Microbiological Quality and Safety of Fresh Rabbit Meat with Special Reference to Methicillin-Resistant S. aureus (MRSA) and ESBL-Producing E. coli

The purpose of this investigation was to evaluate the microbial quality and safety of rabbit meat. A total of 49 rabbit meat samples were taken at the retail level. The mesophiles, staphylococci, Enterobacterales, and Pseudomonas spp. counts were 4.94 ± 1.08, 2.59 ± 0.70, 2.82 ± 0.67, and 3.23 ± 0.76 log CFU/g, respectively. Campylobacter spp. were not detected in any sample. Listeria monocytogenes was isolated from one sample (2.04%) at levels below 1.00 log CFU/g. Multi-resistant S aureus was found in seven samples (14.9%). Methicillin-resistant S. aureus, S. epidermidis, S. haemolyticus, M. caseolyticus, and M. sciuri were found in a sample each (10.20%), and all of them were multi-resistant. Multi-resistant ESBL-producing E. coli were detected in two samples from the same retailer (4.08%). The high resistance found in methicillin-resistant staphylococci and ESBL-producing E. coli is of particular concern, and suggests that special measures should be taken in rabbit meat.


Introduction
Spain, with a production of 40,929 tons in 2022, is the largest producer of rabbit meat in the European Union, followed by France and Italy [1].In Spain, rabbit meat represents the fifth most consumed type of meat after pigs, poultry, cattle, sheep, and goats [1].At the European Union level, it should be noted that there are few producing countries, since the consumption of rabbit meat is linked to cultural factors [1].However, rabbit meat is considered as one of the healthiest types of meat due to its low fat content, high percentage of unsaturated fatty acids, low cholesterol content, high content in easily digestible protein, and B vitamins and minerals contents (mainly calcium, magnesium, and zinc) [2].
Rabbit meat is a highly nutritious substrate with a high water activity (0.99) suitable for the growth of most microorganisms [3].Rabbit meat contamination can occur during the production process, including slaughtering and storage [3].Contamination can originate from the animal, environment, equipment, or workers [3].The bacteria associated with the spoilage of rabbit meat are mainly Pseudomonas, lactic acid bacteria, and Brochothrix thermosphacta [4][5][6].Few studies have been focused on the identification of the microbiota present in rabbit meat [7].
Today, the increase in antimicrobial resistance is considered to be a major threat to human and animal health [13].This threat should be approached from a "One Health" perspective, considering veterinary medicine, human medicine, and the environment, since they are interconnected [14].Therefore, a reduction in the transmission and spread of antibiotic resistance in one of these sectors may affect others [15].
The antimicrobial resistance of bacteria species from food-producing animals could affect human health, as they are potential sources of transmission to humans [16].In fact, there is a serious concern about antimicrobial resistance bacteria present in meat, specifically, extended-spectrum-β-lactamase (ESBL)-producing E. coli and methicillin-resistant S. aureus (MRSA) [17,18].Nevertheless, studies carried out on rabbit meat are scarce and mainly focus on the antimicrobial resistance of E. coli, and S. aureus [19][20][21][22].On the other hand, other methicillinresistant staphylococci (MRS) have been found in meat [23].Recently, staphylococcal species belonging to the S. sciuri group (S.sciuri, S. stepanovicii, S. lentus, S. vitulinus, and S. fleurettii) were reassigned to the genus Mammaliicoccus [24].Consequently, it is also important to evaluate the prevalence of MRS and methicillin-resistant Mammaliicoccus (MRM) in rabbit meat.
The aim of this work was to study the microbiological quality and safety of rabbit meat, together with the prevalence of methicillin-resistant S. aureus, other methicillin-resistant staphylococci, methicillin-resistant Mammaliicoccus, and ESBL-producing E. coli.

Microbiological Quality and Safety of Rabbit Meat
The microbial counts of the 49 rabbit meat samples analysed are shown in Table 1.Mesophiles counts varied between 1.90 and 7.59 log CFU/g, with an average of 4.94 ± 1.08.Mesophiles levels above 7 log CFU/g were obtained only in two samples from the retailer SG (2.04%).Table 1 shows the microbial counts obtained in samples from different retailers.No significant differences (p > 0.05) in mesophiles counts were observed among the samples from different types of retailers or from the same type of retailer (traditional shops, supermarkets, or hypermarkets) (Table 2).The bacteria isolated from Plate count agar in samples from hypermarkets were mainly lactic acid bacteria (30.24%), followed by Micrococcacceae (18.62%)Brochotrix thermosphacta (13.95%),Pseudomonas spp.(11.63%), and Enterobacterales (9.31%).In samples from supermarkets, the predominant bacteria were Pseudomonas spp.(35.48%), followed by lactic acid bacteria (26.24%),Brochotrix thermosphacta (9.93%), Micrococcacceae (7.81%), and Enterobacterales (5.681%), while in samples from traditional shops, the predominant bacteria were Pseudomonas spp.(37.50%) and Micrococcaceae (37.50%) (Table 3).Staphylococci counts below 1 log CFU/g were found in 11 rabbit meat samples (22.45%).The other 38 samples showed counts ranging between 1.30 and 4.13 log CFU/g, with an average number of 2.59 ± 0.70 (Table 1).No significant differences (p > 0.05) in staphylococci counts were observed among samples from different types of retailers or from the same type of retailer (traditional shops, supermarkets, or hypermarkets) (Table 2).
Table 4 shows the Staphylococcus spp., Mammaliicoccus spp., and Macrococcus spp.distribution, with M. vitulinus, S. equorum, and S. saprophyticus being the dominant species in the samples from hypermarkets, supermarkets, and traditional shops, respectively.S aureus was detected in one sample of hypermarket HB and four samples from supermarkets (two from supermarket SC and two from supermarket SG).Methicillin-resistant strains were found in five samples when using chromID MRSA agar, with one from hypermarket HA being identified as S. epidermidis and four from supermarkets being identified as S. aureus (SC), S. haemolyticus (SF), M. sciuri (SD), and M. caseolyticus (SH).
Enterobacterales counts below 1 log CFU/g were found in 17 samples (34.69%).The counts in the other 32 samples ranged between 1.30 and 4.74 CFU/g, with an average number of 2.82 ± 0.67 (Table 1).No significant differences (p > 0.05) in Enterobacterales counts were found among rabbit samples from different types of retailers or from the same type of retailer (Table 2).Table 5 shows the species distribution.Serratia liquefaciens was the dominant species in samples from supermarkets and traditional shops, while Ewingella americana was the predominant species in samples from hypermarkets.E. coli was found in samples from hypermarkets and supermarkets.When using ChromID ESBL, E. coli was found in two samples from supermarket SF.Pseudomonas spp.counts below 1 log CFU/g were found in 14 samples (28.57%).The other 35 samples (71.43%) showed counts between 1.30 and 6.11 log CFU/g, with an average number of 3.23 ± 0.76 (Table 1).Significant differences (p < 0.05) in pseudomonas counts were observed among samples from different supermarkets (Table 2).Table 6 shows the Pseudomonas spp.distribution, with P. libanensis and P. extremorientalis being the dominant species in the samples from hypermarkets and supermarkets, while the dominant species in samples from traditional shops were P. fluorescens and P. libanensis.

Pseudomonas libanensis 40
Pseudomonas extremorientalis Campylobacter spp.were not detected in any sample.L. monocytogenes was only found in one sample from supermarket SG at levels below 1 log CFU/g.L. innocua was isolated from two samples, one from supermarket SG and one from a traditional shop (TJ).
The phenotype of the multi-resistant strains is shown in Table 7. Eight strains were resistant to methicillin, seven from supermarkets (SC, SD, SF, and SG) and one from hypermarket HA.Multi-resistant S. aureus strains were found in samples from supermarkets SC and SG, being resistant to ≥8 antibiotics.Multi-resistant strains of other species were found with a higher prevalence in supermarket SF and hypermarket HA.Strains of M. caseolyticus, S. haemolyticus, and S. lugdunensis showed resistance against 14, 12, and 10 antibiotics, respectively.Multi-resistant S. epidermidis strains were detected in hypermarket HA and supermarket SF.Most of the multi-resistant strains were isolated from supermarkets (19 strains), followed by hypermarkets (4 strains from HA), and 1 strain from a traditional shop (TI).

Discussion
In the present study, mesophiles counts varied between 3.79 ± 0.96 and 6.06 ± 1.02, depending on the retailer where the rabbit samples were purchased.Considering the 49 samples analysed, the average was 4.94 ± 1.08.These results are consistent with those reported by other authors.Thus, Cwiková and Pytel reported mesophiles counts of 5.34 log CFU/g.Similar counts were found by Wang et al. (2021) (4.56 log CFU/g) [25].Rodríguez-Calleja et al. evaluated rabbit meat from two different supermarkets, obtaining mesophiles counts of 5.87 ± 1.03 and 6.60 ± 1.18 log CFU/g [5].We found mesophiles counts above 7 log CFU/g in two samples acquired from a supermarket (SG); these levels are associated with meat spoilage [4,5].Differences in mesophiles counts can be explained by handling, time, and storage conditions.Hygiene and the proper handling of rabbit meat are associated with low levels of contamination [4,10].In addition, other factors such as time and storage conditions also influence meat quality [26].In our work, significant differences were not observed among types of retailers or among the same type of retailer.Rodríguez-Calleja et al. did not find differences in the mesophiles counts between the rabbit samples of the two supermarkets evaluated either [5].
The most common bacteria isolated from rabbit meat were Brochothrix thermosphacta, lactic acid bacteria, and Pseudomonas spp., which is in accordance with the results obtained in the present study [3,6].However, Micrococcaceae can be one of the dominant bacteria in rabbit meat, as shown by the results obtained in samples from traditional shops.
Lower Enterobacterales counts in rabbit meat have been reported by other authors [4,32].Pereira and Malfeito-Ferreira reported Enterobacterales counts of 1.8 ± 1.35 log CFU/g on day 0 of storage (2.82 ± 0.67 log CFU/g in the present work) [4].Also, lower counts were reported by Koné et al., with 1.81 ± 0.10 on day 0, but after 6 days of storage, the levels increased to 4.24 ± 1.55 [32].The differences found can be explained by the hygienic measures taken during meat processing and the storage conditions (time, temperature, and packaging) [9].It should be highlighted that Enterobacterales are used like an indicator of the hygienic conditions during slaughter, because they are related to faecal contamination [33].In this study, the dominant Enterobacterales species varied according to the type of retailer.Ewingella americana, Serratia proteamaculans, and Yersinia intermedia were the predominant species in samples from hypermarkets (23.08-30.77%),while S. liquefaciens was the dominant bacteria in samples from supermarkets and traditional shops (45.16 and 100%, respectively).Also, S. liquefaciens has been identified as the dominant bacteria in other types of meat [17].
We observed that Pseudomonas spp.counts in rabbit meat varied between 1.30 and 6.11 log CFU/g, with an average of 3.23 ± 0.76 log CFU/g.Lower pseudomonas counts were reported by Pereira and Malfeito-Ferreira in rabbit carcasses (2.68 ± 0.85 log CFU/g, ranging between 1.00 and 3.99 log CFU/g) [4].The differences found can be explained by storage conditions (time, temperature, and packaging) [10,27,34].Thus, Nakyinsige et al. reported that pseudomonas counts increase with storage time (3.44 ± 0.16 on day 0, and 5.58 ± 0.08 on day 7 of storage) [34].Similar counts were observed by Rodriguez-Calleja et al. (3.39 ± 1.12 on day 0 of storage) [6].It should be noted that Pseudomonas spp. is responsible for the deterioration of meat due to chromatic alterations related to the enzymatic activity of this bacterium [7].In the present study, 31 Pseudomonas spp.were identified in rabbit meat, with P. libanensis, P. extremorientalis, and P. fluorescens being the dominant species.There have been few works on the identification of Pseudomonas spp. in rabbit meat, and they are focused on the detection of bacteria responsible for spoilage [7].
The presence of Listeria spp. in rabbit meat has also been reperted by other authors, although in a higher percentage (13.7% vs. 6.12% in the present work) [8].Other authors have also isolated L. innocua from rabbit meat [8].We only detected the presence of L. monocytogenes in 2.04% of the rabbit meat samples, lower than the values reported by other authors (7.32-38%) [8,35,36].
We did not detect Campylobacter spp. in any rabbit sample, which is agreement with other studies that have not detected this pathogen in rabbit farms [37,38].
We observed that 25% of the staphylococi strains were multi-resistant, and similar results have been reported by Pipová et al. [22].In the current work, 80% of S. aureus strains showed multi-resistance, with 20% being methicillin-resistant (MRSA).In contrast, other authors did not detect any MRSA isolate from rabbit meat, although it was detected in other types of meat (poultry and pork) [21,39].However, other authors have reported the presence of MRSA in rabbit meat [3,40].Similar to Mosrhdy et al. we observed a high resitance rate against erythromycin (82.4% vs. 70% in the present work) [3].We found higher resistant rates against ciprofloxacin (80%) and norfloxacin (50%) in S. aureus strains than Morshdy et al. (17.6% and 29.4%, respectively) [3].However, these authors observed a high resistance rate against chloramphenicol (88.2%), while we observed that all the S. aureus strains were susceptible.We observed that 60% and 20% of the S. aureus strains isolated from rabbit meat were resistant to gentamicin and mupirocin.However, other authors have reported no resistance to these antibiotics among S. aureus strains isolated from rabbit meat [40].Other authors have also resported that S. aureus strains isolated from rabbit meat were susceptible to chloramphenicol, trimethoprim/sulfamethoxazole, and fusidic acid [40].Regarding the coagulase-negative staphylococci and M. caseolyticus strains, 19.77% were multi-resistant and 8.14% were methicillin-resistant.Also, Pipová et al. reported that 8% of the staphylococci isolated from rabbit meat were methicillinresistant [22].Higher resistance rates to erythromycin (58.4%) and penicillin (51.3%) were reported by Pipova et al. than those in the present work (16.8 and 24.42%, respectively) [22].It should be noted that we found that 10% of the S. aureus strains and 12.79% of the other staphylococci were resistant to mupirocin.Moreover, 2.33% of the coagulase-negative staphylococci were resistant to rifampicin.Both mupirocin and rifampicin are classified as antimicrobials to avoid in animals "Category A" [41].
All the E. coli strains evaluated from the rabbit meat were multi-resistant.The presence of ESBL-producing E. coli was detected in 8.16% of the samples (two samples), all of them from supermarket SF (40% of the samples from supermarket SF).In contrast, Stewardson et al. did not detect ESBL-producing E. coli in rabbit meat [19].Also, Kylie et al. reported high resistance rates in E. coli strains isolated from rabbit meat, especially against tetracycline [20].We observed differences in multi-resistance among retailers, as they were only isolated from hypermarkets HB and all the supermarkets, except supermarket SD.The high rate of multi-resistant strains found is in accordance with those described by Martinez-Laorden et al., which found high rates of multi-resistance for E. coli isolated from turkey meat (71.43-100%) [17].We found that 25% of the E. coli strains were resistant to The presence of Campylobacter spp.and L. monocytogenes in rabbit meat samples was determined as described by Da Silva et al. [43].

Isolation and Identification
From each culture media and sample, between three and five colonies were randomly selected.The appearance of suspected colonies was considered when selective media were employed.Tryptone Soy Agar (Scharlau) was used to purify isolates.The isolates were maintained at −80 • C. Bacterial identification was performed by a Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass-Spectrometry (MALDITOF MS) Biotyper (Bruker, Billerica, MA, USA).

Confirmation of Methicillin Resistance of Mammaliicoccus spp. and Staphylococcus spp.
The methicillin resistance of Mammaliicoccus spp.and Staphylococcus spp.obtained from ChromID MRSA agar, and all the S. aureus strains obtained, was confirmed following the criteria described in the Clinical Laboratory Standards Institute's guidelines [44]  The resistance of Mammaliicoccus and staphylococci was evaluated against twenty-nine antibiotics employing the disk-diffusion technique on Mueller-Hinton agar.The antimicrobials (Oxoid) used and their concentrations have been previously described [43].The antimicrobials were: ceftaroline, cefoxitin penicillin, fusidic acid, clindamycin, tetracycline, minocycline, doxycycline, trimethoprim, enrofloxacin, levofloxacin, ciprofloxacin, norfloxacin, gatifloxacin, gentamicin, trimethoprim-sulfamethoxazole, streptomycin, amikacin, kanamycin, sulfadiazine, tobramycin, erythromycin, tylosin, mupirocin, lincomycin, chloramphenicol, nitrofurantoin, linezolid, rifampicin, tedizolid, and vancomycin.The inhibition zones were recorded after incubation at 37 • C for 18 to 24 h.Depending on the inhibition zones and antibiotic used, the Clinical and Laboratory Standards Institute's guidelines classified the strain as resistant, susceptible, or intermediate (reduced susceptibility) [44].

Phenotypic Confirmation of ESBL-Producing E. coli
One E. coli strain identified by MALDI-TOF was chosen for each different medium and sample for phenotypic confirmation of ESBL.The confirmation was carried out according to the Clinical Laboratory Standards Institute's guidelines [39].

Statistical Analysis
Analysis of variance was conducted using SPSS version 26 software (IBM SPSS Statistics, Armonk, NY, USA).Tukey's test for comparison of means was conducted using the same program.The level of significance was determined at p < 0.05.

Conclusions
This work shows that rabbit meat could be a source of methicillin-resistant S. aureus, methicillin-resistant staphylococci, and ESBL-producing E. coli.Moreover, resistance to critical antibiotics such as mupirocin, rifampicin, aztreonam, meropenem, and tigecycline was detected, being of special concern for consumer's health.These findings highlight the need to take special measures in the frame of One Health.Funding: This research was 65% cofinanced by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra programme (POCTEFA (Programa INTERREG V-A España-Francia-Andorra) 2014-2020) (EFA (España-Francia-Andorra) 152/16).POCTEFA aims to reinforce the economic and social integration of the French-Spanish-Andorran border.Its support is focused on developing economic, social, and environmental cross-border activities through joint strategies favouring sustainable territorial development.Jessica Da Silva Guedes has received funding from the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586.

Author
Contributions: J.d.S.G.: investigation, data curation, formal analysis, visualisation.D.V.-R.: investigation E.G.-F.: funding acquisition, project administration, conceptualisation, methodology, data curation, formal analysis, visualisation, investigation, supervision, writing-original draft preparation, writing-reviewing and editing.All authors have read and agreed to the published version of the manuscript.

Table 2 .
Microbial counts (log CFU/g) in rabbit meat from different retailers.

Table 3 .
Bacteria identified in rabbit meat by type of retailer isolated from Plate Count Agar.

Table 5 .
Enterobacteriacceae isolated from rabbit meat by type of retailer (recovered from McConkey agar).

Table 6 .
Pseudomonas spp.isolated from rabbit meat by type of retailer (recovered from specific media for Pseudomonas).

Table 8 .
Antimicrobial resistance phenotype of Escherichia coli strains isolated from rabbit meat.