Prevalence, Infectious Characteristics and Genetic Diversity of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) in Two Raw-Meat Processing Establishments in Northern Greece

In the present study, we investigated the isolation frequency, the genetic diversity, and the infectious characteristics of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from the incoming meat and the meat products, the environment, and the workers’ nasal cavities, in two meat-processing establishments in northern Greece. The isolated S. aureus strains were examined for their resistance to antimicrobials, carriage of the mecA and mecC genes, carriage of genes encoding for the production of nine staphylococcal enterotoxins, carriage of the Panton–Valentine Leukocidin and Toxic Shock Syndrome genes, and the ability to form biofilm. The genetic diversity of the isolates was evaluated using Pulsed Field Gel Electrophoresis (PFGE) and spa typing. S. aureus was isolated from 13.8% of the 160 samples examined, while only one sample (0.6%) was contaminated by MRSA carrying the mecA gene. The evaluation of the antimicrobial susceptibility of the isolates revealed low antimicrobial resistance. The higher resistance frequencies were observed for penicillin (68.2%), amoxicillin/clavulanic acid (36.4%) and tetracycline (18.2%), while 31.8% of the isolates were sensitive to all antimicrobials examined. Multidrug resistance was observed in two isolates. None of the isolates carried the mecC or lukF-PV genes, and two isolates (9.1%) harbored the tst gene. Eight isolates (36.4%) carried the seb gene, one carried the sed gene, two (9.1%) carried both the sed and sei genes, and one isolate (4.5%) carried the seb, sed and sei genes. Twenty-one (95.5%) of the isolates showed moderate biofilm production ability, while only one (4.5%) was characterized as a strong biofilm producer. Genotyping of the isolates by PFGE indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Ten different spa types were identified, while no common spa type isolates were detected within the two plants. Overall, our findings emphasize the need for the strict application of good hygienic practices at the plant level to control the spread of S. aureus and MRSA to the community through the end products.


Introduction
Staphylococcus aureus is part of the skin and mucous microbiota of humans, with the nasal cavity constituting the most common carriage site. The nasal cavity is regarded as the source of colonization of secondary body sites, such as the hands [1]. S. aureus is also an important versatile pathogen causing various types of infections, ranging from skin and soft-tissue infections to life-threatening septicemia and toxin-mediated diseases, such as 2. Results 2.1. Isolation of S. aureus S. aureus was isolated from 22 of the 160 (13.8%) samples examined from the two meat-processing establishments. The isolation frequency was 23.3% (14/60) and 8.0% (8/100) for plant F and Z, respectively ( Table 1). The isolation frequency of S. aureus in descending order was 37.5% (6/16) from ground meat products, 25% (5/20) from workers, 12.3% (7/57) from equipment, 10.0% (2/20) from incoming pork meat, 8.3% (1/12) from incoming bovine meat, and 4.5% (1/22) from infrastructure. The isolation frequency of S. aureus was 8.1% (3/37) in samples from the incoming meat (bovine, porcine and ovine), and 25.0% (6/24) in samples from the processed raw meat products (ground meat and non-ground meat products), respectively (Table 1).

Carriage of the mecA, mecC, tst, lukF-PV and Enterotoxin Genes
The mecC and lukF-PV genes were not detected in any S. aureus isolate. Two isolates carried the tst gene: one from equipment of plant F and the other from a worker's nasal cavity from plant Z ( Table 2). Ten of the isolates (45.6%) did not carry any of the examined staphylococcal enterotoxin genes, eight of the isolates (36.4%) carried only the seb gene, two (9.1%) carried the sed and sei genes and one (4.5%) carried the seb, sed and sei genes ( Table 2). Of the 12 isolates that carried enterotoxin genes, five (41.7%) were isolated from equipment surfaces, four (33.3%) from workers' nasal cavities and three (16.7%) from meat products. The MRSA isolate (Z112) that originated from a sample of meat product in plant Z carried the mecA gene, but it did not harbor any of the remaining virulence genes examined (Table 2).

Biofilm Formation Ability
All except one of the S. aureus isolates (21/22, 95.5%) were classified as moderate biofilm producers and the remaining isolate was classified as a strong biofilm producer

Carriage of the mecA, mecC, tst, lukF-PV and Enterotoxin Genes
The mecC and lukF-PV genes were not detected in any S. aureus isolate. Two isolates carried the tst gene: one from equipment of plant F and the other from a worker's nasal cavity from plant Z ( Table 2). Ten of the isolates (45.6%) did not carry any of the examined staphylococcal enterotoxin genes, eight of the isolates (36.4%) carried only the seb gene, two (9.1%) carried the sed and sei genes and one (4.5%) carried the seb, sed and sei genes ( Table 2). Of the 12 isolates that carried enterotoxin genes, five (41.7%) were isolated from equipment surfaces, four (33.3%) from workers' nasal cavities and three (16.7%) from meat products. The MRSA isolate (Z112) that originated from a sample of meat product in plant Z carried the mecA gene, but it did not harbor any of the remaining virulence genes examined (Table 2).

Biofilm Formation Ability
All except one of the S. aureus isolates (21/22, 95.5%) were classified as moderate biofilm producers and the remaining isolate was classified as a strong biofilm producer ( Table 2).

Spa Typing
The spa typing of the 22 S. aureus strains revealed ten spa types, one of which was detected for the first time. About half the strains (9/22) belonged to spa type t084, two strains belonged to type t091 and four strains belonged to the new spa type. The sequence of the novel spa type (repeats 07-23-12-12-21-17-13-34-33-34) was confirmed in the whole genome sequence; actually, it is similar to the spa type t2414 (07-23-12-12-21-17-34-33-34) with one additional repeat (repeat 13). The other seven isolates belonged to t012, t197, t499, t774, t891, t899 and t1510 ( Table 2). The MRSA strain belonged to spa type t899 and was not typeable by PFGE. This spa type belongs either to CC398 or to CC9 [19]. The five S. aureus isolates from the nasal cavities of workers were assigned to five different spa types (t084, t197, t891, t1510, 'new').

Pulsed Field Gel Electrophoresis Typing
Nineteen of the staphylococcal isolates were typeable by PFGE revealing 11 distinct pulsotypes (Figure 2). At a similarity level of 91%, most of the S. aureus isolates (57.9%, 11/19) were grouped into the cluster A; isolates from the same processing plant were assigned into separate clusters (A, B, and C). Two isolates from plant F (F22 and F44) were not assigned to any of the three clusters ( Figure 2). Within clusters, common pulsotypes among isolates originating from human (Hu), meat (M), meat product (MP), and equipment (Eq) samples were identified: within cluster A, pulsotype P1 was shared by Hu, M, MP and Eq isolates, while within cluster C, pulsotype P8 was shared by Hu, MP and Eq isolates. was not typeable by PFGE. This spa type belongs either to CC398 or to CC9 [19]. The five S. aureus isolates from the nasal cavities of workers were assigned to five different spa types (t084, t197, t891, t1510, 'new').

Pulsed Field Gel Electrophoresis Typing
Nineteen of the staphylococcal isolates were typeable by PFGE revealing 11 distinct pulsotypes (Figure 2). At a similarity level of 91%, most of the S. aureus isolates (57.9%, 11/19) were grouped into the cluster A; isolates from the same processing plant were assigned into separate clusters (A, B, and C). Two isolates from plant F (F22 and F44) were not assigned to any of the three clusters ( Figure 2). Within clusters, common pulsotypes among isolates originating from human (Hu), meat (M), meat product (MP), and equipment (Eq) samples were identified: within cluster A, pulsotype P1 was shared by Hu, M, MP and Eq isolates, while within cluster C, pulsotype P8 was shared by Hu, MP and Eq isolates. * MP = meat product, M = incoming meat, Eq = equipment, Hu = human

Next-Generation Sequencing
Analysis of the whole genome sequence of the two isolates (Z77 and Z112) showed that Z77, which was sensitive to all antibiotics tested, did not carry any antimicrobial re-

Next-Generation Sequencing
Analysis of the whole genome sequence of the two isolates (Z77 and Z112) showed that Z77, which was sensitive to all antibiotics tested, did not carry any antimicrobial resistance gene. Regarding the isolate Z112, which was resistant to several antibiotics, genes conferring resistance to beta-lactams (mecA, blaZ), chloramphenicol (fexA), tetracycline (tetM) and trimethoprim (dfrC) were detected; however, no gene associated with resistance to tobramycin was detected (Table 3). Table 3. Results of the analysis of the whole genome sequences of the two S. aureus isolates subjected to NGS. Spa typing confirmed the type t899 in the Z112 isolate, while it was not possible to be defined in the Z77 isolate. The Z112 isolate belonged to sequence type ST398 and the Z77 isolate to ST97, respectively. Among the toxin genes we found, hlgA, hlgB, and hlgC were encoding for gamma-hemolysin components A, B and C, respectively, while lukD and lukE encode for leucotoxins D and E; the exoenzyme genes aur, splA, splB, and splE encode for the aureolysin and serine protease-like proteins A, B and E, respectively; finally, sak and scn genes encode for the host immune defense proteins staphylokinase and staphylococcal complement inhibitor.

Isolation, Antimicrobial Susceptibly and Biofilm Formation Ability of S. aureus and MRSA Isolates
Considering that bibliographic data on the prevalence of staphylococci in the products and in the environment of meat-processing establishments are limited, the main objective of this study was to report the prevalence of S. aureus and MRSA in two meat-processing establishments in northern Greece. In general, compared to meat-processing establishments, a higher detection frequency of S. aureus and MRSA has been reported for beef, pork and poultry meat products at the retail level [20]. In the present study, S. aureus was isolated from 13.8% of the samples examined, while only one sample of ground meat (0.6%) was contaminated with MRSA. These detection frequencies are lower than the estimates of 29.2% and 3.2% for S. aureus and MRSA, respectively, which were reported in a global meta-analysis of different meat products [20]. In samples taken from equipment surfaces of the meatprocessing plants, the overall S. aureus isolation frequency was 12.3%, being comparable with a previous Spanish study, which reported a prevalence of 15.5% on equipment surfaces of a pork-processing plant [21]. However, further data concerning the S. aureus isolation frequency from equipment surfaces are scarce in the literature. In the personnel of the two meat-processing establishments, the isolation frequency of S. aureus was 25%. This finding is not surprising, since the detection of S. aureus in the population is around 20% and 60% in persistent and occasional nasal carriers, respectively [22]. Personnel in slaughterhouses and meat-processing plants are among the groups of workers with the highest incidence of occupational injuries. Injuries that lead to the disruption of skin-tissue integrity [23,24] as well as the frequent exposure to materials of animal origin (nasal secretions, gastrointestinal contents, etc.) favor the contamination of workers with S. aureus [11,25]. On the other hand, none of the samples from workers tested positive for MRSA. Drougka et al. [8] reported the isolation of MRSA from 4.2% of slaughterhouse workers in Greece, where the prevalence in the general population is low (0.94%) [26]. In our study, the lack of detection of MRSA in the samples obtained from workers at the examined facilities is likely due to the small number of samples examined. However, even the mere presence of S. aureus and MRSA on the surface of meat constitutes a potential risk for their transmission to humans, particularly to those who work in meat-processing companies [6].
To gain insight into the identification of specific phenotypic and genomic characteristics of S. aureus isolated from the meat-processing plants, we investigated their antimicrobial susceptibility, biofilm formation capacity and carriage of enterotoxin genes. The highest resistance frequency was observed to P (68.2%), A/C (36.4%), TET (18.2%) and TOB (13.6%). Seven distinct antimicrobial resistance profiles were revealed, with the profiles 'P' (22.7%) and 'P, A/C' (22.7%) being the most frequent. Finally, two MDR isolates (F40 and Z112) were revealed. The presence of antimicrobial-resistant strains of S. aureus and MRSA has been reported in various foods as well as in meat [8,27] and meat products [28,29] and multiple profiles of antimicrobial resistance are often observed among the S. aureus isolates [30]. The high resistance frequencies of S. aureus and MRSA isolates from livestock, meat and food handlers to P and TET, ranging from 60.9% to 100%, and from 5.6% to 89.1% respectively, have been reported by numerous studies worldwide (USA [29], Lithuania [31], Greece [8,32], Italy [33], Nigeria [34], China [35] and Korea [36]). The frequent resistance to penicillins and tetracyclines is apparently related to the widespread use of these antimicrobials due to their low cost, ease of access (without a prescription) and administration. We also observed a high (80%) resistance frequency to P among the S. aureus isolates originating from the workers' nasal cavities (in four out of five positive workers) ( Table 2). These findings are similar to those previously reported (70% [37], 48% [38], 57.9% [39]). On the other hand, the sensitivity of the workers' S. aureus isolates to TET in the present study may be due to the infrequent use of tetracyclines in human clinical practice [40].
Previous studies have shown the presence (2.9%) of MDR MRSA isolates in turkey and pork meat [41]. Interestingly, the single MRSA isolate in our study (from ground pork meat) was characterized as MDR, showing resistance to six antimicrobial classes. Overall, the evaluation of antimicrobial susceptibility revealed a high frequency of resistance to specific antimicrobials as well as variable ARPs among the S. aureus isolates. Therefore, our findings emphasize the need for preventive measures to control the dispersion of antimicrobial resistance along the entire food production chain [42] with strict application of proper hygiene and industrial practices.
All S. aureus isolates were characterized by moderate biofilm-forming capabilities in polystyrene microtiter plates. In line with our findings, previous studies have reported the ability of biofilm formation by S. aureus isolates from raw meat [43], from several food-processing facilities [44,45] and from food of animal origin [35]. The ability of S. aureus to produce biofilms and their coexistence with saprophytic microorganisms within these biofilms result in persistent contamination sources in food-processing facilities [16,46].
Four of the five (80%) S. aureus strains isolated from the workers' nasal cavities were enterotoxigenic and five of the seven (71%) were from the equipment surfaces. In a study conducted in Hong Kong, 40% of the S. aureus isolates from the nasal cavities of meat handlers in mass-catering kitchens carried at least one enterotoxin gene, with sea (20%) and seb (11%) being the most frequently detected [49]. Workers carrying enterotoxigenic strains of S. aureus are recognized as the main source of their spread in food either through direct contact or indirectly through respiratory secretions [50].
The application of good hygiene practices during food processing and the effort to control and limit the population of staphylococci to low levels in foods are the most important measures for the prevention of staphylococcal intoxications [51].

Carriage of Genes Encoding for the Toxic Shock Syndrome and Panton-Valentine Leukocidin Toxins
The tst gene was detected in a S. aureus strain isolated from a worker's nasal cavity and in one sample obtained from processing equipment. This low-isolation frequency (9.1%) is in line with the results of other studies. A zero-detection frequency was reported for isolates recovered from workers of a slaughterhouse in Greece and food handlers in Lebanon, respectively [8,52]. A low prevalence (5.3%) was also reported in samples taken from the hands of workers in restaurants in Spain [53]. These tst gene detection frequencies in workers are considerably lower compared to those reported for the general population in several countries, ranging from 15.8% to 40.0% (Brazil 15.8% [54], Madagascar 21.0% [55], Iran 22.8% [56], Spain 28.3% [57] and Egypt 40.0% [58]). There are limited reports on tst gene carriage among S. aureus isolates from processing surfaces. Sahin et al. [59] reported one S. aureus strain (isolated from surface samples in food-processing establishments in Turkey) carrying the tst gene, while Sospedra et al. [53] did not recover any S. aureus isolates carrying the tst gene from surfaces and equipment in restaurants in Spain.
In the present study, none of the S. aureus isolates carried the lukF-PV gene. Weese et al. [60] reported that none of the MRSA isolates from raw meat and its preparations in Canada carried this gene. On the contrary, Gutierrez et al. [45] reported that 10.1% of the S. aureus isolates from ground beef and pork chops in Colombia carried the lukF-PVL gene.
Given (a) the limited pertinent available literature internationally, (b) the fact that food handlers and processing surfaces are important sources of food contamination, and (c) the severity of clinical manifestations associated with TSST-1 and PVL intoxications, the low detection frequency of S. aureus isolates carrying these genes does not justify complacency.

Genetic Diversity of S. aureus Isolates
Another scope of this study was to reveal the genetic diversity of the S. aureus isolates to better understand the transmission routes and infection sources of this pathogen in meatprocessing establishments. Ten different spa types were identified based on spa-typing; among them, five spa types (t197, t499, t774, t899 and t1510) were detected for the first time in Greece. Most of the strains belonged to the spa type t084 (40.9%), four (18.2%) to a new spa type and two (9.1%) to t091 (Figure 2). According to the Ridom Spa Server, the global spread of spa types t084 and t091 is quite high (1.74% and 0.99%, respectively). The most prevalent spa type (t084) was detected in all types of samples from plant F (Figure 2). Methicillin-sensitive S. aureus (MSSA) strains of this type have been isolated from several sources globally [57,61,62]. The spa type t091 represents the basic type of the clonal complex CC7 and has been detected as either MSSA or MRSA from various sources [57,[63][64][65][66]. In addition, the spa type t012, which was identified in one isolate in our study, displays high global spread (1.53%). The spa types t197, t499, t774, t891, t899 and t1510 show a relatively low global spread, ranging between 0.02% and 0.31%. Interestingly, S. aureus isolates from workers belonged to different spa types (t084, t197, t891, t1510 and to a new type), which are usually isolated from human clinical samples [67][68][69]. The only MRSA isolate in the present study belonged to spa type t899. To our knowledge, and according to the data available in the Ridom Spa Server, this spa type is detected for the first time in Greece, and its global spread is 0.31%, mainly in Italy and other European countries. Originally, this spa type was related with MLST type ST9, but recently, it is most often related with ST398, which is the predominant LA-MRSA lineage in Europe [19,70]. Although t011 and t034 still constitute the main spa types associated to CC398, isolates belonging to spa type t899 have been reported as the cause of sporadic illness in humans, and they are increasingly being described in the human and veterinary medical literature [71][72][73][74][75].
PFGE genotyping of the isolates revealed the existence of three PFGE clusters (A, B and C) (Figure 2). Each of them comprised enterotoxigenic isolates; cluster A included isolates from plant F only, whereas clusters B and C contained isolates from plant Z only. The assignment of isolates from the same processing plant into separate clusters indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Furthermore, the findings of indistinguishable S. aureus pulsotypes from meat products, workers and equipment surfaces indicates probable cross-contamination pathways. These findings, in combination with the fact that isolates with no common spa type were detected from the two plants, supports the hypothesis that there is no universal spread of certain spa types and there is also a distinct genetic population of S. aureus in each processing plant. These facts emphasize the need for the strict application of good hygienic practices at the processing plant level in order to control the spread of foodborne and particularly multi-drug resistant pathogens to the community through the end products.

Next-Generation Sequencing
NGS was performed on two isolates, one MSSA belonging to the novel spa type, and one MRSA belonging to spa type t899. NGS analysis revealed that the isolate of the new spa type belonged to sequence type ST97 and the MRSA t899 isolate belonged to LAassociated ST398 ( Table 3). The ST97 strain did not have any enterotoxin gene but carried the antibiotic efflux genes mgr (resistance to quinolones, tetracyclines), arlR (fluoroquinolones, disinfection agents and antiseptics), mepR (tetracycline, glycycycline), norC (hydrophobic quinolones such as moxifloxacin and norfloxacin), sdrM (norfloxacin, ethidium bromide) and sepA (resistance to disinfectants and dyes). The ST398/t899 carried the same with ST97 antibiotic efflux genes plus the norA (resistance to hydrophilic quinolones such as ciprofloxacin and norfloxacin) and the antimicrobial resistance genes mecA and blaZ (resistance to beta-lactamases), the tetM (tetracycline), the fexA (phencicol antibiotics) and the dfrC (diaminopyrimidine antibiotic) [76]. In the international literature, it has been reported that the ST97 S. aureus clone is shared by a great number of different spa types. Zhang et al. [77] isolated 82 S. aureus ST97 from bovine clinical mastitis in China, which shared 15 different spa types (t359, t237, t4682, t521, t730, t16314, t16315, t224, t6297, t2756, t131, t1234, t267, UK-1). In a study contacted in Czech Republic, Pomorska et al. [78] reported that S. aureus isolates t267 and t359 from the blood of hospitalized patients belonged to the ST97 clone. Sato et al. [79] isolated five MRSA ST97 strains from pigs of one farm in Japan identified as spa type t1236. To our knowledge, this is the first time that the ST97 clone of S. aureus has been isolated in Greece.

Sample Collection
In the present study, we examined a total of 160 samples from two meat-processing establishments located in northern Greece (60 and 100 samples from establishment F and Z, respectively). The distance between the two establishments is more than 200 km. Thirtyseven samples were collected from the incoming meat (bovine, ovine, porcine), 24 samples from the final meat products (ground meat and non-ground meat products), 79 samples from the environment (22 from infrastructure surfaces that do not come in contact with meat and 57 from equipment surfaces) and 20 samples were obtained from the nasal cavities of workers (Table 1).
Surface sampling was performed by swabbing a minimum area of 100 cm 2 (or the maximum available area, in case of smaller tools) using swabs moistened in buffered peptone water (LAB M, Lancashire, United Kingdom). A bi-lateral nasal (anterior nares) swab sample was taken from all workers (who participated voluntarily).
All samples were collected aseptically using sterile swabs along with single-use, screwcapped tubes filled with Stuart transport medium (Deltalab, Barcelona, Spain). Samples were transported to the laboratory under refrigerated conditions in less than four hours from the time of sampling.

Isolation and Identification of Staphylococcus aureus
Upon arrival to the laboratory, each sample was immediately transferred to a test tube filled with 10 mL of Tryptone Soy Broth (TSB, LAB M) supplemented with 6.5% (w/v) NaCl (Merck, Darmstadt, Germany) and 0.3% (w/v) yeast extract (LAB M). Following an 18 h incubation at 37 • C, 10 µL of the pre-enriched broth was surface-plated onto Baird-Parker Agar (LAB M) supplemented with egg-yolk tellurite (LAB M), and the plates were incubated aerobically at 37 • C for 48 h. Up to four presumptive S. aureus colonies (black colonies surrounded by an opaque zone and a clearance zone around the opaque zone) from each plate were sub-cultured on Tryptone Soya Agar (LAB M) for 24 h at 37 • C and were then subjected to Gram staining, along with mannitol fermentation testing and catalase-testing [80]. Furthermore, all suspect colonies were subjected to a rapid test (Microgen Staph Rapid Test; Microgen Bioproducts, Surrey, UK) for the detection of the coagulase enzyme and of the protein A, assisting the tentative identification to the species level (S. aureus). Among them, one presumptive S. aureus isolate (Gram-positive, catalasepositive, mannitol-positive, coagulase-positive and protein A-positive) per sample was randomly chosen and stored under freezing conditions (−80 • C) in cryotubes containing TSB with 20% glycerol for further processing.

Molecular Characterization of Staphylococcus aureus
For species confirmation of the presumptive S. aureus isolates, a PCR test detecting the coa and the species-specific nuc genes was performed. Genomic DNA was extracted by using a DNA purification protocol for Gram-positive bacteria (Pure Link Genomic DNA kit; Invitrogen, Carlsbad, CA, USA), while PCR conditions [81] and primers [82,83] previously described were used (Supplementary Table S1).

Detection of Virulence Genes and Methicillin Resistance Genes
Multiplex PCRs were used for the detection of virulence genes contributing to the pathogenicity of S. aureus and for the detection of methicillin resistance genes. Two separate sets of primers and multiplex PCR reactions were used to assess the carriage of genes that encode nine types of SEs (sea, seb, sec, seh and sej; sed, see, seg, sei) and the carriage of the tst gene, which encodes for the production of the toxic shock syndrome toxin-1 (TSST-1) [84]). The carriage of the mecA and mecC genes, which confer resistance to methicillin and the carriage of the lukF-PV gene, which encodes for the Panton-Valentine Leukocidin toxin (PVL) were sought also via multiplex PCR using the primers and conditions described by Stegger et al. [85] (Supplementary Table S1).

Assessment of Biofilm-Formation Ability
The ability of the S. aureus isolates to produce biofilms was evaluated following the protocol of the microtiter plate method, as described by Wang et al. [88]. Staphylococcal strains were characterized according to Borges et al. [89] as no biofilm producers, weak biofilm producers, moderate biofilm producers, or strong biofilm producers.

Pulsed Field Gel Electrophoresis Analysis
PFGE analysis of the S. aureus isolates was performed according to a standardized protocol [90] with the SmaI endonuclease (New England Biolabs, Beverly, MA, USA) using a CHEF-DR III apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA). DNA from Salmonella enterica serotype Braenderup H9812, which was digested with XbaI, was used as a reference size standard, while PFGE patterns were analyzed using the FPQuest software (Bio-Rad Laboratories Inc. Pty Ltd.). PFGE profiles were compared using the Dice correlation coefficient with a maximum position tolerance of 1.5% and an optimization of 1.5%, while the Unweighted Pair Group Method using Averages (UPGMA) was used for clustering analysis and the generation of a dendrogram.

Spa Typing
For amplification of the Staphylococcus protein A (spa) repeat region, the extracted DNA (from Section 4.3) was subjected to a PCR using the primers described by Airesde-Sousa et al. [91] according to the protocol described in the Ridom Spa Server website (http://spaserver.ridom.de/ accessed on 2 March 2021). PCR products were sequenced in a 3130 genetic analyzer (Thermo Fisher Scientific, South San Francisco, CA, USA). For the analysis of spa sequences, the software Ridom StaphTypeTM (Ridom GmbH, Wórzburg, Germany) was used [92].

Next-Generation Sequencing and Analysis of the Whole Genome
Two isolates were selected for further analysis by next-generation sequencing: S. aureus isolate was assigned to the novel spa type (Z77) and the multi-resistant MRSA isolate (Z112). DNA was extracted using the DNA extraction kit (Qiagen, Hilden, Germany), and its concentration was measured using the Qubit double strand (ds) DNA HS assay kit (Q32851, Life Technologies Corporation, Grand Island, NY, USA). NGS was performed using the Ion PGM Hi-Q View Sequencing kit on an Ion Torrent PGM Platform (Life Technologies Corporation).
The procedures of shearing, purification, ligation, barcoding, size selection, library amplification and quantitation, emulsion PCR and enrichment were performed according to the manufacturer's instructions. PCR products were loaded on an Ion-316™ chip kit V2BC. The Ion PGM Hi-Q View (200) chemistry (Ion PGM Hi-Q View Sequencing kit) was applied. The sequence reads were de novo assembled and annotated using Geneious Prime version 2021.2.1. Sequences from the whole genome of the samples Z77 and Z112 were submitted to ENA and received the accession numbers ERS13643805 and ERS13643806, respectively.
MLST analysis was performed using the free web-based S. aureus database in PubMLST (https://pubmlst.org/saureus/ accessed on 10 March 2021) [93]. Antimicrobial resistance genes were identified using the online databases Resfinder version 4.1 [94] and Comprehensive Antibiotic Resistance Database (CARD) [76], with the selection criteria set to perfect (100% identity) and strict (>95% identity) hits only to the reference sequences in CARD [95]. Virulence genes and plasmid content were identified using the Viru-lenceFinder 2.0 and PlasmidFinder version 2.1 from the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/ accessed on 15 March 2021) [96]. The threshold for %ID and for the minimum length were set to 90% and 60%, respectively.

Conclusions
Despite the limitations of the present study (two meat processing establishments and a relatively low number of examined samples), interesting conclusions can be drawn from the results. This study revealed the low prevalence of MRSA and MDR S. aureus, no common palsotypes among the isolates from the two establishments and high genetic diversity by spa typing. Although the prevalence of MRSA was low, meat-processing establishments are a very important spot for controlling pathogen spread in the community. Thus, the implementation of rules of good hygiene practice in combination with biosecurity measures are crucial factors for achieving this goal. However, further data are needed to monitor the spread of MRSA at meet the meat processing establishments level.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens11111370/s1, Table S1: Primers used in this study for the detection of molecular characterization genes, toxin genes and methicillin resistance genes.

Funding:
The current work was supported in part by the European Union's Horizon 2020 grant VEO (grant number 874735).

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, in compliance with the Greek legislation, and the protocol was approved by the Ethical Committee of the Veterinary Research Institute, ELGO Dimitra.