Prevalence and Characterization of Antibiotic-Resistant Staphylococcus aureus Recovered from Pasteurized Cheese Commercialized in Panama City Markets

Laboratory of Experimental and Applied Microbiology, University of Panama, Panama, Panama Panamanian Food Safety Authority, Panama City, Panama Laboratorio Nacional para La Investigación en Inocuidad Alimentaria (LANIIA) Del Centro de Investigación en Alimentación y Desarrollo (CIAD), Sinaloa, Mexico Panama Canal Authority, Panama City, Panama Sistema Nacional de Investigación (SNI) SENACYT, Panama City, Panama


Introduction
Cheese, characterized by its snow-white color, crumbly texture, and a lightly salted flavor is an essential ingredient of Panamanian cuisine. Cheese is rich in nutrients, including vitamins, proteins, lactose, fats, minerals, and water. erefore, cheese favors the growth and survival of bacterial enteric pathogens such as Listeria monocytogenes (L. monocytogenes), Salmonella, Shiga-toxin producing Escherichia coli (STEC), and Staphylococcus aureus (S. aureus) [1][2][3][4]. Hand manipulation of processed cheese along with its high moisture content and barely acidic pH are factors that contribute to the presence and growth of foodborne pathogens [5,6]. In Panama, cheese is either sold to wholesalers or directly to end consumers, usually in small markets and/or through door-to-door services, using no labels and undergoing minimum food-safety precautions in its transportation and storage [5,7,8]. e lack of hazard analysis and critical control points (HACCP) or good manufacturing practices (GMP) and good hygiene practices (GHP) at the processing plant, along with hand manipulation by its personnel, increase the risk of cheese contamination [9,10].
S. aureus is an important foodborne pathogen that causes a wide spectrum of infections in humans, from mild skin infections, bacteremia, systemic disease, or osteomyelitis to more life-threatening infections, such as toxic shock syndrome and staphylococcal food poisoning (SFP) [11,12]. S. aureus is one of the main causative agents of SFP associated with cheese consumption [13]. In the United States of America (USA) and according to the last report available from 2017, the annual incidence of foodborne outbreaks was 841 resulting in 14,481 illnesses, including 827 hospitalizations and 20 deaths, with 2 of them reported as SFP outbreaks [14]. An estimated 77 million people in the Americas suffer an episode of foodborne illness each year, where children under the age of 5 represent 40%, with 125,000 deaths a year [15]. For example, in Ecuador, a total of 19,487 cases were reported in 2019, 12,203 of which were caused by bacteria, eventhough the specific causative agents were not indicated except for Salmonella and Shigella outbreaks [16]. In Panama, 277,286 illnesses associated with foodborne pathogens were reported in 2019 [17]. However, no updated data about the microorganisms responsible of these outbreaks is available since 2002, when twenty-eight percent (28%) of the investigated foodborne outbreaks were related to SFP, while E. coli was responsible for sixty-two percent (62%) [18].
Antibiotic-resistant S. aureus has been isolated from milk and cheese in many parts of the world [19][20][21][22][23]. To exacerbate the food-safety breach of S. aureus in cheese, some strains are able to elude antibiotic treatments. ese strains, very often referred to as antibiotic resistant strains, have shown multiple antibiotic resistance patterns [21,24].
is resistance to various antibiotic groups can be mediated by the bacterial chromosome, plasmids, and transposons or gene cassettes that are incorporated into integrons [25,26], thus making the gene transfer among bacterial strains an easier process. Antibiotic resistance among bacteria is a major public health issue as a result of their persistent circulation in the environment and its consequent contamination of cheese [27]. Besides monitoring the antibiotic resistance of S. aureus in cheese, it is of great importance to prevent the spread of multidrug-resistant strains, which may have multiple undesirable consequences. erefore, the aim of this study was to evaluate S. aureus concentration levels and its antibiotic resistance pattern, present in locally produced and commercialized pasteurized cheese in Panama City.

Study Area and Sample Collection.
A total of five brands of pasteurized fresh white cheese sold in Panama City were analyzed. Five samples of each pasteurized fresh white cheese brand were collected from different independent local supermarkets, where all these cheeses are wrapped and kept under refrigeration at 4°C. Sample collections were done in five field visits in a row and were placed in sterile bags, numbered, and brought in the cold chain to the Laboratory of Experimental and Applied Microbiology (LAMEXA) of the University of Panama and stored at 4°C until processing. e analysis was carried within 24 h upon collection.

2.2.
Isolation and Identification of S. aureus. S. aureus was isolated and counted using the method described in the bacteriological analytical methods [28]. Briefly, 25 g of each sample was weighed aseptically into sterile stomacher bags (Seward Medical Stomacher Bags © Seward, Germany) containing 225 mL of sterilized 0.1% (w/v) peptone water (Difco, Mexico City, MEX) and homogenized for 2 min, followed by serial 10-fold dilutions until 1 × 10 −6 . Bacteriological analysis was performed by plating 0.1 mL of each dilution into triplicates on Baird-Parker agar (BP, Oxoid, Basingstoke, Hampshire, UK), supplemented with egg yolk tellurite emulsion (Oxoid, Basingstoke, Hampshire, UK). All the analyses were conducted under aseptic conditions. e plated cultures were then incubated at 37°C for 48 h. Colonies that exhibited the characteristics of S. aureus morphology (circular black colonies with an opaque zone within an outer clear zone) were considered for further confirmation.

Coagulase Test and Enumeration of S. aureus.
All presumptive S. aureus isolates were subjected to the coagulase test as described by Koneman et al. [29]. e number of S. aureus was reported as CFU/g of a tested sample, taking into account the total colonies counted (TC), the number of coagulase-confirmed colonies (CC), the selected presumptive colonies (SC), the dilution (D), and inoculated volume (V) onto BP agar. e formula employed is

Assessment of the Microbiological Quality of Pasteurized
Cheese. According to guidelines set by the Laboratory Methods in Food Microbiology (LMFM) [30], the microbiological quality of pasteurized cheese can be grouped into two different categories based on their bacterial counts of S. aureus: (1) acceptable and (2) unacceptable/potentially hazardous. A cheese sample with S. aureus counts, greater or equal to 1 × 10 3 CFU/g, is considered unhealthy for human consumption; therefore, it fits into category (2).

Antibiotic Susceptibility Testing. Coagulase-confirmed
S. aureus colonies were tested for susceptibility to different antibiotics, using the disk-agar diffusion method in accordance to the Clinical and Laboratory Standards Institute [31]. Inoculums from each S. aureus isolate were grown aerobically in 5 mL of Müller-Hinton broth (Bioxon, Mexico City, MEX) and were incubated at 37°C to reach a turbidity equal to a MacFarland 0.5 standard.  [31]. Details of the antimicrobials used in this study are given in Table 1.
2.6. Statistical Analysis. Statistical differences were determined by performing a variance analysis with a significant level of 0.05%. e software SPSS Statistic 22 [32] was used for the analysis, and a p value ≤0.01 was considered statistically significant.

Bacteriological Counts and Microbiological Quality of Pasteurized Cheese.
A total of 25 pasteurized cheese samples from five different brands (five samples per brand) were screened for the presence of S. aureus ( Figure 1). All the suspected S. aureus colonies were subjected to the coagulase test for confirmation. Coagulase-positive colonies were counted, and the levels of contamination of each brand were determined, giving the following results: the five samples analyzed from brand 1 were positive for the presence of S. aureus with a mean value of 6 × 10 3 CFU/g; two positive samples were detected in brand 2 (mean value of 2 × 10 4 CFU/g); four positive samples in brand 3 (mean value of 1.6 × 10 4 CFU/g); and 1 positive sample in brand 5 (mean value of 3.6 × 10 4 CFU/g). us, according to the Laboratory Methods in Food Microbiology, these four brands of pasteurized cheese are considered unsatisfactory for human consumption. On the other hand, brand 4 did not show any contamination by S. aureus, and therefore, its consumption is safe.

Antibiotic Susceptibility of S. aureus Isolates.
Nine antimicrobial agents of veterinary and human health relevance, from different antibiotic classes, were tested. A total of one hundred S. aureus isolates were analyzed for antimicrobial susceptibility. Forty-nine of them were resistant to at least one of the antibiotics tested. A summary of the percentage of S. aureus strains, resistant to these antibiotics, is provided in Table 1. From all the examined brands, we were able to detect S. aureus isolates showing susceptibility to some of the following six antibiotics: GEM, VA, OX, OFX, TE, and RA (Table 1). By contrast, fifty-five percent (55%) of the S. aureus isolates tested showed no susceptibility to E. An intermediate resistance to CHL and CC was observed in fifty-one percent (51%) and thirty-seven (37%) of the isolates, respectively. However, a significant resistance to CC was observed in thirty-five (35%) of the S. aureus isolates ( Table 1). Although it is not one of the highest levels of resistance encountered in the study, the result is still startling, considering CC as one of the most common antibiotics used to treat nonlife-threatening human infections with S. aureus. Susceptibility to two or more of the nine antibiotics tested was observed in forty-two percent (42%) of the S. aureus isolates (21 out of 49). A total of fourteen distinct antibiotic multiresistance profiles were detected ( Table 2). Pattern A showed 4 phenotype resistance profiles to two antibiotics (A1-A4) and was expressed by seven isolates (14%): A1 with four isolates showing resistance to E-TE; pattern B1-B6 showed resistance to three antibiotics (20%, 9/49) including the resistance profile to CC; pattern C1, C2, and D1 exhibited resistance to four antibiotics (4%, 2/49) and to five antibiotics (2%, 1/49), respectively. One out of the forty-nine S. aureus isolates showed resistance to eight out of the nine antibiotics being tested. e results showed a multidrug resistance profile to more than two agents from different antibiotic classes in twenty S. aureus isolates out of forty-nine representing forty-two percent (42%). Multidrug resistance is frequently found in strains of human origin, whereas in veterinary medicine, it is occasionally reported [33,34]. e brands with the highest antibiotic resistance profiles were 2 (8 antibiotic resistances) and 5 (6 resistances), followed by 1 and 3 with 5 and 2 antibiotic resistances, respectively.

Discussion
S. aureus is a foodborne pathogen that has been linked to various types of foodborne outbreaks related to the consumption of cheese, which have caused a significant impact on health, economic, and trade issues. Regarding to the analyzed five pasteurized cheese brands, four of them were not suitable for human consumption due to the high levels of bacteria found [35], according to guidelines set by the Laboratory Methods in Food Microbiology (LMFM) [30]. A cheese sample with S. aureus counts, greater or equal to 1 × 10 3 CFU/g, is considered unhealthy for human consumption, concluding that only brand 4 fits the criteria. e coagulase test is a tool to discriminate Staphylococcus pathogenic species [36]. In the present study, the total prevalence of coagulase-positive staphylococci was eighty percent (80%). e rate of contamination is higher than that observed in the survey conducted by Normano et al. [27], in which the total prevalence of coagulase-positive staphylococci in dairy products was only 17.3%. Nusrat et al. [37] showed that forty-seven percent (47%) of cheese samples harbored S. aureus. e presence of coagulase-positive Staphylococcus in the cheese analyzed in this study, elaborated with pasteurized milk, may be an indicative of sanitary deficiencies in the storage and retailing of the product [27]. On the other hand, it has been demonstrated that S. aureus may enter via contaminated raw milk due to subclinical mastitis in cows, and due to improper food handling, it may promote its colonization throughout the processing plant and consequently contaminate the final product [9,38,39]. e robustness of S. aureus in food increases the risk of staphylococcal enterotoxins production because the bacteria can only be eliminated through a thermal process, but the toxins may remain and cause SFP [40,41]. e diagnostic criterion for SFP is based upon the detection and recovery of 1 × 10 5 CFU/g of S. aureus [11,42]. Coagulase-positive colonies counted were 6 × 10 3 CFU/g for brand 1 and 3.6 × 10 4 CFU/g for brand 5 (Figure 1), and thus, both meet the diagnostic criterion for SFP that is based upon the detection and recovery of less than 1 × 10 5 CFU/g of S. aureus. However, since values are high, especially for brand 5, it would be worthy to analyze the presence of S. aureus toxins that would represent a risk for public health. e lack of critical control points in the production process of cheese may also contribute to the survival and outgrowth of the pathogen [10], although we cannot exclude that handling conditions at the supermarket level might have also an impact. us, these results indicate an urgent need for implementation of food hygiene standards in the food industry.
On the other hand, the use and inappropriate prescriptions of antibiotics in the public, private, and agricultural sectors are considered the main factors of the increase in bacterial antimicrobial resistance [43,44]. High levels of

Class of antibiotic Antibiotic Abbreviation
Inhibition zone (mm) a Antibiotic profile antibiotic resistance were found in this study. A high percentage of resistance to erythromycin was observed, and this may be attributed to its frequent use in the treatment of human infections. In fact, both nasal and hand contamination of food from food handlers with multiresistant and virulent S. aureus has been reported. For example, antibiotic resistant and enterotoxin gene-positive S. aureus have been isolated from nasal swabs and hand fingerprints of food handlers in central Iran, Hong Kong, Portugal, or Malaysia [45][46][47][48].
e results of the present study demonstrated that S. aureus isolates from pasteurized cheese in Panamanian markets were resistant to antibiotics from the macrolides class. ese results are in agreement with Montoya's [49], which showed S. aureus resistance to macrolides such as erythromycin and clindamycin from the lincosamide class. Multidrug resistance was observed in forty-two percent (42%) of the S. aureus isolates, including all the antibiotic classes used in the present study (Table 2), as reported by Rivera-Salazar et al. [21]. Whether the differences in the antibiotic resistance profile observed in our work compared with those reported by Nusrat et al. [37] are due to different cheese characteristics (such humidity or acidity), manufacturing, distribution, or handling cannot be addressed, since authors do not describe these data. It would be interesting to study if the antibiotic resistance profiles of S. aureus isolates from food handlers as a source of dissemination would explain these differences between regions.

Conclusions
e occurrence of S. aureus in pasteurized cheese sold in Panama City's retail markets possesses a risk for consumers of these food items.
e low microbiological quality of pasteurized cheese in Panama City markets suggests that the bacterium is likely to be found in other Panamanian markets with similar or even poorer sanitary conditions, justifying worker-vendor training and further research on the persistence of the pathogen, as well as the application of effective corrective actions at the market level. Consequently, continuous surveillance of S. aureus in Panamanian retail markets will help identify pathogenic sources and promote the development of better practices for processing and selling food. It should also improve the legislative framework that governs the presence of pathogens in food. Lastly, these data confirm that good hygienic practices and temperature control are critical for S. aureus control and prevention in food at retail markets.

Data Availability
No data were used to support this study.

Conflicts of Interest
e authors declare that they have no conflicts of interest.