Prevalence and Multidrug Resistance Pattern of Methicillin Resistant S. aureus Isolated from Frozen Chicken Meat in Bangladesh

Infections by methicillin-resistant Staphylococcus aureus (MRSA) are continuously expanding within the community. Chicken meat is usually contaminated by MRSA, and this contaminated chicken meat is an important source of foodborne infections in humans. In this study, a cross-sectional supershop survey was conducted to determine the prevalence and antimicrobial resistance pattern of MRSA in 113 domestic frozen chicken meat samples purchased from nine branded supershops available in five divisional megacities of Bangladesh. The study also focused on the determination of methicillin resistance gene in MRSA isolates. S. aureus was identified by standard culture-based and molecular methods, and subjected to antimicrobial susceptibility testing. MRSA was screened by cefoxitin disk diffusion test. Methicillin resistance gene was identified by PCR. Of samples, 54.9% were positive for S. aureus, and, of these, 37.1% isolates were identified as MRSA. All the isolates were multidrug resistant (MDR): 52.2% were resistant to 6–8 antimicrobial classes, and 47.8% isolates to 9–12 classes. Three (3.2%) isolates of S. aureus were possible extensively drug resistant. The highest rates of resistance were observed against cefoxitin (100%), followed by nalidixic acid, ampicillin and oxacillin (97.7%), colistin (91.3%), amoxicillin-clavulanic acid and amoxicillin (87%), penicillin-G and cloxacillin (82.6%), oxytetracycline (78.3%), and cefixime (73.9%). Screening of methicillin resistance gene revealed that 43.5% isolates of MRSA were positive for mecA gene. The high prevalence of MDR MRSA in frozen chicken meat samples in this study emphasizes the need for better sanitary education of food handlers in hygienic practices focusing on their potential role as reservoirs and spreaders of MRSA.


Introduction
Foodborne diseases (FBDs) are a significant general well-being concern worldwide [1]. The World Health Organization (WHO) characterizes FBD as "disease of infectious or toxic nature caused by, or thought to be caused by, the consumption of food or water" [1]. Staphylococcus aureus (S. aureus) is one of the most important causes of FBD, causing an expected 241,000 illnesses each year in the United States [2]. S. aureus is an opportunistic pathogen in human and animals, and is the third largest cause of food related illness throughout meat had been widely consumed for its high value in protein and nutrient substance including low cost and availability. However, chicken meat is usually contaminated by antibiotic resistant strains of S. aureus, especially MRSA, mostly due to improper handling by food-handlers and poor sanitation practices, and thus poses a great risk in the food safety [12]. Contaminated chicken meat is one of the significant causes of FBDs in humans worldwide. Research has been conducted in Bangladesh on S. aureus contamination and AMR patterns in raw chicken meat from live bird markets [19][20][21]. Nowadays, city dwellers of Bangladeshi consumers prefer to buy their all essential daily commodities including frozen chicken meat and meat products from supershops because of easy availability, and this frozen chicken meat takes minimal processing for cooking, and also saves consumers time. However, contamination of frozen chicken meat with S. aureus, especially MRSA, has an important concern of food safety and public health hazards in Bangladesh. One inland study reported S. aureus contamination in preprocessed raw chicken meat collected from three supershops of Dhaka city [22], and another study reported the contamination of processed raw meat with MRSA [23]. However, prevalence and MDR pattern of MRSA as well as detection of methicillin resistance gene isolated from frozen chicken meat samples have not been investigated thoroughly and are not well documented in Bangladesh. Therefore, particular attention is required to be paid regarding studying the prevalence and AMR patterns of MRSA isolated from frozen chicken meat covering more outlets of available branded supershops to take effective measures in limiting the contamination of frozen chicken meat with MRSA and to protect consumers' health. Therefore, the objective of the present study was to determine the prevalence of MRSA and their AMR pattern isolated from frozen chicken meat of different supershops across different divisional megacities in Bangladesh. This study also focused on the determination of methicillin resistance gene in MRSA isolates. Such information is useful for better understanding of the risk of exposure to MRSA through food, particularly chicken meat.

Sample Collection
From April to December 2019, a cross-sectional supershop survey was conducted in 40 outlets of nine different supershops in five divisional megacities (Dhaka, Chattogram, Sylhet, Rajshahi, and Mymensingh) of Bangladesh. A total of 113 domestic frozen chicken meat samples (82 broilers, 31 cockerels) including whole chicken or meat cuts (breast, drumstick, leg, and wing muscle) were purchased from these outlets. Each supershop had its own branded frozen chicken meat. On purchase, each sample was placed in a separate sterile tightly sealed plastic bag and kept in a cold box at a temperature lower than 4 • C for transportation. On arrival at the laboratory, frozen chicken meat in the sealed plastic bag was placed in a cool area until it defrosted. In addition, data on brand name, source of chicken, processing and packaging of meat, and special labels (e.g., green or organic chickens) were also collected.

Isolation and Identification of S. aureus
Isolation and identification of S. aureus were performed according to the EN ISO 6888-1 standard procedure of the International Organization for Standardization [24]. For pre-enrichment of bacteria, a 25 g portion from each piece of meat sample was chopped into very small fine pieces, homogenized with 225 mL of buffered peptone water, and incubated at 37 • C for 18-24 h. After pre-enrichment in buffered peptone water, 1 mL of the culture was mixed with 5 mL of nutrient broth and incubated for 24 h at 37 • C. Then, a loopful of culture broth was streaked onto Mannitol Salt Agar in duplicate and incubated at 37 • C for 24 h. Three presumptive S. aureus yellow color colonies with yellow zones from each selective agar plate were picked, and then subcultured to obtain a pure culture. Gram staining, catalase, and coagulase tests were performed with the pure culture. All presumptive S. aureus isolates were subjected to DNA extraction using the "boiling" method as described earlier [25]. A duplex PCR was carried out for the confirmation of S. aureus with two sets of genus-and species-specific primers. Primers used were Staph756F (5 -AACTCTGTTATTAGGGAAGAACA-3 ) and Staph750R (5 -CCACCTTCCTCCGGTTTGTCACC-3 for the amplification of 756 bp from 16S rRNA gene, and Nuc450-F (5 -AGTATATAGTGCAACTTCAACTAAA-3 ) and Nuc450-R (5 -ATCAGCGTTGTCTTCGCTCCAAATA-5 ) for the amplification of 450 bp from nuc (thermonuclease) gene [26,27]. The PCR reaction mixture (25 µL) consisted of 12.5 µL of OneTaq Quick-Load PCR master mix with standard buffer (New England BioLabs Inc., Ipswich, MA, USA), 1.5 µL (15 pmol) each of forward and reverse primers, 2 µL of DNA template, and 4.5 µL of nuclease-free water. The thermal profile consisted of initial denaturation at 94 • C for 5 min, 30 cycles of separation at 94 • C for 1 min, primer annealing at 55 • C for 1 min, and extension at 72 • C for 2 min with a final elongation at 72 • C for 10 min. After amplification, PCR products were analyzed by gel electrophoresis on 1.5% UltraPure™ Agarose gel was stained with ethidium bromide (5 µg/mL) including a 100-bp DNA ladder (New England BioLabs Inc., Ipswich, MA, USA) which served as a molecular weight marker. The resulting band of PCR product was visualized under UV transilluminator and photographed. All PCR-confirmed S. aureus isolates were stored on nutrient broth containing 50% (v/v) glycerol at −20 • C for further study.

Screening of Methicillin-Resistant S. aureus (MRSA)
Phenotypically, MRSA were detected by Cefoxitin Disk Diffusion method as per CLSI guidelines [28]. Briefly, for each isolate, a minimum of four to five S. aureus colonies isolated from an overnight growth were transferred to nutrient broth. Bacterial suspensions in nutrient broth at a density equivalent to a 0.5 McFarland standard was inoculated onto Mueller-Hinton agar plate in duplicate with cefoxitin (30 µg), oxacillin (1 µg), cloxacillin (5 µg), and methicillin (5 µg) disk. The plates were incubated at 37 • C for 24 h. The isolates of S. aureus that showed resistance to cefoxitin (≤21 mm zone diameter) were considered MRSA.

Detection of Methicillin Resistance Gene
A uniplex PCR targeting methicillin resistance gene (mecA) in S. aureus was standardized, and used in this study with specific primer as described earlier [27]. The sequence of the forward primer was MecA1 (5 -GTAGAAATGACTGAACGTCCGATAA-3 ) and of the reverse primer was MecA2 (5 -CCAATTCCACATTGTTTCGGTCTAA-3 ) for the amplification of 310 bp. Each PCR reaction mixture was constituted in a final reaction mixture of 25 µL made up of 12.5 µL PCR master mix (New England BioLabs Inc., Ipswich, MA, USA), 1.5 µL (15 pmol) each of forward and reverse primers, 7.5 µL of nuclease-free water, and 2 µL of DNA template. Amplification was performed by using a Veriti 96-well thermal cycler (Thermo Fisher Scientific) with this thermal profile: heating at 94 • C for 5 min, followed by 30 cycles of denaturation at 94 • C for 1 min, primer annealing at 55 • C for 1 min, extension at 72 • C for 2 min, and a final extension step for 10 min at 72 • C. Positive (mecA gene) and negative (sterile phosphate buffer saline) controls were included in each run of PCR. After amplification, PCR product was subjected to electrophoresis on 1.5% UltraPure™ agarose gel containing ethidium bromide (0.5 µg/mL). The resulting band of PCR product was examined under UV-transilluminator and photographed. A 100-bp molecular weight standard ladder was included on each run.

Data Analyses
Data were extracted, entered into a spreadsheet (Microsoft Excel) and transferred into SPSS software v22.0 (IBM Corp., Armonk, NY, USA) for statistical analysis. Descriptive statistics were used to compute the prevalence of S. aureus and resistance percentage. The significant differences in prevalence of S. aureus and resistance percentage among different brands, sampling area, chicken types, production types, and meat types were determined using chi-square test (Z-test for proportions) and Fisher's exact test (wherever appropriate). The association between resistance phenotypes (outcome) and their corresponding methicillin resistance genes was analyzed by binary logistic regression. The level of significance was set at p ≤ 0.05. An UpSet plot was constructed to show the antimicrobial resistance pattern of methicillin-resistant S. aureus and methicillin-susceptible S. aureus by using online tools (https://asntech.shinyapps.io/intervene/ (accessed on 18 March 2021)).

Prevalence and Distribution of Methicillin-Resistant S. aureus and Methicillin-Susceptible S. aureus
Among 113 frozen chicken meat samples, the overall prevalence of S. aureus was 54.9% (n = 62) ( Table 1). All isolates of S. aureus produced expected product size of 450 bp by PCR ( Figure 1). Of the 62 S. aureus isolates, 23 (37.1%) were phenotypically identified as methicillin-resistant S. aureus (MRSA) based on cefoxitin disc diffusion test, and the remaining 39 (62.9%) isolates were methicillin-susceptible S. aureus (MSSA) ( Table 1).  The distribution of MRSA and MSSA related to brands, divisions, chicken types, production types, and meat types is also summarized in Table 1. Among the nine brands, the prevalence of S. aureus from brand 7 (100%) was highest than those from brand 4 (11.3%). S. aureus was not recovered from frozen chicken meat of brand no. 8 and brand no. 9. The prevalence of MRSA among different brands varied from 36.8% to 50%, whereas the prevalence of MSSA varied from 50% to 100%. A significant difference was observed in the prevalence of MSSA among brands. Regarding division-wise distribution, the prevalence of MRSA was significantly higher in Chattogram division (66.7%) than Sylhet (33.3%) and The distribution of MRSA and MSSA related to brands, divisions, chicken types, production types, and meat types is also summarized in Table 1. Among the nine brands, the prevalence of S. aureus from brand 7 (100%) was highest than those from brand 4 (11.3%).
S. aureus was not recovered from frozen chicken meat of brand no. 8 and brand no. 9. The prevalence of MRSA among different brands varied from 36.8% to 50%, whereas the prevalence of MSSA varied from 50% to 100%. A significant difference was observed in the prevalence of MSSA among brands. Regarding division-wise distribution, the prevalence of MRSA was significantly higher in Chattogram division (66.7%) than Sylhet (33.3%) and Dhaka divisions (38.3%), while the highest prevalence of MSSA was found in Mymensingh and Rajshahi divisions (100%) than Chattogram division (33.3%). Chicken type-wise distribution revealed that there was significant difference in the prevalence of both MRSA and MSSA between broiler and cockerel chickens. MRSA prevalence in broiler and cockerel chickens were 45.2% and 20%, respectively, whereas the MSSA prevalence was 54.8% and 80%, respectively. When looking at the production type-wise distribution of MRSA and MSSA, there was no significant difference between production types. Meat type-wise distribution revealed that 100% of leg muscles were contaminated with MRSA, however, 73.9% of whole chicken pool sample were contaminated with MSSA.

Antimicrobial Resistance Pattern
Of the 62 S. aureus isolates, two isolates (3.2%) were possible extensively drug resistant (pXDR); it showed resistance to 11-12 of 14 antimicrobial classes. Regarding brand-wise distribution, it was observed that the pXDR S. aureus isolates were mostly observed in brands 1 and 3.

Phenotypic and Genotypic Association of Antimicrobial Resistance
In case of MRSA, among the seven isolates resistant to cefotaxime, six isolates (85.7%) carried the mecA gene (Table 3). For the 15 isolates resistant to gatifloxacin and azithromycin, the mecA gene was detected in nine isolates (60%). Eight (8/12, 66.7%) of the ciprofloxacin and ofloxacin resistant isolates also carried the mecA gene. However, these phenotypes were positively associated (OR > 1) with the presence of the mecA gene. For MSSA, a certain percentage of isolates (10/31, 32.3%) resistant to penicillin-G harbored the mecA gene (Table 3). Penicillin-G resistance was negatively associated with the presence of the mecA gene (OR = 0.1, p = 0.02). Table 3. Association between antimicrobial resistance phenotypes and methicillin resistance mecA gene in MRSA and MSSA isolated from frozen chicken meat.

Phenotypic and Genotypic Association of Antimicrobial Resistance
In case of MRSA, among the seven isolates resistant to cefotaxime, six isolates (85.7%) carried the mecA gene (Table 3). For the 15 isolates resistant to gatifloxacin and azithromycin, the mecA gene was detected in nine isolates (60%). Eight (8/12, 66.7%) of the ciprofloxacin and ofloxacin resistant isolates also carried the mecA gene. However, these phenotypes were positively associated (OR > 1) with the presence of the mecA gene. For MSSA, a certain percentage of isolates (10/31, 32.3%) resistant to penicillin-G harbored the mecA gene (Table 3). Penicillin-G resistance was negatively associated with the presence of the mecA gene (OR = 0.1, p = 0.02). Table 3. Association between antimicrobial resistance phenotypes and methicillin resistance mecA gene in MRSA and MSSA isolated from frozen chicken meat.

Discussion
The present study provides the first comprehensive evidence on the extent and distribution of methicillin-resistant S. aureus (MRSA) and their AMR profile including methicillin resistance gene (mecA) isolated from frozen chicken meat in Bangladesh. Around 55% of frozen chicken meat samples were found positive for S. aureus, and of them 37% were identified as MRSA. The occurrence of MRSA in frozen chicken meat is consistent with earlier reports where 33.3% S. aureus isolates were identified as MRSA from processed raw meat samples in Bangladesh [23]. Compared with studies from other countries, a variable occurrence of MRSA (8.1-89%) in frozen chicken meat was reported in China and Egypt [10,11]. Variable occurrence may be due to differences in handling and management practices of frozen chicken meat samples and geographical location [10]. The present study demonstrated that the occurrence of MRSA as well as methicillin-susceptible S. aureus (MSSA) varied among different brands of frozen chicken meat. The highest distribution of MRSA was observed in the Chattogram division than Sylhet and Dhaka divisions of Bangladesh, which is in contrast with a previous report, where a comparatively lower percentage of MRSA contamination was observed in processed raw meat samples in Dhaka division of Bangladesh [23]. On the contrary, the occurrence of MSSA was higher in Mymensingh and Rajshahi divisions than Chattogram division. The chicken types (broiler vs. cockerel) had significant effect on the occurrence of MRSA and MSSA, however, production types (organic vs. non-organic) had no significant effects. Within the present study, a variable degree of contamination with MRSA was found among different types of meat samples. Contamination of frozen chicken meat with high occurrence of MRSA could be attributed to poor hygienic practices of meat handlers, and cross-contamination during slaughtering, or processing and packaging of chicken meat [10]. In spite of the fact that the nasal area is viewed as the primary site of colonization with S. aureus, these organisms are also present in the gastrointestinal tract of chickens [31]. During slaughtering, carcasses may get contaminated by the contents of the intestinal tract, from the slaughtering environment or even by MRSA-infected handlers who have direct contact with carcasses or meat [32].
Considering the obvious importance of S. aureus as foodborne pathogen, and the worldwide emergence of MDR in this foodborne bacterium, we screened the AMR profiles of S. aureus isolated from frozen chicken meat. An important finding of concern in this study is that all the isolates of MRSA and MSSA were MDR (resistant to three or more classes of antimicrobials), of which a significant proportion of the isolates were resistant to 6-8 and 9-12 antimicrobial classes. A previous report also documented that 100% MRSA isolates were MDR [23]. MDR MRSA from retail chicken meat were also reported from different parts of the world, 64.3% in Nigeria [6], 45.7% in India [33], and 44.4% in China [34]. The high occurrence of MDR MRSA and MSSA in frozen chicken meat was related with the type of brands, which might be due to brand level differences in production, handling, processing, and packaging including the type of antimicrobials used. Though all the branded supershops collected live broiler chickens from their contract farms, it was assumed that the farming practices in terms of biosecurity, hygiene, and use of antimicrobials were different among the farms. Of note, the current study additionally observed that 3.2% of S. aureus isolates were possible extensively drug-resistant (pXDR). A report from India depicts the development of an extensively drug-resistant S. aureus isolates in humans [35]. The high percentage of MDR and existence of pXDR may be due to the result from random chromosomal mutations and transfer of resistance genes via conjugation and transformation of the resistance transfer factor and resistance determinants or could be considered preliminary evidence suggesting the extensive use of antimicrobial agents in veterinary and medical practices for the control of bacterial diseases [7]. Reports from Bangladesh revealed that multiple antimicrobials are used indiscriminately in broiler chickens throughout the production cycle, which play a vital role in the emergence of antibiotic-resistant bacteria [36,37]. Another conceivable clarification is that the high percentage of MDR might be credited to the cross-contamination during slaughtering, handling and processing [23,32]. Therefore, strict regulations including good production practices, responsible use of antimicrobials, and hygienic measures in slaughtering and processing are essential to reduce the carcass contamination with drug-resistant S. aureus.
Clinical management of Staphylococcal disease depends on antimicrobial treatment which frequently fails due to forceful resistance of organisms to antimicrobials. We found that all the isolates in this study were resistant to at least five antimicrobial agents, and 43.4% isolates of MRSA, and 18% isolates of MSSA were resistant to 20-30 antimicrobial agents. There was a high percentage of antimicrobial-resistance among MRSA and MSSA, which is in disagreement with the previous report in China, in which 2.3% of S. aureus isolates in frozen chicken meat were resistant to 16-24 antimicrobials [11]. This may be due to the fact that the indiscriminate uses of antimicrobial agents in poultry production for therapy, prophylaxis and growth promotion, along with poor biosecurity and waste management systems accelerate the emergence of antimicrobial resistant pathogens, which may be implicated in foodborne antimicrobial resistant bacterial infection in humans [38].
Although antimicrobial use is a fundamental technique for control of S. aureus infection, especially for MRSA, attributable to its formidable ability to adapt to variable environmental conditions, this organism has an extraordinary capability to quickly get resistant to essentially all antimicrobials [39]. In the current study, we observed that all the MRSA isolates were resistant to cefoxitin. This prevalence was higher than the finding of earlier study in Bangladesh, where the author reported 33.3% isolates showed resistance to cefoxitin [23]. In the current study, we observed that all the MRSA isolates were resistant to cefoxitin. This prevalence was higher than the finding of earlier study in Bangladesh, where the author reported 33.3% isolates showed resistance to cefoxitin [11]. This prevalences were higher than the finding of earlier study in Central Africa [40]. S. aureus is well known to express the highest resistance to penicillin antimicrobial class, and penicillin resistance by Gram-positive bacteria has been reported since 1940 [39]. Nalidixic acid and oxytetracycline resistance were also commonly observed among MRSA and MSSA isolates. This prevalences were higher than the findings of earlier studies on frozen chicken meat in China and Central Africa [11,40]. This is not surprising, because oxytetracycline is one of the most commonly used antibiotics for treatment of infections in poultry and humans without basic programs and restrictive policies on the use of these antibiotics, therefore, very frequent occurrences of resistance in MRSA and MSSA are probably a consequence of this. On the other hand, quinolone resistance among S. aureus emerged quickly, very conspicuously among the methicillin-resistant strains. As a result, the capacity to use fluoroquinolones as antistaphylococcal agents was dramatically diminished. The explanations for the difference in rates of quinolone resistance between MRSA and MSSA strains are unclear. One causative issue is probably the antibiotic selective pressure, particularly within the hospital setting, leading to the choice and spread of the more antibiotic resistant MRSA strains [41]. Of note, in the present study, resistance to colistin, last-resort antimicrobials used for human therapy, was detected in 91% MRSA and 87% MSSA isolates, which might be due to the chromosomal mutations through amino acid substitution [42]. Furthermore, environmental, meat processing, and human hygiene related factors may have an effect on AMR of S. aureus isolated from frozen chicken meat samples. On the other hand, MRSA isolates in this study showed relatively low resistance to vancomycin, and MSSA isolates to meropenem and cefaclor, which may be due to the fact that these antimicrobials do not have veterinary preparations, and are not available for veterinary use, and also are not routinely used in clinical setting in Bangladesh.
MRSA has become a serious concern in food safety, and constitutes a major health care problem [6]. S. aureus isolates were designated as MRSA based on the presence of the methicillin resistance gene mecA. Interestingly, the frequency of the mecA gene in both MRSA and MSSA recovered from frozen chicken meat reached around 44% in the current study. Our result contrast sharply with data published in Egypt and China, reporting that 5.6% and 8.6%, respectively, of the S. aureus isolates from frozen chicken meat were mecA gene positive, suggesting that the presence of mecA gene is the principal evidence for the detection of MRSA isolates [10,11]. However, harboring mecA gene is not sufficient for methicillin resistance, because some S. aureus isolates that contain the mecA gene are still shown to be susceptible to methicillin [43].
This study also described the association between AMR phenotypes and presence of mecA gene in MRSA and MSSA isolated from frozen chicken meat samples. Among the 23 MRSA and 39 MSSA isolates, we observed that the occurrence of methicillin resistance gene (mecA) was found not only in phenotypically resistant isolates but also in phenotypically nonresistant isolates. The association between the AMR and the presence of mecA gene in MRSA and MSSA might be because of colocalization of resistance gene on the same genetic elements, and the possible coselection of many resistance genes by a single antimicrobial [44]. However, resistance genes can be linked to genetic elements, and the use of a particular antimicrobial can select for resistance not only to its own, but also potentially to a variety of other antimicrobials [44]. Since methicillin resistance gene alone is insufficient to confer resistance, further mechanisms are likely associated with the resistance phenotype of MRSA and MSSA strains.
It would be worthwhile if samples were taken from more outlets of various supershops, however, frozen chicken meat were purchased from most of the renowned supershops situated in five divisional megacities of Bangladesh; in this manner, the information is illustrative of the entire of Bangladesh.

Conclusions
To our knowledge, this is the first thorough study on the prevalence of MRSA in frozen chicken meat samples from different supershops of Bangladesh, and is the only study describing the presence of MDR, pXDR and mecA gene in frozen chicken meat. This study reported a relatively high prevalence of MRSA and high rates of MDR amongst the isolates, thus indicating the potential role of chicken meat in the dissemination of MDR MRSA strains in Bangladesh and highlighting the health risks for consumers. Therefore, periodic surveillance of antimicrobial resistance of these organisms in foods of animal origin in different geographical areas is needed. The high prevalence of MDR MRSA in frozen chicken meat samples emphasizes the need for better sanitary education of food handlers in hygienic practices focusing on their potential role as reservoirs and spreaders of MRSA. Hygienic measures should be taken to ensure the safety of food products, and a proper risk assessment should be conducted to further clarify the possible health hazard for consumers. Finally, the proper application of good manufacturing practice, good hygiene practice, and well-designed hazard analysis of a critical control point program in the slaughterhouses and processing units should be implemented.