillin-resistant; however, the one that is most often employed is the Kirby-Bauer method, which uses oxacillin and cefoxitin [25]. However, conventional culturing methods demand a lot of time; thus, methods based on polymerase chain reaction (PCR) and hybridization assays have been increasingly used as rapid methods for detection of MRSA [41]. The combination of methods has been widely employed for the detection of MRSA.

Resistance mechanism

The mecA gene, responsible for methicillin resistance in S. aureus, is the reason for these groups of microorganisms to be considered resistant to all beta-lactam antibiotic [42]. Methicillin resistance in S. aureus is mediated by the mecA gene, which encodes a new penicillin-binding protein (PBP), PBP-2a [43]. In MRSA strains, exposure to methicillin renders the four high-affinity binding proteins (PBPs) present inactive, whereas PBP-2a has low affinity to methicillin, allowing the growth of the cell, because it assumes the functions of the PBPs [43]. This resistance allows the biosynthesis of the cell wall, which is the target of β-lactam antibiotics, and occurs even in the presence of often inhibitory concentrations of antibiotics [44]. The regulation of the phenotype of resistance to methicillin and the production of PBP-2a are carried out by other genes, mecR1 and mecl; in addition, antibiotics with high PBP-2a affinity have shown effectiveness against MRSA in vivo [43].

These binding proteins decrease the ability of β-lactam antibiotics to act on bacteria [45]. In addition to the mecA gene, the mecC gene (previously called mecLGA251) has been recently described. This gene has been identified in strains isolated from, for example, milk collected from 465 herds in England in 2007 [46], and in cattle that would be submitted to slaughter between October 2011 and January 2012, mainly in Belgium and in France [47].

The resistance mechanism acquired by S. aureus can be divided in two categories: the mutation of a bacterial gene in the chromosome or the acquisition of a resistance gene from other bacteria through genetic exchange (conjugation, transduction, or transformation) [48].

The mobile genetic elements are related to PBP-2a, the penicillin-binding protein encoded by the mecA gene, which is found in SCCmec, the genetic element that encodes methicillin resistance; the resistance expression is controlled through transduction by a proteolytic signal, which corresponds to a sensor protein (mecR1) and a repressor (mecl) [44]. Molecular and biochemical mechanisms concerning methicillin resistance in S. aureus have been subject of studies, including regulatory events and those related to the structure of proteins [44].

The type of SCCmec can confer resistance to multiple antibiotics [13, 49, 50]. It transports site-specific recombinases, called cassette chromosome recombinases (ccr), which are responsible for the mobility of the elements. In S. aureus, three types of ccr genes have already been identified, ccrA, ccrB, and ccrC [51].

It is important to understand the origin and evolution of MRSA clones. The acquisition and diversity of different SCCmecs are crucial [52], and it is through that methicillin-susceptible S. aureus (MSSA) becomes MRSA. SCCmec carries either the mecA or mecC gene, regulatory genes, ccrAB or/and ccrC site-specific recombinase genes, and a variety of accessory genes encoding for a new specific penicillin-binding protein (PBP2a) [53, 54]. The SCCmec element contains three J regions, besides the mec and ccr gene complexes, they were first report as the L-C, C-M, and I-R regions but were later changed to J regions. These regions constitute non-essential components of the cassette and may carry additional antimicrobial resistance determinants [55].

According to International Working Group on the Staphylococcal Cassette Chromosome elements (IWG-SCC), there are 11 official types of SCCmec recognized [56]. Some studies even mention the existence of 12 SCCmec [54] or even 13 SCCmec [52]. Traditionally, types I to III have been associated with MRSA strains from clinical isolates, while community (CA-) or livestock-associated (LA-) strains tend to harbor smaller and supposedly more mobile type IV SCCmec types and V [57, 58]. But it is currently possible to find several types of Staphylococcus SCCmec isolated from different types of food [21, 59, 60].

Methods used for identification and characterization of MRSA

There are several methods for identification of MRSA strains, phenotypically or genotypically. Strains can be screened on MRSA agar, after 18 to 24 h of incubation at 37 °C, colonies that appear pink are considered MRSA [59]. Other typical way are Kirby-Bauer standard disk-diffusion methods, which are commonly used to determine antimicrobial susceptibility according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [25, 59, 61,62,63] or even the broth microdilution technique [64].

Some studies use resistance to oxacillin [59, 65] or cefoxitin [62, 64] or both [25] to determine if the strain is MRSA. It is also possible to determine MIC of S. aureus strains by the E-test according to the protocol suggested by the manufacturer [61].

Identification by polymerase chain reaction (PCR) [41, 59, 62,63,64,65,66,67,68] is also widely used. Specie confirmation can be performed using nuc gene; mecA gene is used to identify the methicillin-resistant strain [59, 62, 63, 65, 67, 68]. Other alternative for detection of MRSA is the use of kits based on PCR, a multiplex PCR and real-time PCR, to detect each SCCmec type and the chromosomal orfX-SCCmec junction [69]. In some cases when S. aureus is resistance to cefoxitin but is negative for the mecA gene, they can be tested for mecC gene, which also characterizes S. aureus as MRSA [67].

After confirmation of the mecA gene, it is important to submit the strains to additional molecular characterization. The techniques that are generally used are as follows: staphylococcal protein A (spa) typing [59, 61, 66,67,68], multilocus sequence typing (MLST) [59, 61, 64, 66, 67, 70], pulsed field gel electrophoresis (PFGE) [64,65,66], Staphylococcal cassette chromosome mec (SCCmec) typing [59, 66].

Methicillin-resistant Staphylococcus aureus isolated from food

MRSA has already been isolated from food, indicating that they are present as contaminants in the food production chain [6, 51, 71]. Recently, studies have been performed focused on transmission of MRSA, since its diffusion among food-producing animals and food has increased [2, 7, 72, 73].

The presence of this group of microorganisms was reported mainly in meats such as pork, beef, lamb, chicken, rabbit, and turkey, and also in dairy products, e.g. milk and cheese [51]. This means that the food production chain is a channel of transmission between resistant microorganisms and humans [4]. In this way, the monitoring of genetic characteristics of MRSA is important to better understand its genetic evolution [72].

The following table presents a data survey on studies conducted in different countries where MRSA was isolated from various kinds of food.

As shown in Table 1, a survey of MRSA research over the past 16 years has shown a great occurrence of this type of bacteria in meat, milk, and dairy products, becoming increasingly clear that MRSA is more present in products of animal origin or food-producing animals.

Table 1 Incidence of methicillin-resistant Staphylococcus aureus (MRSA) in foods in different countries
Full size table

MRSA in Brazil

The data survey on the incidence of MRSA in food has been happening also in Brazil. Researches in different regions have been showing the incidence of MRSA in food and food-producing animals, with an emphasis on milk and dairy products and meat and meat products. Furthermore, the importance of researches conducted with samples of food processing environments and food handlers should also be highlighted [65, 89, 90]. Next, studies shall be presented to demonstrate the incidence of S. aureus and specifically MRSA in Brazilian territory. S. aureus is still a common contaminant in food in Brazil; it has been reported as one of the most prevalent causes of FBD in the country according to the Secretary of Health Surveillance [91], and it is commonly identified as the cause of diseases in food-producing animals.

In the Northeast of the country, Soares et al. [92] analyzed the presence of S. aureus in samples of food handlers in public schools of Camaçari, Bahia State. Swabs were collected out of the hands of 166 handlers, 53.3% of the samples were positive for the presence of S. aureus. The results indicated that the food handlers were using inadequate sanitary practices and rethink their training to ensure proper hygiene was essential [92]. Still in the Northeast, a study by Ferreira et al. [93] aimed to evaluate the presence of MRSA in food handlers of public hospitals in the city of Salvador, Bahia. The researchers collected swabs from the nostrils and hands of 140 food handlers in 10 public hospitals; 50% of the handlers had S. aureus on their hands or nostrils and 28.6% had MRSA. These authors also concluded that there is great deficiency in the hygiene of food handlers, which could cause infection in patients [93].

André et al. [94] held in Goiânia a study conducted from February 2004 to March 2005, in which 24 milk samples, 24 samples of Minas Frescal cheese, and 92 samples of food handlers (46 of their hands and 46 of their nostrils) were collected, totaling 140 samples [94]. From these samples, 63 isolates of S. aureus were obtained, corresponding to 32.6% of the nasal swabs, 30.4% of the hand swabs, 67.7% of the milk swabs, and 70.8% of the cheese swabs. The researchers conducted the disk-diffusion test in agar to check the resistance of the strains. Disks impregnated with erythromycin (15 μg), ciprofloxacin (5 μg), tetracycline (30 μg), gentamicin (10 μg), vancomycin (30 μg), oxacillin (1 μg), and penicillin (10 μg) were used. The results showed that 23% of the isolates had resistance to some antibiotic and 5.5% were possibly MRSA [94]. Rodrigues et al. [21] analyzed Staphylococcus spp. isolates from three cheese processing plants including samples of raw milk, food handler, and cheese; a total of 100 isolates were characterized of which 88% were S. aureus and mecA gene was identified in six (6%) strains. In addition, Brazilian producers of milk derivatives were evaluated for the presence and diversity of S. aureus; interestingly, only 7.4% of the samples was positive for S. aureus, and no MRSA was found [89]. Corroborating these results, Silveira-Filho et al. [95] did not detect MRSA in samples of milk and milk derivatives collected in the Northeastern region of the country.

Costa et al. [20] carried out the isolation of S. aureus and identification of MRSA. Samples of different types of meats served in 10 hospitals of the city of Salvador, Bahia, were collected, and a total of 114 raw meat samples (30 chicken samples, 30 beef samples, 24 pork samples, and 30 fish samples) were analyzed. Of the 114 raw meat samples, 28.1% were positive for MRSA. S. aureus was also isolated from 63 cooked meat samples (15 chicken samples, 15 beef samples, 15 pork samples, and 18 fish samples); of these, 9.5% contained MRSA. The high prevalence of MRSA in meat, mainly in food prepared for consumption, emphasizes the need for the best food handling practices in hospitals [20], and also for the best practices in the handling of animals.

A survey conducted with 552 milk samples from 15 dairy farms in the state of Paraíba identified 65 samples which tested positive for S. aureus, and of these, 20 had MRSA and none isolate was resistant to vancomycin [96]; in this case, 30.7% of the samples had MRSA. Recently, an outbreak of bovine mastitis caused by S. aureus in a Brazilian dairy farm was analyzed. Guimarães et al. [97] evaluated 115 milk samples from the herd affected by the disease and found that 53% of the samples had Staphylococcus spp.; of these, 98.4% were positive for S. aureus, and the presence of the mecA gene was identified in 48.3% of the S. aureus isolates. In total, 12.2% of the cases of mastitis were caused by MRSA; this high percentage raises concern for animal and human health [97]. On the other hand, Silva et al. [31] identified methicillin-sensitive S. aureus in milk samples from cows with mastitis. They obtained 56 MSSA isolates from 1484 milk samples of 518 cows in 11 different farms located in Brazil. In the same study, the researchers conducted molecular characterization, gene research, reinforcing the importance that these instruments have for characterizing S. aureus [31].

Rabello et al. [98] developed a research in the state of Rio de Janeiro in which they identified 227 S. aureus isolates in milk samples of cows with subclinical mastitis, with the exception of two that had clinical mastitis. The samples were collected from 18 herds distributed in 9 cities of Rio de Janeiro from July 2001 to July 2004. PCR analysis was performed to amplify 16S rRNA gene for bacteria species identification. The characterization of the strains was important to determine the cause of infection and develop control measures [98].

The study conducted by Monte [65] included 110 staphylococci isolated from 147 samples of 21 semi-extensive dairy farms in the Northeast of Brazil. Of these, 40 of them were S. aureus, most of them isolated from milk samples, two of which presented the mecA gene, indicative of MRSA. The other 70 isolates were coagulase negative, most of them from swabs (52.4%) and environmental samples (29.5%), 14 of these isolates were positive mecA.

Alves et al. [90] collected 64 samples from three dairy products from Minas Frescal artisanal cheese production located in the Midwest region of Goiás, Brazil. These samples include processing environments, raw materials, and final product. Those were confirmed by PCR amplification of the 16S rRNA gene, MLST, and antimicrobial susceptibility test, 33 isolates were confirmed as S. aureus, but only one was identified as MRSA (strain isolated from brine).

Given the above, it is possible to verify that the incidence of S. aureus and MRSA is common in food and in the food production chain, and that researches are still required for a better understanding of the distribution of MRSA and its diversity, with emphasis on analyses that allow verification of similarity of the strains and thus determinate or suggest their origin and, consequently, indicate preventive and control measures.

Conclusion

The use of antibiotics, and more specifically the indiscriminate use of antibiotics, in the treatment of infections caused by Staphylococcus generated a very serious public health problem: the resistance of Staphylococcus spp. to antibiotics. In this review, information about MRSA was gathered from studies published around the world. Herein, the incidence of MRSA in food samples, food handlers, food-producing animals, and food processing environments was presented. The situation is critical since VISA and VRSA are being reported in the treatment of infections caused by MRSA, revealing the difficulty to treat infections and the need for new antibiotics. The need for caution in the use of antibiotics in both human and animal health is emphasized.

In Brazil, researches support that S. aureus is widely present in the food production chain and in final products, thus representing a potential risk to public health. However, researches on MRSA in food in certain regions of the country are still scarce, which may be concealing a reality that is different from the one presented here. Finally, it is worth noting that studies on MRSA are still required because of their significance to public health.