Prevalence of Methicillin-Resistant Staphylococcus aureus in Raw Hamburgers and Ready-to-Eat Sandwiches Commercialized in Supermarkets and Fast Food Outlets in Brazil

Although well known, Staphylococcus aureus is a bacterium that remains widely studied because of its high pathogenic potential and its ability to develop resistance to antibiotics routinely used in clinical practice. The present study investigated the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in hamburgers and sandwiches sold in supermarkets and fast food outlets in Salvador, BA, Brazil. Fifty samples of frozen raw hamburgers (25: beef and 25: chicken) and 50 samples of ready-to-eat sandwiches (25: beef and 25: chicken) were collected and investigated for the presence of MRSA. MRSA was present in 32% of the hamburgers and 8% of the sandwiches. The frequency of MRSA was higher in the samples containing chicken meat. However, the statistical analysis showed no association between MRSA presence and the type of meat investigated (P > 0.05). The high prevalence of MRSA in hamburgers and the presence of the microorganism in ready-to-eat sandwiches are worrying and indicate the need for better control during food preparation to prevent the spread of bacteria.


Introduction
With the reduced time available for food preparation and/or consumption, in recent years, there have been changes in individuals' eating habits, motivated especially by urbanization processes and the professionalization of women, substantially favoring the consumption of processed products and food prepared away from home.Thus, the search for ready-to-eat meals produced on a large scale and with reduced cost has culminated in an increased number of fast-foods outlets and individuals eating in cafeterias and trailers [1]- [3].
Staphylococcus aureus is considered the third-most important cause of food-borne disease in the world [4] and has two aggravating characteristics, toxin production and antimicrobial resistance.This pathogen is considered an excellent indicator of thermal processing inefficiency, inadequate hygienic conditions during food production/preparation, or inadequate cooling after food preparation [5].
Regarding antimicrobial resistance, S. aureus is one of the most microorganisms implicated in cases of nosocomial infection worldwide.The methicillin-resistant Staphylococcus aureus (MRSA) was considered a nearly exclusively responsible pathogen in hospital-acquired infections between 1930 and 1980; however, at the end of 1990, MRSA infections among healthy individuals began to emerge [6] [7].
S. aureus resistance to antibiotics is due to the production of a penicillinase, an enzyme that inactivates the beta-lactâmico ring of the penicillin molecule, making the antibiotic ineffective against S. aureus.Methicillin resistance presented by S. aureus is mediated by the presence of the mecA gene, which encodes the 2a protein (PBP2a) found in the cell wall of the microorganism [7]- [10].
In particular, strains of MRSA harboring genes encoding the Panton-Valentine leukocidin (PVL) toxin are of concern from a public health perspective because they are usually capable of causing severe skin and soft tissue infections and occasionally necrotizing pneumonia, which is associated with high mortality [11].
This study aimed to investigate the occurrence of methicillin-resistant S. aureus (MRSA) in sandwiches and hamburgers marketed in Salvador, BA, and evaluate the association between MRSA isolates and investigated meat.

Sampling and Laboratory Procedures
Fifty samples of frozen raw hamburgers (25 beef and 25 chicken) and 50 samples of ready-to-eat sandwiches (25 beef and 25 chicken) were collected from supermarkets and fast food outlets, respectively, in the city of Salvador, Bahia, in northeastern Brazil, from September 2013 through December 2013.
To detect MRSA, approximately 500 g of frozen hamburger and 200 g of ready-to-eat sandwiches were aseptically collected in supermarkets and fast food outlets, respectively, and transported to the laboratory for analysis.Prior to analysis, hamburger samples were defrosted in the original package under refrigeration for 24 hours.For sandwiches, the meat (beef or chicken) and bread portions were separated, and the bread was discarded.Approximately 25 g of each food was added to 225 ml of Tryptic Soy Broth (TSB, Difco, Detroit, MI, USA) supplemented with 6.5% NaCl and homogenized in a Stomacher (240 bpm; ITR model 1204, series 126, Esteio, RS, Brazil) for 2 min in a class II biosafety cabinet (Labconco Corporation, Labconco Purifier Class IIb, Total Exhaust, model 36210-04, certified ISO 9002, Kansas City, MO, USA).This homogenate was then incubated at 37˚C for 16 -20 h.Next, a 1-ml aliquot was added to 9 ml of Phenol Red Mannitol Broth (PHMB, HiMedia, São Paulo, SP, Brazil) containing ceftizoxime (5 μg/ml) and aztreonam (75 μg/ml) (Sigma-Aldrich Brazil Ltda, São Paulo, SP, Brazil).From the culture obtained in PHMB, the surface of the hypertonic mannitol agar (HMA, HiMedia, São Paulo, SP, Brazil) was inoculated with a sterile loop.The plates were incubated for 24 h at 37˚C and examined for typical colonies, Gram, catalase and coagulase.For confirmation, a maximum of five typical colonies per plate were purified on Tryptic Soy Agar (TSA, Difco, Detroit, MI, USA) and subjected to antibiotic susceptibility testing using the disc diffusion method [12].

Statistical Analysis
The Pearson's chi-square (X 2 ) (SPSS 17.0 for Windows) was used to determine significant differences (P < 0.05) in MRSA contamination between the investigated meats.

Results and Discussion
A total of 34 hamburger samples were positive for coagulase-positive Staphylococci, 16 (64%) hamburgers made with beef and 18 (72%) made with chicken meat.For the sandwiches, 7 samples were contaminated, 5 (20%) sandwiches made with beef and 2 (8%) made with chicken meat (Table 1).
The MRSA prevalence results in the samples are summarized in Table 2. MRSA strains were isolated from 32% of the hamburger samples and 8% of the sandwiches.
Although the hamburger samples made with chicken meat had the highest prevalence of MRSA, no association between MRSA contamination and type of meat investigated was observed (P > 0.05).
The resistant/susceptibility profile of the coagulase-positive Staphylococci isolated from the samples is demonstrated in Table 3 and Table 4.In chicken meat samples from sandwiches, resistance equal or higher than 50% was observed for penicillin G (66.7%), erythromycin (50.0%) and oxacillin (66.7%) (Table 3).For isolates from hamburgers made with beef or chicken meat, resistance was always lower than 50% (Table 4).
In Japan, only two strains of S. aureus, both isolated from chicken thigh muscle parts, have been identified as MRSA, suggesting that food handlers have accidentally transmitted the pathogen [14].Improper handling is currently the primary source of contamination of foodstuffs, and proper hand hygiene, with warm water and soap and antiseptic, would prevent such contamination [20].
Here, the presence of MRSA in ready-to-eat sandwiches indicates that the beef and chicken meat were      insufficiently cooked or that cross-contamination occurred after cooking in sandwich preparation by an asymptomatic food handler or utensils.In Brazil, Costa [21] reported that some preparations of meats ready for consumption were contaminated with MRSA.The author considered the occurrence of contamination after the preparation of the meat dishes by asymptomatic carriers or contaminated utensils used in the distribution of meals, once the thermal processing was sufficient to eliminate the microorganism.
According to the results of the present study, 66.7% of the coagulase-positive Staphylococci isolated from samples of chicken sandwiches were resistant to penicillin, a drug used in human and veterinary medicine.For beef sandwiches, this resistance was observed in only 23.1% of the isolates.
In Brazil, the resistance of the S. aureus to penicillin varies from 20% to 100%, whereas the percentage of resistance to other antimicrobials is lower [22].In several countries, however, large-scale studies have shown that resistance of the microorganism to penicillin is approximately 60%, and studies from 11 different countries have demonstrated that the prevalence of samples resistant to various antibacterials routinely used to treat mastitis was generally low [23].
Susceptibility to vancomycin, the drug of choice for MRSA treatment in humans, was observed in all isolates from samples of beef sandwiches and beef hamburgers, whereas 75% of the isolates from chicken sandwiches were susceptible (Table 3).These results indicate that selecting vancomycin for MRSA infection treatment is suitable.
Cefoxitin was recently shown to be a marker of resistance to MRSA.However, the results demonstrated that 100% of the isolates of sandwiches samples were susceptible to this marker (Table 3).For hamburger samples, similar results were found (Table 4).
Fessler et al. [17] demonstrated that all MRSA isolates were resistant to oxacillin, and 62.5% exhibited multidrug resistance.Hanson et al. [18] showed that MRSA isolated from samples of pork, beef, chicken, and turkey were resistant to oxacillin and were multidrugresistant.Van Loo et al. [15] found similar results to those of Hanson et al. [18]; i.e., samples of pork and beef were contaminated with MRSA, and all isolates were resistant to oxacillin and cefoxitin.Recently, Lin et al. [24] found that S. aureus ST398 isolated from retail foods were most frequently resistant to penicillin (100%), followed by trimethoprim, erythromycin, ampicillin (each 86.7%), clindamycin (80.0%), and tetracycline (26.7%).All ST398 isolates were susceptible to amikacin, chloramphenicol, cefoxitin, gentamicin, oxacillin, and vancomycin.
According to Hososaka et al. [25], some clinical isolates of S. aureusare mec A-positive and susceptible to oxacillin.The presence of this gene was verified in strains of S. aureus identified as methicillin-susceptible (MSSA) that were isolated from hamburgers [26].
Due to the growing problem of microbial resistance, the outlook for the use of antimicrobial drugs is uncertain.Consequently, research intending to increase antibacterial activity and characterize the interaction mechanism with antibiotics is needed.

Conclusion
The high prevalence of MRSA among the tested foods, primarily in raw hamburger made from chicken meat, and the presence of the microorganism in prepared ready-to-eat sandwiches highlights the necessity of enforcing hygienic practices within the food industry and fast food outlets.Better knowledge of the transmission routes of MRSA in the food chain, "from the farm to the fork", is necessary to provide tools for preventing the spread of MRSA, clarify the possible health hazards for consumers related to the presence of MRSA in foods, and determine adequate measures for MRSA control.

Table 1 .
Frequency of isolation of coagulase-positive Staphylococci in hamburger and sandwich samples.

Table 2 .
Frequency of MRSA isolation in hamburger and sandwich samples.

Table 3 .
Antimicrobial activity of antibiotics against positive-coagulase Staphylococci isolated from sandwiches.

Table 4 .
Antimicrobial activity of antibiotics against coagulase-positive Staphylococci isolated from hamburgers.