Microbiological Quality and Safety of Fresh Quail Meat at the Retail Level

The objective of this study was to evaluate the microbiological quality and safety of 37 fresh quail meats. Mesophiles, Pseudomonas spp., Enterobacteriaceae, and staphylococci counts were 5.25 ± 1.14, 3.92 ± 1.17, 3.09 ± 1.02, and 2.80 ± 0.64 log CFU/g, respectively. Listeria monocytogenes was detected in seven samples (18.92%). Campylobacter jejuni was detected in one sample (2.70%). Clostridium perfringens was not detected in any sample. The dominant bacteria were Pseudomonas spp. (30.46%), Micrococcaceae (19.87%), lactic acid bacteria (14.57%), and Enterobacteriaceae (11.92%). Brochotrix thermosphacta and enterococci were isolated to a lesser extent, 7.28% and 1.99%, respectively. The dominant Enterobacteriaceae found were Escherichia coli (42.53%). ESBL-producing E. coli was detected in one sample (2.70%), showing resistance to 16 antibiotics. Sixteen different Staphylococcus spp. and three Mammaliicoccus spp. were identified, the most common being S. cohnii (19.86%) and M. sciuri (17.02%). S. aureus and S. epidermidis were also found in one and four samples, respectively. Methicillin-resistant M. sciuri and S. warneri were found in 13.51% and 10.81% of quail samples, respectively. These bacteria showed an average of 6.20 and 18.50 resistances per strain, respectively. The high resistance observed in ESBL-producing E. coli and methicillin-resistant S. warneri is of special concern. Measures should be adopted to reduce the contamination of quail meat.


Introduction
The consumption of quail meat (Coturnix coturnix) has gradually increased in the last decades, although it is less consumed than chicken and turkey meat [1,2].The most common quail for human consumption is Japanese quail (Coturnix coturnix japonica), which is nowadays distributed worldwide [3,4].The highest quail meat producers are in the USA and Europe, mainly in France and Spain [4][5][6].The increase in quail production is based on its high growth rates, resistance to diseases, good adaptation, small size, and low investment and maintenance costs [7][8][9].
Quail meat is recognized as a healthy meat because of its high protein content, low fat and cholesterol levels, fatty acid profile, and content of vitamins (pyridoxine, niacin, thiamin, pantothenic acid, and riboflavin) and minerals (copper, iron, manganese, and zinc) [10][11][12].It is also considered an alternative source of protein for human consumption, especially in developing countries [4,13].Aside from their economic viability, quail meat products are gaining popularity as delicatessen products [4].
Most studies on quail deal with production, nutrition, and meat composition [14][15][16].Information on the microbiological quality of quail meat is scarce, and most of the studies focus on the microbial count of mesophiles, Enterobacteriaceae, Staphylococcus spp., and Pseudmomonas [17,18].There are no studies on the identification of the microbiota present in quail meat.The most important factor that causes poultry meat spoilage and reduces shelf life is bacterial growth.Mesophiles, Pseudomonas spp., lactic acid bacteria (LAB), Enterobacteriaceae, and Micrococcaceae are often used in poultry meat as indicators of processing hygiene and microbiological quality and safety [19][20][21].Therefore, it is relevant to study the microbiota present in quail meat.Foodborne pathogens such as Campylobacter spp., Salmonella, Clostridium perfringens, Listeria monocytogenes, and Staphylococcus aureus have been found in poultry meat [21,22].While chicken and turkey meat has been involved in outbreaks of Salmonella, Bacillus cereus, Clostridium perfringens, Campylobacter spp., Staphylococcus aureus, and Listeria monocytogenes, data on quail meat are not available [23].The main sources of microbiological contamination of poultry meat are the gastrointestinal tract of birds (Enterobacteriaceae, enterococci, Lactobacillus spp., Clostridium spp.), the feathers and skin of birds (Staphylococcus spp., Acinetobacter spp.), and the processing environment (Pseudomonas spp., Acinetobacter spp., Carnobacterium spp., Lactobacillus spp., and Listeria spp.[22,[24][25][26][27]. There is a great deal of concern about extended-spectrum-βlactamase (ESBL)-producing E. coli in poultry meat [20].However, there is no information available on its prevalence in quail meat.On the other hand, various authors have observed that E. coli isolated from quail meat has a significant level of antimicrobial resistance [28].
Staphylococci are common bacteria on poultry skin [29].Some species, such as Staphylococcus aureus, are well-known pathogens; other species are considered commensals [26].There is a special concern about methicillin-resistant S. aureus (MRSA) and its presence in meat since this pathogen has been related to hospital-acquired infections [30].In fact, some studies have shown a prevalence of MRSA of 29% in quails at the slaughterhouse level [31], which is higher than in chickens [32,33].Other methicillin-resistant staphylococci (MRS) have been found in animals and food [34].It should be noted that in 2020, the staphylococcal species belonging to the S. sciuri group (S.sciuri, S. lentus, S. fleurettii, S. vitulinus, and S. stepanovicii) were reassigned to the genus Mammaliicoccus [35].Mammaliicoccus sciuri has often been isolated from birds [36].Therefore, it is relevant to study the prevalence of MRSA, MRS, and methicillin-resistant Mammaliicoccus (MRM) in quail meat.
The purpose of this study was to evaluate the microbiological quality and safety of quail meat, as well as the prevalence of ESBL-producing E. coli, methicillin-resistant S. aureus, methicillin-resistant staphylococci, and methicillin-resistant Mammaliicoccus.

Quail Carcasses and Microbiological Determinations
Thirty-seven fresh quail carcasses produced in Spain were collected at random in Logroño (Spain) from traditional shops, supermarkets, and hypermarkets in 2020.The samples were collected in a preliminary study where the presence of antibiotic residues in commercial meat was evaluated [37].The quantity of samples was selected in accordance with the trade model statistics and availability [38].A total of 19 samples were collected in two different hypermarkets (H1 and H2), 12 in three different supermarkets (S1, S2, and S3), and 6 in two traditional shops (T1, T2).The samples were taken to the university facilities under refrigeration and kept at 4 • C for no longer than 1 h before analysis.

Isolation and Identification
A total of three to five typical colonies were selected from each quail meat sample and culture medium.Strains were purified on tryptone soy agar (Oxoid, Hampshire, UK).The purified strains were kept at −80 • C. Bacterial identification was conducted using the MALDI-TOF Biotyper technology (Bruker, Daltonik, Bremen, Germany).

Phenotypic Confirmation of ESBL-Producing E. coli
Phenotypic confirmation of ESBL-producing E. coli was conducted in accordance with the Clinical Laboratory Standards Institute's guidelines [40].This test was applied to all the E. coli strains selected from ChromID ESBL agar, MacConkey agar, and PCA agar.One E. coli strain identified by MALDI-TOF was chosen for each different medium and sample.

Confirmation of Methicillin Resistance of Staphylococcus spp. and Mammaliicoccus spp.
The methicillin resistance of Staphylococcus spp.and Mammaliicoccus spp.isolated from ChromID MRSA agar, besides all the S. aureus strains isolated, was confirmed in accordance with the Clinical Laboratory Standards Institute's guidelines [40].

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.

Results
Counts of the different bacteria in the 37 quail samples analyzed are shown in Table 2. Data on the microbial counts and presence of L. monocytogenes in quail samples analyzed from each retailer are shown in Table 3.  Mesophile counts were below 7 log CFU/g in all the samples analyzed and varied between 3.2 and 6.83 log CFU/g, with an average value of 5.25 ± 1.14 log CFU/g (Table 2).Significantly higher microbial loads (p < 0.05) were obtained in carcasses from hypermarkets and traditional businesses than in samples from supermarkets.No significant differences (p > 0.05) in mesophile populations were detected among carcasses collected in the same type of retailer (traditional shops, supermarkets, or hypermarkets) (Table 3).
Pseudomonas counts below 1 log CFU/g were obtained in 12 quail carcasses (32.431%).The counts in the other 25 carcasses varied between 2 and 6.48 log CFU/g, with an average figure of 3.92 ± 1.17 log CFU/g (Table 2).Significantly higher pseudomonas counts (p < 0.05) were obtained in carcasses from hypermarkets and traditional businesses than in samples from supermarkets.No significant differences (p > 0.05) in pseudomonas populations were detected among carcasses collected by the same type of retailer in the cases of traditional shops and hypermarkets (Table 3).Table 5 shows the Pseudomonas spp.distribution in fresh quail samples.The most common species were P. libanensis (41.76%) and P. extremorientalis (23.53%), followed by P. fluorescens (15.29%).A total of 85 pseudomonas were identified from thirteen different species.Enterobacteriaceae counts below 1 log CFU/g were obtained in 7 quail carcasses (18.92%).The counts in the other 30 carcasses varied between 1.3 and 5.72 log CFU/g, with an average figure of 3.09 ± 1.02 log CFU/g (Table 2).No significant differences (p > 0.05) in Enterobacteriaceae counts were observed among carcasses from different types of retailers or from the same type of retailer in the case of traditional shops and hypermarkets (Table 3).
Table 6 shows the Enterobacteriaceae distribution in fresh quail samples.The most often isolated Enterobacteriaceae was E. coli (42.53%).In fact, E. coli was detected in 19 samples (51.35%).A higher percentage of samples with the presence of E. coli was observed in samples from hypermarkets (57.89%) and supermarkets (50%) than in those from traditional shops (33.33%).Yersinia enterocolitica was found in two samples from hypermarket H1, while Yersinia frederikenii was found in one sample from hypermarket H2 and one sample from supermarket S2.E. coli was only detected in one sample from supermarket S2 when using ChromID ESBL.This strain was confirmed phenotypically as ESBL-producing.None of the strains of E. coli isolated from MacConkey agar or PCA agar were confirmed as ESBL-producing.
Staphylococci counts were below 1 log CFU/g in 4 carcasses (10.81%), three of them from hypermarket H1 (27.27%) and one from supermarket S2 (16.67%).The counts in the other 33 meat samples varied between 1.3 and 3.94 log CFU/g, with an average figure of 2.80 ± 0.64 log CFU/g (Table 2).No significant differences (p > 0.05) in staphylococci counts were detected among carcasses from different types of retailers or from the same type of retailer (traditional shops, supermarkets, or hypermarkets) (Table 3).Table 8 shows the Micrococcaceae distribution in fresh quail samples.The species most often isolated were S. cohnii (19.86%) and M. sciuri (17.02%).S. aureus was detected in one sample from hypermarket H1. S. epidermidis was found in 4 samples: 1 from hypermarket H1, 2 from supermarket S1, and 1 from traditional shop T2.Sixteen different Staphylococcus spp.and three Mammaliicoccus spp.were identified.The species found varied depending on the purchase establishment.M. sciuri was not found in any sample from supermarkets, but it was isolated from all the hypermarkets and traditional shops.M. sciuri was the dominant species in samples from hypermarket H2 and traditional shop T2.S. cohniii was not found in any sample from traditional shops, but it was isolated from all the hypermarkets and supermarkets.S. cohnii was the dominant staphylococcus in samples from hypermarket H1 and supermarkets S1 and S2.
Table 7. Antimicrobial resistance phenotype of E. coli isolated from quail meat. 3ESBL-producing strain. 4strains isolated from the same sample, but in different mediums. 5strains isolated from the same sample, but different medium.Methicillin-resistant strains were recovered from 8 samples when using chromID MRSA agar.The strains isolated were identified as M. sicuri in 5 samples (13.51%) and S. warneri in 4 samples (10.81%).One sample from traditional shop T2 contains both M. sicuri and S. warneri.We did not detect any methicillin-resistant S. aureus (MRSA) in quail meat.However, methicillin-resistant M. sciuri was isolated in two samples from hypermarket H1 (18.18%), two from traditional shop T2 (50%), and one from supermarket S2 (16.67%).Methicillin-resistant S. warneri was isolated from one sample from hypermarket H2 (12.5%), two samples from traditional shop T1 (100%), and one from traditional shop T2 (25%).The antimicrobial resistance phenotypes of methicillin-resistant staphylococci (MRS) and methicillin-resistant Mammaliicoccus (MRM) are shown in Table 9.All the strains were multiresistant.It is worth noting that all the methicillin-resistant S. warneri showed resistance to at least 16 antibiotics, all of them resistant not only to cefoxitin and penicillin but also to clindamycin, tetracycline, amikacin, streptomycin, sulfadiazine, and mupirocin.L. monocytogenes was detected in seven (18.92%) samples (Table 3).All the L. monocytogenes positive samples were from the hypermarket H1, which means that this pathogen was present in 63.64% of the samples from this hypermarket (Table 3).L. monocytogenes counts were below 2 log CFU/g in 5 samples, while the other 2 samples showed counts of 2.15 and 2.94 log CFU/g.

Medium of Isolation
Campylobacter jejuni was only detected in one sample (2.70%) from hypermarket H1 (9.09%) (Table 3).Clostridium perfringens was not detected in any sample.

Discussion
We found mesophile counts of 5.25 ± 1.14 log CFU/g in quail samples.Similar counts have been reported by Piras et al. (4.90 log CFU/g) and Naeem et al. (5.17 ± 0.11) in quail meat [41,42].Other authors have also found similar figures in turkey meat [20].
Pseudomonas spp., Micrococcaceae, lactic acid bacteria, Enterobacteriaceae, Brochotrix thermosphacta, and enterococci are often present in poultry meat [21,42,43].As Piras et al. observed, the dominant bacteria in fresh quail meat were pseudomonas, followed by staphylococci and, to a lesser degree, lactic acid bacteria and Enterobacteriaceae [40].Pseudomonas has been noted as the principal spoilage bacteria in poultry meat [20,24,44].Also, pseudomonas has been reported as the predominant bacteria in chicken meat [45].However, other studies have pointed out that the prevalent bacteria in turkey meat are lactic acid bacteria [20].As in the current study, other authors have also isolated Acinetobacter spp.from chicken, which is related to cross-contamination during processing [46].However, the species reported in chickens are A. lwoffii, A. johnsonii, and A. guillouiae [43], while we found the following species: Acinetobacter harbonensis, Acinetobacter albensis, and Acinetobacter calcoaceicus.Also, Chryseobacterium spp.and Microbacterium spp.have been isolated from chicken and turkey [19,20,47,48].
We found pseudomonas counts below 1 log CFU/g in 32.41% of the samples.The other samples displayed counts of 3.92 ± 1.17, being in the range between 2 and 6.48 log CFU/g.Lower pseudomonas counts have been reported by Naeem et al. in quail meat [42].Pseudomonas spp.are relevant spoilage bacteria.Other studies have also shown that the most common Pseudomonas spp. in turkey meat are P. libanensis and P. extremorientalis [20].A total of 13 different species of Pseudomonas were isolated in the current work, while lower Pseudomonas spp.have been reported in chicken meat by other authors (9 species) [49].It should be noted that the principal contamination source for this bacterium is the processing environment [25].
We found Enterobacteriaceae counts below 1 log CFU/g in 18.92% of the quail carcasses.The other samples displayed counts of 3.09 ± 1.02, being in the range between 1.3 and 5.72 log CFU/g.Lower Enterobacteriaceae counts in quail meat have been reported by other authors [41,42].In the present work, the most common Enterobacteriaceae isolated were E. coli.We observed a prevalence of E. coli in fresh quail meat of 51.35%, while other authors have reported lower prevalence rates (27.77%)[50].However, other authors have reported higher prevalence rates [28,51].It seems that the prevalence rates of E. coli are higher in quail meat than in turkey meat [20,49].It should be noted that high counts of E. coli (9.79 log CFU/g) have been reported in the quail gut microbiota, possibly due to carcass contamination during slaughtering [52].Hafnia spp., Pantoea spp., Serratia spp., Yersinia spp., Ewingella americana, and Buttiauxella spp.have been often isolated from chicken and turkey meat [19,53].
In the present study, L. monocytogenes was detected in 18.92% of the samples.A lower prevalence of L. monocytogenes has been found by Rahimi et al. in quail meat (5.2%) [65].Other authors have also found a similar prevalence of L. monocytogenes in chicken meat [66], while a higher prevalence has been reported by other authors in chicken and turkey meat [67].Contamination of poultry with L. monocytogenes mainly occurs during processing; thus, its prevalence depends on the hygienic practices during processing, especially in the portioning operations [22].It should be noted that all the L. monocytogenes-positive samples were from the same retailer (hypermarket H1), with this pathogen present in 63.64% of the samples from this hypermarket.These findings suggest the relevance of meat handling and cross-contamination and highlight the importance of maintaining good hygienic practices.
In the present study, Campylobacter jejuni was detected in one quail meat sample (2.70%).A higher prevalence of Campylobacter has been reported in quails at the farm level and slaughterhouse [3,68].Meat can be contaminated with Campylobacter spp.during slaughter and processing [69].As El-Dengawy and Nassar reported, we did not detect Clostridium perfringens in quail meat [70].
We identified 19 different genera in quail meat, a higher figure than that reported by other authors in chicken meat (15 genera) [49].Moreover, we isolated bacteria considered to be recognized foodborne pathogens as well as opportunistic pathogens.

Conclusions
This study highlights that quail carcass microbiota can be a source of both opportunistic or emerging pathogens and recognized foodborne pathogens.Moreover, quail carcasses can be a source of ESBL-producing E. coli, methicillin-resistant staphylococci (MRS), and methicillin-resistant Mammaliicoccus (MRM).The presence of ESBL-producing E. coli and multi-resistant S. warneri in quail carcasses is of special concern, and additional measures should be adopted in the context of the One Health approach.Resistance to critical antibiotics in accordance with the European Medicine Agency (EMA) criteria (such as rifampicin, linezolid, mupirocin, ceftaroline, and fluoroquinolones) has been found in S. warneri strains, while resistance to carbapenems, glycylcyclines, fluoroquinolones, and cephalosporins of the third generation has been found in E. coli strains.

Table 1 .
Media, temperature, and incubation times are used for microbiological determinations.
1 Number of samples.

Table 3 .
Microbial counts (log CFU/g) and the presence of Listeria monocytogenes found in quail meat from different retailers.

Table 4 .
Bacteria identified in fresh quail meat isolated from Plate Count Agar.

Table 5 .
Pseudomonas spp.isolated from fresh quail meat (recovered from chromogenic agar for Pseudomonas).
1Number of samples in which E. coli was isolated.

Table 9 .
Antimicrobial resistance phenotypes of methicillin-resistant staphylococci and Mammaliicoccus isolated from quail meat.