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Article

Microbial Load and Antibiotic Resistance Patterns of Escherichia coli and Enterococcus faecalis Isolates from the Meat of Wild and Domestic Pigeons

by
Jorge Cordero
1,2,
Carlos Alonso-Calleja
1,2,
Camino García-Fernández
1,2 and
Rosa Capita
1,2,*
1
Department of Food Hygiene and Technology, Veterinary Faculty, University of León, E-24071 León, Spain
2
Institute of Food Science and Technology, University of León, E-24071 León, Spain
*
Author to whom correspondence should be addressed.
Foods 2019, 8(11), 536; https://0-doi-org.brum.beds.ac.uk/10.3390/foods8110536
Submission received: 14 September 2019 / Revised: 14 October 2019 / Accepted: 17 October 2019 / Published: 1 November 2019
(This article belongs to the Section Food Microbiology)
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Abstract

:
An expansion in the consumption of pigeon meat has occurred in recent years. However, little is known about microbial load and antibiotic resistance of this foodstuff. The hygiene status and the antibiotic resistance patterns (disc diffusion; Clinical and Laboratory Standards Institute, CLSI) of Escherichia coli and Enterococcus faecalis isolates from wild and domestic pigeon carcasses were investigated. Average microbial loads (log10 cfu/cm2) ranged from 1.40 ± 1.17 (fecal coliforms) to 3.68 ± 1.40 (psychrotrophs). The highest (p < 0.05) microbial loads were observed in domestic pigeons. No substantial differences were found between isolates from domestic and wild pigeons with regard to the prevalence of antibiotic resistance. Of the E. coli strains, 20.00% were susceptible, 25.00% showed resistance or reduced susceptibility to one antimicrobial and 55.00% were multi-resistant. Among the E. faecalis isolates, 2.22% were susceptible and 97.78% were multi-resistant. The greatest prevalence of resistance or reduced susceptibility among E. coli was observed for amoxicillin-clavulanic acid (20.00% strains), ampicillin (26.67%), streptomycin (55.00%) and tobramycin (20.00%). The prevalence of resistance or reduced susceptibility among E. faecalis ranged from 31.11% (trimethoprim/sulfamethoxazole) to 97.78% (erythromycin). Meat from pigeons is a major reservoir of antibiotic-resistant bacteria. The need for the correct handling of this foodstuff in order to reduce risks to consumers is underlined.

Graphical Abstract

1. Introduction

Each year, over two million wild pigeons are hunted in Spain for human consumption. This amounts to approximately 1256 tons of meat [1]. In addition, there are farms that breed pigeons and, in 2004, these produced approximately 57 tons of meat. Moreover, this activity is on an upward trend in Spain, as the number of such farms rose from 535 in 2007 to 1717 in 2019 [2].
The expansion in the production of pigeon meat that has occurred in recent years justifies an interest in its hygiene status. Aerobic plate counts (APCs), psychrotrophs, Enterobacteriaceae, fecal coliforms, enterococci, Micrococcaceae, lactic-acid bacteria (LAB) and Brochothrix thermosphacta have been used in the meat and poultry industries for assessing microbiological safety, processing hygiene, potential shelf-life and the keeping quality of products [3,4,5].
Escherichia coli and Enterococcus spp. are common bacteria in the intestinal tract of human beings and animals. Most strains of such bacteria are not pathogenic and are seen solely as indicators of fecal contamination. However, between 10% and 15% of E. coli strains are pathogenic and are able to cause a wide range of food-borne or water-borne illnesses [6,7]. In 2017, E. coli was responsible for 48 (0.9%) out of a total of 5079 outbreaks of food-borne diseases in the European Union [8]. On the other hand, enterococcal species have emerged as major causes of nosocomial- and community-acquired infections because of their capacity to acquire virulence traits [9]. Both E. coli and Enterococcus spp. have a capacity to acquire antibiotic resistance genes, based on the efficient horizontal transfer mechanisms these microorganisms have developed over the course of time. Hence, strains of these bacterial groups have been recognized as reservoirs of antimicrobial resistance genes. This is a worrying fact in the context of public health, since there is a high likelihood of the transfer of genes to pathogenic bacteria. On the other hand, this characteristic also allows these bacterial groups to be used as sentinels for resistance to antibiotics, and for tracking the evolution of antibiotic resistance in different environments [10].
Resistance to antibiotics has grown at an alarming rate in recent years, so as to become one of the greatest threats to public health and one of the main challenges for medicine in the twenty-first century [11]. Food-producing animals are a possible source of antibiotic-resistant bacteria that can be transferred to humans along the food chain [12]. Contacts with species which transfer multidrug-resistant bacteria to humans provide a biological mechanism for the increase noted in antibiotic resistance genes in human populations [13,14]. In this context, the main concerns about meat are the possibility of the bacteria surviving as a consequence of insufficient cooking and the potential for cross-contamination of other foods through inappropriate handling. In addition, wild animals are of importance for antibiotic resistance in two ways. Firstly, they can constitute a reservoir of antibiotic-resistant bacteria and resistance genes, with the potential for long-distance dissemination throughout the environment. Secondly, human activities can influence the gut microbiota of wild species, which could mirror the spectrum of antibiotic resistance in humans, as well as in the environment [9].
Monitoring resistance to antibiotics is essential in obtaining information about the size of this problem and the way it is trending, as well as in planning any control measures introduced and checking their effectiveness. However, little is known about resistance to antibiotics in strains from domestic and wild pigeons in Spain. The aim of this research was to learn and compare the microbiological quality of meat from domestic and wild pigeons in northwestern Spain. In addition, the patterns of resistance to antibiotics in strains of E. coli and Enterococcus faecalis isolated from such products were determined.

2. Materials and Methods

2.1. Samples

Forty pigeon carcasses were analyzed. Of these, 12 samples were domestic pigeons (Columba livia) reared in captivity, and 28 were wild pigeons (Columba palumbus) that had been hunted. Wild birds were obtained directly from hunters. All of the samples came from six localities in the Provinces of León and Zamora in northwestern Spain. Immediately after death, the birds were plucked and eviscerated in a closed room under aseptic conditions. None of the pigeon samples had the intestine perforated. The carcasses were transported individually in an ice chest to the laboratory, where they were kept under refrigeration (4 °C). From death to analysis, a maximum of 24 h elapsed.

2.2. Microbiological Determinations

The breasts of the carcasses were used as sampling points, and only regions which were not wounded by lead shot were used. Each sample was prepared by excising a piece of skin 10 cm2 in area with a sterile knife blade and a template. The samples were placed in a sterile stomacher bag containing 90 mL of sterile 0.1% (w/v) peptone water (Oxoid Ltd., Hampshire, UK) and homogenized (Masticator IUL, Barcelona, Spain) for two minutes. Decimal dilutions in sterile 0.1% (w/v) peptone water were prepared from this homogenate. Table 1 shows the culture media, incubation conditions and references used for each of the microbial groups studied. All inoculations were carried out in duplicate. Plates with between 25 and 250 colonies (spread plate technique) or between 30 and 300 colonies (pour plate technique) were counted, and average counts were calculated.

2.3. Isolation and Identification of Escherichia coli and Enterococcus spp. Strains

A total of three to five typical colonies in violet red bile agar (VRBA) and in kanamycin aesculin azide (KAA) agar were taken for later identification. Strains were streaked onto plates of tryptone soy agar (TSA, Oxoid, Hampshire, UK) and then incubated for 24 h at 44 °C (strains from VRBA) or 42 °C (strains from KAA) in order to obtain pure cultures. Presumed E. coli were examined for colony and cell morphology, for Gram staining, and for oxidase and catalase activities. Strains corresponding to Gram-negative, catalase-positive and oxidase-negative bacilli were identified with the aid of a miniaturized E. coli test system (Liofilchem s.r.l., Teramo, Italy) in accordance with the manufacturer’s instructions. A total of 60 E. coli strains (32 from domestic pigeons and 28 from wild) were selected for further antibiotyping.
Presumed enterococci were identified on the basis of Gram staining, catalase production, growth at 10 °C and at 45 °C, and growth in the presence of 6.5% NaCl. Gram-positive catalase-negative cocci able to grow under these conditions were inoculated into microtubes of API 20 Strep (bioMérieux, Marcy L’Étoile, France) in accordance with the manufacturer’s instructions. Data interpretation was carried out using the Analytical Profile Index (API) database (V7.0) with apiweb™ identification software (bioMérieux, Marcy L’Étoile, France). A total of 45 E. faecalis strains, 16 from domestic pigeons and 29 from wild, were selected for further antibiotyping. All strains were stored at −50 °C in tryptone soy broth (TSB) with 20% glycerol.

2.4. Antibiotic Susceptibility Determination

The strains were investigated to determine their susceptibility to antibiotics on Muller–Hinton agar (MH, Oxoid, Hampshire, UK) using the disc diffusion method. The susceptibility of E. coli strains was tested against a panel of 16 antimicrobials of clinical importance: amoxicillin-clavulanic acid (AMC; 30 µg), ampicillin (AMP; 10 µg), ceftazidime (CAZ; 30 µg), cefotaxime (CTX; 30 µg), cefoxitin (FOX; 30 μg), imipenem (IPM; 10 µg), aztreonam (ATM; 30 µg), nalidixic acid (NA; 30 mg), ciprofloxacin (CIP; 5 µg), chloramphenicol (C; 30 µg), gentamicin (CN; 10 µg), amikacin (AK; 30 μg), streptomycin (STR; 10 µg), tobramycin (TOB; 10 µg), tetracycline (TE; 30 µg) and trimethoprim/sulfamethoxazole (SXT; 25 µg). The E. faecalis strains were investigated to determine their susceptibility to 14 antibiotics: ampicillin (AMP; 10 µg), ciprofloxacin (CIP; 5 µg), chloramphenicol (C; 30 µg), gentamicin (CN; 10 µg), kanamycin (K, 120 µg), streptomycin (STR; 10 µg), erythromycin (E; 15 µg), fosfomycin (FOS; 50 µg), nitrofurantoin (F; 300 µg), rifampicin (RD; 5 µg), teicoplanin (TEC, 30 µg), vancomycin (VA, 30 µg), tetracycline (TE; 30 µg), and trimethoprim/sulfamethoxazole (SXT; 25 µg). All the discs used were purchased from Oxoid (Hampshire, UK).
After incubation at 37 °C for 18 to 24 h, inhibition halos were measured and scored as susceptible, intermediate (reduced susceptibility) or resistant according to the Clinical and Laboratory Standards Institute (CLSI) [19] guidelines. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as reference strains for antibiotic disc control.

2.5. Statistical Analysis

Microbial counts were transformed to log10 cfu/cm2. Data were compared by analysis of variance (ANOVA) techniques, using Duncan’s multiple range test to separate averages. The prevalence of resistant strains in wild and domestic birds was compared using Fisher’s Exact Test. Significance was determined at the 5% (p < 0.05) level. All the tests were carried out with the Statistica® 8.0 package (Statsoft Ltd., Tulsa, OK, USA).

3. Results

3.1. Microbial Loads

Microbial counts obtained from pigeon meat can be seen in Table 2. Average counts (log10 cfu/cm2) varied between 1.40 ± 1.17 for fecal coliforms and 3.68 ± 1.40 for psychrotrophs. Figures for domestic pigeons ranged from 1.47 ± 2.17 log units for B. thermosphacta to 3.84 ± 2.04 log units for psychrotrophs. There would appear to be no studies relating to the meat of farmed pigeons that could be used in comparisons with these results. In earlier investigations of chicken carcasses and drumsticks obtained from slaughterhouses or retail outlets, counts higher than those in the present research were observed [4,5,20,21,22]. The low microbial counts found in meat from domestic pigeons as compared with chicken meat may be due to the fact that the pigeons studied were slaughtered individually, since they were killed at farms for own consumption, and were plucked, eviscerated and refrigerated immediately after death, with microbiological analyses performed within 24 h of slaughter. It has been demonstrated that microbial counts increase significantly as storage time lengthens [23].
Microbial counts obtained on meat from wild pigeons varied between 1.10 ± 1.11 log10 cfu/cm2 for fecal coliforms and 3.61 ± 1.04 log10 cfu/cm2 for psychrotrophs. These figures are similar to microbial counts reported by El-Ghareeb et al. [24] on the skin of the breast area of pigeons hunted in Egypt. The present results are also in agreement with values observed by El-Dengawy and Nassar [25] for wild quail carcasses in Egypt, and by Paulsen et al. [23] for hunted pheasants in the Slovak Republic. In contrast, higher microbial loads were observed by Mousa et al. [26] in frozen quail in Egypt. These authors reported values of 6.71 log10 cfu/g, for psychrotrophs, 3.93 log10 cfu/g for enterobacteria and 3.86 log10 cfu/g for coliforms. It should be noted, however, that comparations between reports should be considered with caution because the different ways to report microbial load (ufc/cm2 or ufc/g).
It must be pointed out that the evisceration and refrigeration of the carcasses of the wild pigeons tested were performed immediately after they were hunted. This may have had an influence over the low counts found. In a number of studies, the evisceration of game has often been carried out no earlier than several days after death. This raises concerns from the viewpoint of food hygiene, especially when the intestines have been perforated and fecal material released into the body cavity [27]. Moreover, microbiological analyses were carried out within a period not exceeding 24 h at most after the animal was shot, so that it was to be expected that no substantial microbial growth would have occurred.
The lowest levels (p < 0.05) for APC, Enterobacteriaceae, fecal coliforms, Micrococcaceae and LAB were observed in wild pigeons. The microbiological condition of the carcasses of hunted animals depends on several factors. Among other considerations, the health of the animal, the time before chilling, and the conditions under which carcasses are eviscerated, transported, stored, and processed have a critical influence over carcass hygiene. Moreover, small game animals such as pigeons are usually hunted with shotguns, ensuring death from multiple lead shots hitting the body, occasionally penetrating deeper tissues and intestines [27,28]. The low levels of microorganisms recorded in the meat of hunted pigeons in the present study indicate that the product had been correctly handled. Moreover, the findings suggest that wild pigeons had less superficial contamination than domestic pigeons that had been reared on farms with a high density of animals. On these same lines, several authors have observed that the higher the stocking density, the higher the microbiological counts found in poultry meat. This occurs mainly because close proximity in livestock farms facilitates surface contamination with fecal material [29].
Psychrotrophs are the microorganisms of choice for assessing the microbiological quality of refrigerated meat. Higher average figures were observed for psychrotrophs, incubated at 7 °C, than for APC, incubated at 30 °C. These findings coincide with those from earlier work relating to poultry and poultry preparations stored under refrigeration [21]. In the work being presented here, samples remained under refrigeration for approximately 24 h from death to analysis. The fact that higher levels of psychrotrophs were observed suggests that storage temperatures were low enough.

3.2. Antibiotic Susceptibility in E. coli Strains

It would appear that the present study was the first to demonstrate the presence of several antimicrobial resistances in E. coli and E. faecalis recovered from pigeon meat in northwestern Spain. A total of 60 E. coli isolates, 32 from domestic pigeon meat and 28 from wild pigeon meat, were screened for susceptibility to a panel of 16 antimicrobial substances of veterinary and human clinical significance. Twelve strains (20.00% of the total) were sensitive to all the antibiotics, 15 strains (25.00%) showed resistance or reduced susceptibility to one antibiotic, and 33 strains (55.00%) were multi-resistant (showing resistant or reduced susceptibility to two or more antibiotics). The presence of antibiotic-resistant E. coli strains in meat is a frequent finding [12].
The average number of resistances per E. coli strain was 0.57. If the strains with resistance and those with reduced susceptibility are grouped together, the number of resistances per strain was 2.03. The number of resistances was 0.19 for isolates from domestic pigeons and 1.00 for those from wild birds. If the grouping of resistance and reduced susceptibility mentioned above is applied, the figures are 1.53 resistances per strain in domestic birds and 2.61 in wild. The average number of resistances per E. coli strain observed in the present study was lower than the figures recorded by Logue et al. [30] in the United States. These researchers found that strains of enterobacteria originating in poultry meat were resistant to 4.0 antimicrobials on average.
Clinical treatment for infections is rendered complex when strains are resistant to antibiotics. Infections caused by multi-resistant bacteria are associated with high morbidity and mortality rates, together with an increase in the costs of treating infections [31,32]. Bacteria resistant to antibiotics can cause infections in humans through the consumption of foodstuffs when cross-contamination or incomplete cooking occur. Thus, the presence of multi-resistant bacteria in food is a cause for great concern [10].
Table 3 and Figure 1 show the percentages of E. coli strains that were susceptible, intermediate or resistant to each of the antibiotics used. All E. coli strains were susceptible to imipenem (IPM) and trimethoprim-sulfamethoxazole (SXT). Resistance was observed in E. coli strains relating to amoxicillin-clavulanic acid (AMC) (3.33% of strains), ampicillin (AMP) (6.67%), cefotaxime (CTX) (1.67%), cefoxitin (FOX) (5.00%), aztreonam (ATM) (8.33%), nalidixic acid (NA) (1.67%), gentamicin (CN) (8.33%), amikacin (AK) (6.67%), streptomycin (STR) (5.00%), tobramycin (TOB) (6.67%) and tetracycline (TE) (3.33%). Resistance or reduced susceptibility was observed in the case of AMC (20.00% of strains), AMP (26.67%), CAZ (5.00%), CTX (13.33%), FOX (6.67%), ATM (15.00%), NA (5.00%), CIP (1.67%), C (5.00%), CN (11.67%), AK (11.67%), STR (55.00%), TOB (20.00%) and TE (6.67%). High levels of resistance to such antimicrobials have also been reported in E. coli from domestic birds [33,34,35,36,37,38,39] and from various kinds of wild birds and mammals living in close proximity to humans, including pigeons [14,40,41,42,43,44,45,46,47,48,49,50,51,52,53].
It should be stressed that at least 20% of strains showed resistance or reduced susceptibility to amoxicillin-clavulanic acid, ampicillin, streptomycin and tobramycin, which are classified as “critically important antimicrobials for human medicine” [54]. According to the World Organization for Animal Health, these four antimicrobial compounds are categorized as “critically important veterinary antimicrobials” [55].
Data for resistance (including reduced susceptibility) in E. coli from domestic and wild pigeons are compared in Table 3 and Figure 2. Wild pigeons showed a higher (p < 0.05) percentage of resistant or intermediate strains than domestic pigeons in the case of FOX and AK. Similar (p > 0.05) percentages of resistant or intermediate E. coli strains were found in domestic and wild pigeons for all the remaining antibiotics. This is a surprising result, since it was to be expected that domestic birds would show higher levels of antibiotic resistance than wild pigeons. Contact with anthropogenic factors, such as human refuse or livestock farming, may encourage the colonization of birds by resistant bacteria, while also potentially allowing exposure to antimicrobial medication, antimicrobial residues, or resistant genes. This would contribute to the development and maintenance of antibiotic resistance in the microbiota of domestic animals [10,13,56,57]. The very slight differences noted here between domestic and wild pigeons in respect to resistance to antibiotics in isolates of E. coli underlines the impact of both livestock and human densities on the environment. It has been reported that a number of sites of human activities, such as human and veterinary clinical establishments, farms, landfills, or waste-water treatment facilities, may be the scene of interactions with wildlife, which may have a direct association with the antibiotic resistance profiles of bacteria of domestic and wild animals in any given geographic location [9]. It must be pointed out that the wild pigeons included in the present study came from zones close to human settlements and cattle farms, and so there would be a strong likelihood of contact and exchange of bacteria between pigeons, people and farm animals.

3.3. Antibiotic Susceptibility in E. faecalis Strains

Forty-five E. faecalis strains, 16 from domestic pigeons and 29 from wild, were screened for susceptibility to 14 antibiotics. Forty-four strains (97.78% of the total) were multi-resistant, having resistance or reduced susceptibility to two or more antibiotics, while just one strain (2.22%) was sensitive to all the antibiotics. The number of resistances per strain was 5.58. If strains with resistance and those with reduced susceptibility are taken together, the number of resistances per strain would rise to 10.49. The number of resistances per strain was similar in wild and in domestic pigeons. Thus, the figure for strains from domestic pigeons would be 5.38 if just resistance were to be considered, but 10.56 if both resistance and reduced susceptibility were considered together. For wild pigeons, the corresponding values would be 5.69 and 10.45, respectively. These data are similar to those previously recorded for Gram-positive bacteria in chicken meat, where an average of 6.35 antimicrobials was found for Staphylococcus aureus strains [5].
It should be noted that the average number of resistances per strain observed in the work being reported here was much higher for E. faecalis than for E. coli isolates. It has been suggested that E. faecalis has a striking ability to acquire and transfer antibiotic resistance genes [9]. Hence, E. faecalis is the enterococcal species that shows the highest average number of antimicrobial resistances per isolate [9,58,59,60].
The considerable prevalence of resistant or intermediate E. faecalis strains observed in the work being reported here is worrying, since the resistances detected would probably undermine the usefulness as a therapeutic option of several antibiotics used in both human and veterinary medicine. E. faecalis isolates showed resistance to ampicillin (AMP (22.22% of strains), ciprofloxacin (CIP) (55.56%), chloramphenicol (C) (2.22%), gentamicin (CN) (66.67%), kanamycin (K) (71.11%), streptomycin (STR) (88.89%), erythromycin (E) (35.56%), fosfomycin (FOS) (15.56%), nitrofurantoin (F) (62.22%), rifampicin (RD) (57.78%), teicoplanin (TEC) (2.22%), vancomycin (VA) (13.33%), tetracycline (TE) (62.22%) and trimethoprim-sulfamethoxazole (SXT) (2.22%). Resistance or reduced susceptibility was observed for AMP (62.22% of strains), CIP (93.33%), C (77.78%), CN (86.67%), K (84.44%), STR (93.33%), E (97.78%), FOS (53.33%), F (84.44%), RD (77.78%), TEC (35.56%), VA (75.56%), TE (95.56%) and SXT (31.11%), as shown in Table 4 and Figure 3. These high percentages are of concern, because the above-mentioned antibiotics are categorized as “critically important antimicrobials” in the case of ampicillin, ciprofloxacin, gentamicin, kanamycin, streptomycin, erythromycin, fosfomycin, rifampicin, teicoplanin and vancomycin, as “highly important antimicrobials” in respect to chloramphenicol and tetracycline, or as “important antimicrobials” in the case of nitrofurantoin and trimethoprim-sulfamethoxazole, for human medicine [54]. Moreover, ampicillin, ciprofloxacin, gentamicin, kanamycin, streptomycin, fosfomycin, tetracycline and trimethoprim-sulfamethoxazole are classified as “veterinary critically important antimicrobials”, and rifampicin as a “veterinary highly important antimicrobial” [55]. High levels of resistance to such antimicrobials have also been reported in enterococcal strains from various domestic and wild animal species, including pigeons [9,14,45,46,51,61,62,63].
The great number of resistant strains in foods of animal origin mentioned in numerous publications would appear to be related to the use of antibiotics in animal production and clinical practice [10]. Thus, in the present study, a considerable prevalence of resistance was observed to antibiotics widely used in animal production [64,65,66]. It should be noted, however, that a high prevalence of resistance was also observed to substances whose use has been prohibited in food-producing animals for some decades, for instance, chloramphenicol or nitrofurantoin. Mechanisms of cross-resistance and co-resistance may have contributed to the persistence over time of genes for resistance to these substances, as previously suggested [10,35].
Data for resistance or reduced susceptibility in E. faecalis from domestic and wild pigeons may be seen in Table 4 and Figure 4. Wild pigeons showed a higher (p < 0.05) percentage of resistant or intermediate strains than domestic pigeons in the case of C and RD. In contrast, the highest percentage of resistance or reduced susceptibility to TEC was observed for isolates from domestic pigeons. Similar (p > 0.05) percentages of resistant or intermediate E. coli strains were seen in strains from both domestic and wild pigeons in the case of all the other antibiotics.

4. Conclusions

Pigeon meat had low levels of all the microbial groups studied in comparison with the counts recorded in previous work with meat from other species of birds. The highest microbial loads were shown by pigeons reared in captivity, possibly as a consequence of high animal densities on farms. One worrying fact is the considerable prevalence of resistance to antibiotics observed among strains of E. coli and especially of E. faecalis isolated from pigeon meat. No substantial differences were noted between the levels of the prevalence of resistance to antibiotics in bacteria taken from the meat of domestic or of wild pigeons, which highlights the strong correlation between human activity and the spread of antibiotic resistance into wildlife.
The results from the present study provide evidence that bacteria from pigeon meat pose major potential risks, both direct and indirect, to consumers, in view of the considerable rates of resistance or of reduced susceptibility to antibiotics that were found. These results underline the importance of the careful handling of pigeon meat during preparation, avoiding cross-contamination and ensuring thorough cooking. The extensive prevalence of resistance to antibiotics found is a worrying fact from the viewpoint of food safety and public health, pointing to a need to take measures to reduce the rates of resistance to antibiotics among the bacteria present in pigeons.

Author Contributions

Conceptualization, C.A.-C. and R.C.; Data Curation, J.C.; C.A.-C.; C.G.-F. and R.C.; Formal Analysis, J.C.; C.G.-F. and R.C.; Funding Acquisition, C.A.-C. and R.C.; Investigation, J.C.; C.A.-C.; C.G.-F. and R.C.; Methodology, J.C. and C.A.-C.; Project Administration, C.A.-C. and R.C.; Resources, C.A.-C. and R.C.; Software, R.C.; Supervision, C.A.-C.; Validation, C.A.-C.; C.G.-F. and R.C.; Visualization, C.A.-C.; Writing—Original Draft, J.C. and R.C.; Writing—Review and Editing, C.A.-C. and R.C.

Funding

Ministerio de Ciencia, Innovación y Universidades: RTI2018-098267-R-C33; Consejería de Educación, Junta de Castilla y León: LE164G18.

Acknowledgments

The authors wish to thank the Ministerio de Ciencia, Innovación y Universidades (Spain; RTI2018-098267-R-C33) and the Junta de Castilla y León (Spain; LE164G18).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Game Meat—Production and Trade in the UNECE Region, FAO, Geneva. Available online: https://www.unece.org/fileadmin/DAM/timber/meetings/2018/20180321/game-meat-draft-2018-03.pdf (accessed on 14 September 2019).
  2. El Sector de la Avicultura de Carne en Cifras. Principales Indicadores Económicos. 2018. Ministerio de Agricultura, Pesca y Alimentación. Available online: https://www.mapa.gob.es/es/ganaderia/temas/produccion-y-mercados-ganaderos/indicadoresaviculturacarneparapublicar2018_tcm30-419674.pdf (accessed on 14 September 2019).
  3. European Commision. Commission regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Off. J. Eur. Union 2005, L 338, 1–26. [Google Scholar]
  4. Del Río, E.; Panizo-Morán, M.; Prieto, M.; Alonso-Calleja, C.; Capita, R. Effect of various chemical decontamination treatments on natural microflora and sensory characteristics of poultry. Int. J. Food Microbiol. 2007, 115, 268–280. [Google Scholar] [CrossRef]
  5. Buzón-Durán, L.; Capita, R.; Alonso-Calleja, C. Microbial loads and antibiotic resistance patterns of Staphylococcus aureus in different types of raw poultry-based meat preparations. Poult. Sci. 2017, 96, 4046–4052. [Google Scholar] [CrossRef]
  6. Kaper, J.B.; Nataro, J.P.; Mobley, H.L. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2004, 2, 123–140. [Google Scholar] [CrossRef]
  7. Brzuszkiewicz, E.; Thurmer, A.; Schuldes, J.; Leimbach, A.; Liesegang, H.; Meyer, F.D.; Boelter, J.; Petersen, H.; Gottschalk, G.; Daniel, R. Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC). Arch. Microbiol. 2011, 193, 883–891. [Google Scholar] [CrossRef] [PubMed]
  8. EFSA; ECDC. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018, 16, e05500, 262. [Google Scholar] [CrossRef]
  9. Guerrero-Ramos, E.; Cordero, J.; Molina-González, D.; Poeta, P.; Igrejas, G.; Alonso-Calleja, C.; Capita, R. Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain. Food Microbiol. 2016, 53, 156–164. [Google Scholar] [CrossRef] [PubMed]
  10. Capita, R.; Alonso-Calleja, C. Antibiotic-resistant bacteria: A challenge for the food industry. Crit. Rev. Food Sci. Nutr. 2013, 53, 11–48. [Google Scholar] [CrossRef] [PubMed]
  11. Antimicrobial Resistance. Tackling the Burden in the European Union. Organization for Economic Coordination and Development (OECD). Available online: https://www.oecd.org/health/health-systems/AMR-Tackling-the-Burden-in-the-EU-OECD-ECDC-Briefing-Note-2019.pdf (accessed on 14 September 2019).
  12. EFSA; ECDC. The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2017. EFSA J. 2019, 17, e05598, 278. [Google Scholar] [CrossRef]
  13. Allen, S.E.; Boerlin, P.; Janecko, N.; Lumsden, J.S.; Barker, I.K.; Pearl, D.L.; Reid-Smith, R.J.; Jardine, C. Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in southern Ontario, Canada. Appl. Environ. Microbiol. 2011, 77, 882–888. [Google Scholar] [CrossRef]
  14. Santos, T.; Silva, N.; Igrejas, G.; Rodrigues, P.; Micael, J.; Rodrigues, T.; Resendes, R.; Goncalves, A.; Marinho, C.; Goncalves, D.; et al. Dissemination of antibiotic resistant Enterococcus spp. and Escherichia coli from wild birds of Azores Archipelago. Anaerobe 2013, 24, 25–31. [Google Scholar] [CrossRef] [PubMed]
  15. Jay, J.M. A review of aerobic and psychrotrophic plate count procedures for fresh meat and poultry products. J. Food Prot. 2002, 65, 1200–1206. [Google Scholar] [CrossRef] [PubMed]
  16. Cousin, M.A.; Jay, J.M.; Vasavada, P.C. Psychrotrophic microorganisms. In Compendium of Methods for the Microbiological Examination of Foods; Downes, F.P., Ito, K., Eds.; American Public Health Association: Washington, DC, USA, 2001; pp. 159–166. [Google Scholar]
  17. Baird, R.M.; Corry, J.E.J.; Curtis, G.D.W. Pharmacopeia of culture media for food microbiology. Int. J. Food Microbiol. 1987, 5, 221–222. [Google Scholar]
  18. Anonymous. The Oxoid Manual; Oxoid: Basingstoke, UK, 1990. [Google Scholar]
  19. CLSI. M100 Performance Standars for Antimicrobial Susceptibility Testing, 29th ed.; Clinical and Laboratory Standards Institute: Pennsylvania, PA, USA, 2019. [Google Scholar]
  20. Capita, R.; Alonso-Calleja, C.; García-Fernández, M.C.; Moreno, B. Microbiological quality of retail poultry carcasses in Spain. J. Food Prot. 2001, 64, 1961–1966. [Google Scholar] [CrossRef]
  21. Álvarez-Astorga, M.; Capita, R.; Alonso-Calleja, C.; Moreno, B.; García-Fernández, M.C. Microbiological quality of retail chicken by-products in Spain. Meat Sci. 2002, 62, 45–50. [Google Scholar] [CrossRef]
  22. Capita, R.; Alonso-Calleja, C.; García-Arias, M.T.; Moreno, B.; García-Fernández, M.C. Methods to detect the occurrence of various indicator bacteria on the surface of retail poultry in Spain. J. Food Sci. 2002, 67, 765–771. [Google Scholar] [CrossRef]
  23. Paulsen, P.; Smulders, F.J.M.; Hilbert, F. Salmonella in meat from hunted game: A central European perspective. Food Res. Int. 2008, 45, 609–616. [Google Scholar] [CrossRef]
  24. El-Ghareeb, W.R.; Morshdy, A.M.A.; Winkelmayer, R.; Paulsen, P. Microbiological condition and shelf life of meat from hunted game birds. Eur. J. Wildl. Res. 2009, 55, 317–323. [Google Scholar] [CrossRef]
  25. El-Dengawy, R.A.; Nassar, A.M. Investigation on the nutritive value and microbiological quality of wild quail carcasses. Nahrung 2001, 45, 50–54. [Google Scholar] [CrossRef]
  26. Mousa, M.M.; Salem, N.I.E.; Khalifa, A.M.A.; Abd-El-Hady, H.A.M.; El Gamel, A.M. Quality assessment of frozen quail in Kafr El Sheikl Governorate. Alex. J. Vet. Sci. 2016, 48, 57–61. [Google Scholar]
  27. Gill, C.O. Microbiological conditions of meats from large game animals and birds. Meat Sci. 2007, 77, 149–160. [Google Scholar] [CrossRef] [PubMed]
  28. Costa, H.; Mafra, I.; Oliveira, M.B.P.P.; Amaral, J.S. Game: Types and composition. In The Encyclopedia of Food and Health; Caballero, B., Finglas, P., Toldrá, F., Eds.; Academic Press: Oxford, UK, 2016; Volume 3, pp. 177–183. [Google Scholar]
  29. Patria, C.A.; Afnan, R.; Arief, I.I. Physical and microbiological qualities of kampong-broiler crossbred chickens meat raised in different stocking densities. Media Peternakan 2016, 39, 141–147. [Google Scholar] [CrossRef]
  30. Logue, C.M.; Sherwood, J.S.; Olah, P.A.; Elijah, L.M.; Dockter, M.R. The incidence of antimicrobial-resistant Salmonella spp. on freshly processed poultry from US Midwestern processing plants. J. Appl. Microbiol. 2003, 94, 16–24. [Google Scholar] [CrossRef] [PubMed]
  31. Cosgrove, S.E. The relationship between antimicrobial resistance and patient outcomes: Mortality, length of hospital stay, and health care costs. Clin. Infect. Dis. 2006, 42 (Suppl. 2), S82–S89. [Google Scholar] [CrossRef]
  32. Tumbarello, M.; Spanu, T.; Di Bidino, R.; Marchetti, M.; Ruggeri, M.; Trecarichi, E.M.; De Pascale, G.; Proli, E.M.; Cauda, R.; Cicchetti, A.; et al. Costs of bloodstream infections caused by Escherichia coli and influence of extended-spectrum-beta-lactamase production and inadequate initial antibiotic therapy. Antimicrobiol. Agents Chemother. 2010, 54, 4085–4091. [Google Scholar] [CrossRef]
  33. Guerra, B.; Junker, E.; Schroeter, A.; Malorny, B.; Lehmann, S.; Helmuth, R. Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J. Antimicrob. Chemother. 2003, 52, 489–492. [Google Scholar] [CrossRef]
  34. Koo, H.; Woo, G. Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products. Int. J. Food Microbiol. 2011, 145, 407–413. [Google Scholar] [CrossRef]
  35. Álvarez-Fernández, E.; Cancelo, A.; Díaz-Vega, C.; Capita, R.; Alonso-Calleja, C. Antimicrobial resistance in E. coli isolates from conventionally and organically reared poultry: A comparison of agar disc diffusion and Sensi Test Gram-negative methods. Food Control 2013, 30, 227–234. [Google Scholar] [CrossRef]
  36. Capita, R.; Álvarez-Fernández, E.; Fernández-Buelta, E.; Manteca, J.; Alonso-Calleja, C. Decontamination treatments can increase the prevalence of resistance to antibiotics of Escherichia coli naturally present on poultry. Food Microbiol. 2013, 34, 112–117. [Google Scholar] [CrossRef]
  37. Ojer-Usoz, E.; González, D.; Vitas, A.I.; Leiva, J.; García-Jalón, I.; Febles-Casquero, A.; Escolano, M. Prevalence of extended-spectrum-beta-lactamase producing Enterobacteriaceae in meat products sold in Navarra, Spain. Meat Sci. 2013, 93, 316–321. [Google Scholar] [CrossRef]
  38. Hussain, A.; Shaik, S.; Ranjan, A.; Nandanwar, N.; Tiwari, S.K.; Majid, M.; Baddam, R.; Qureshi, I.A.; Semmler, T.; Wieler, L.H.; et al. Risk of transmission of antimicrobial resistant Escherichia coli from commercial broiler and free-range retail chicken in India. Front. Microbiol. 2017, 8, 2120. [Google Scholar] [CrossRef] [PubMed]
  39. Davis, G.S.; Waits, K.; Nordstrom, L.; Grande, H.; Weaver, B.; Papp, K.; Horwinski, J.; Koch, B.; Hungate, B.A.; Liu, C.M.; et al. Antibiotic-resistant Escherichia coli from retail poultry meat with different antibiotic use claims. BMC Microbiol. 2018, 18, 174. [Google Scholar] [CrossRef] [PubMed]
  40. Ishiguro, N.; Oka, C.; Sato, G. Isolation of citrate-positive variants of Escherichia coli from domestic pigeons, pigs, cattle, and horses. Appl. Environ. Microbiol. 1978, 36, 217–222. [Google Scholar] [PubMed]
  41. Kimpe, A.; Decostere, A.; Martel, A.; Haesebrouck, F.; Devriese, L.A. Prevalence of antimicrobial resistance among pigeon isolates of Streptococcus gallolyticus, Escherichia coli and Salmonella enterica serotype Typhimurium. Avian Pathol. 2002, 31, 393–397. [Google Scholar] [CrossRef]
  42. Costa, D.; Poeta, P.; Saenz, Y.; Vinue, L.; Rojo-Bezares, B.; Jouini, A.; Zarazaga, M.; Rodrigues, M.J.; Torres, C. Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal. J. Antimicrob. Chemother. 2006, 58, 1311–1312. [Google Scholar] [CrossRef]
  43. Radhouani, H.; Poeta, P.; Igrejas, G.; Goncalves, A.; Vinue, L.; Torres, C. Antimicrobial resistance and phylogenetic groups in isolates of Escherichia coli from seagulls at the Berlengas nature reserve. Vet. Rec. 2009, 165, 138–142. [Google Scholar] [CrossRef]
  44. Silva, V.L.; Nicoli, J.R.; Nascimento, T.C.; Diniz, C.G. Diarrheagenic Escherichia coli strains recovered from urban pigeons (Columba livia) in Brazil and their antimicrobial susceptibility patterns. Curr. Microbiol. 2009, 59, 302–308. [Google Scholar] [CrossRef]
  45. Radimersky, T.; Frolkova, P.; Janoszowska, D.; Dolejska, M.; Svec, P.; Roubalova, E.; Cikova, P.; Cizek, A.; Literak, I. Antibiotic resistance in faecal bacteria (Escherichia coli, Enterococcus spp.) in feral pigeons. J. Appl. Microbiol. 2010, 109, 1687–1695. [Google Scholar] [CrossRef]
  46. Radhouani, H.; Igrejas, G.; Gonçalves, A.; Pacheco, R.; Monteiro, R.; Sargo, R.; Brito, F.; Torres, C.; Poeta, P. Antimicrobial resistance and virulence genes in Escherichia coli and enterococci from red foxes (Vulpes vulpes). Anaerobe 2013, 23, 82–86. [Google Scholar] [CrossRef]
  47. Hasan, B.; Islam, K.; Ahsan, M.; Hossain, Z.; Rashid, M.; Talukder, B.; Ahmed, K.U.; Olsen, B.; Abul Kashem, M. Fecal carriage of multi-drug resistant and extended spectrum beta-lactamases producing E. coli in household pigeons, Bangladesh. Vet. Microbiol. 2014, 168, 221–224. [Google Scholar] [CrossRef]
  48. Stenzel, T.; Bancerz-Kisiel, A.; Tykałowski, B.; Śmiałek, M.; Pestka, D.; Koncicki, A. Antimicrobial resistance in bacteria isolated from pigeons in Poland. Pol. J. Vet. Sci. 2014, 17, 169–171. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Chindamba, L.; Korsten, L. Antibiotic resistance in Escherichia coli isolates from roof-harvested rainwater tanks and urban pigeon faeces as the likely source of contamination. Environ. Monit. Assess. 2015, 187, 405. [Google Scholar] [CrossRef] [PubMed]
  50. Blanco-Peña, K.; Esperón, F.; Torres-Mejía, A.M.; de la Torre, A.; de la Cruz, E.; Jimenez-Soto, M. Antimicrobial resistance genes in pigeons from public Parks in Costa Rica. Zoonoses Public Health 2017, 65, e23–e30. [Google Scholar] [CrossRef] [PubMed]
  51. Zigo, F.; Takac, L.; Zigova, M.; Takacova, J.; Vasi, M. Occurrence of antibiotic-resistant bacterial strains isolated in carrier pigeons during the race season. J. Chem. Pharm. Sci. 2017, 10, 10–13. [Google Scholar]
  52. Hata, A.; Shibahara, T.; Yamamoto, H.; Fujitani, N. Antimicrobial resistance of Enterobacteriaceae in feral pigeons living in the Kanto region of Japan. Int. J. Anal. Bio-Sci. 2018, 6, 10–18. [Google Scholar]
  53. Ngozi, A.; Agabus, N.; Eucharia, O.; Onyinyechi, U.-I.; Abraham, E.; Chika, E.; Ifeanyichukwu, I. A three-year study on the prevalence and antibiotic susceptibility pattern of Escherichia coli isolated from cloacal swabs of wild and domestic birds in Ebonyi State, Nigeria. EC Microbiol. 2018, 14, 266–273. [Google Scholar]
  54. World Health Organization. Critically Important Antimicrobials for Human Medicine, 6th ed.; World Health Organization: Geneva, Switzerland, 2019; Available online: https://apps.who.int/iris/bitstream/handle/10665/312266/9789241515528-eng.pdf?ua=1 (accessed on 14 October 2019).
  55. World Organization for Animal Health. OIE List of Antimicrobial Agents of Veterinary Importance; World Organization for Animal Health: Paris, France, 2019; Available online: https://www.oie.int/fileadmin/Home/eng/Our_scientific_expertise/docs/pdf/AMR/A_OIE_List_antimicrobials_July2019.pdf (accessed on 14 October 2019).
  56. Navarro-González, N.; Casas-Díaz, E.; Porrero, C.M.; Mateos, A.; Domínguez, L.; Lavín, S.; Serrano, E. Food-borne zoonotic pathogens and antimicrobial resistance of indicator bacteria in urban wild boars in Barcelona, Spain. Vet. Microbiol. 2013, 167, 686–689. [Google Scholar] [CrossRef]
  57. Radhouani, H.; Silva, N.; Poeta, P.; Torres, C.; Correia, S.; Igrejas, G. Potential impact of antimicrobial resistance in wildlife, environment and human health. Front. Microbiol. 2014, 5, 23. [Google Scholar] [CrossRef] [Green Version]
  58. Gomes, B.C.; Esteves, C.T.; Palazzo, I.C.V.; Darini, A.L.C.; Felis, G.E.; Sechi, L.A.; Franco, B.D.G.M.; De Martinis, E.C.P. Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods. Food Microbiol. 2008, 25, 668–675. [Google Scholar] [CrossRef]
  59. Pesavento, G.; Calonico, C.; Ducci, B.; Magnanini, A.; Lo Nostro, A. Prevalence and antibiotic resistance of Enterococcus spp. isolated from retail cheese, ready-to-eat salads, ham, and raw meat. Food Microbiol. 2014, 41, 1–7. [Google Scholar] [CrossRef]
  60. Valenzuela, A.; Omar, N.B.; Abriouel, H.; López, R.L.; Ortega, E.; Cañamero, M.M.; Gálvez, A. Risk factors. Risk factors in enterococci isolated from foods in Morocco; determination of antimicrobial resistance and incidence of virulent traits. Food Chem. Toxicol. 2008, 46, 2648–2652. [Google Scholar] [CrossRef] [PubMed]
  61. Poeta, P.; Costa, D.; Igrejas, G.; Rodrigues, J.; Torres, C. Phenotypic and genotypic characterization of antimicrobial resistance in faecal enterococci from wild boars (Sus scrofa). Vet. Microbiol. 2007, 125, 368–374. [Google Scholar] [CrossRef] [PubMed]
  62. Silva, N.; Igrejas, G.; Figueiredo, N.; Gonçalves, A.; Radhouani, H.; Rodrigues, J. Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus). Sci. Total Environ. 2010, 408, 4871–4876. [Google Scholar] [CrossRef] [PubMed]
  63. Guerrero-Ramos, E.; Molina-González, D.; Blanco-Morán, S.; Igrejas, G.; Poeta, P.; Alonso-Calleja, C.; Capita, R. Prevalence, antimicrobial resistance, and genotypic characterization of vancomycin-resistant enterococci in meat preparations. J. Food Prot. 2016, 79, 748–756. [Google Scholar] [CrossRef] [PubMed]
  64. De Briyne, N.; Atkinson, J.; Pokludová, L.; Borriello, S.P. Antibiotics used most commonly to treat animals in Europe. Vet. Rec. 2014, 175, 325. [Google Scholar] [CrossRef]
  65. Cameron, A.; McAllister, A.A. Antimicrobial usage and resistance in beef production. J. Anim. Sci. Biotechnol. 2016, 7, 68. [Google Scholar] [CrossRef]
  66. Roth, N.; Käsbohrer, A.; Mayrhofer, S.; Zitz, U.; Hofacre, C.; Doming, K.J. The application of antibiotics in broiler production and the resulting antibiotic resistance in Escherichia coli: A global overview. Poult. Sci. 2019, 98, 1791–1804. [Google Scholar] [CrossRef]
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Figure 1. Percentage of Escherichia coli strains susceptible, intermediate or resistant to each antibiotic tested. Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); ceftazidime (CAZ); cefotaxime (CTX); cefoxitin (FOX); imipenem (IPM); aztreonam (ATM); nalidixic acid (NA); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); amikacin (AK); streptomycin (STR); tobramycin (TOB); tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
Figure 1. Percentage of Escherichia coli strains susceptible, intermediate or resistant to each antibiotic tested. Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); ceftazidime (CAZ); cefotaxime (CTX); cefoxitin (FOX); imipenem (IPM); aztreonam (ATM); nalidixic acid (NA); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); amikacin (AK); streptomycin (STR); tobramycin (TOB); tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
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Foods 08 00536 g002 550
Figure 2. Percentage of Escherichia coli strains from domestic and wild pigeons with resistance or reduced susceptibility to each antibiotic tested. Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); ceftazidime (CAZ); cefotaxime (CTX); cefoxitin (FOX); imipenem (IPM); aztreonam (ATM); nalidixic acid (NA); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); amikacin (AK); streptomycin (STR); tobramycin (TOB); tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
Figure 2. Percentage of Escherichia coli strains from domestic and wild pigeons with resistance or reduced susceptibility to each antibiotic tested. Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); ceftazidime (CAZ); cefotaxime (CTX); cefoxitin (FOX); imipenem (IPM); aztreonam (ATM); nalidixic acid (NA); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); amikacin (AK); streptomycin (STR); tobramycin (TOB); tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
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Foods 08 00536 g003 550
Figure 3. Percentage of Enterococcus faecalis strains susceptible, intermediate or resistant to each antibiotic tested. Ampicillin (AMP); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); kanamycin (K); streptomycin (STR); erythromycin (E); fosfomycin (FOS); nitrofurantoin (F); rifampicin (RD); teicoplanin (TEC); vancomycin (VA), tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
Figure 3. Percentage of Enterococcus faecalis strains susceptible, intermediate or resistant to each antibiotic tested. Ampicillin (AMP); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); kanamycin (K); streptomycin (STR); erythromycin (E); fosfomycin (FOS); nitrofurantoin (F); rifampicin (RD); teicoplanin (TEC); vancomycin (VA), tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
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Foods 08 00536 g004 550
Figure 4. Percentage of Enterococcus faecalis strains from domestic and wild pigeons with resistance or reduced susceptibility to each antibiotic tested. Ampicillin (AMP); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); kanamycin (K); streptomycin (STR); erythromycin (E); fosfomycin (FOS); nitrofurantoin (F); rifampicin (RD); teicoplanin (TEC); vancomycin (VA), tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
Figure 4. Percentage of Enterococcus faecalis strains from domestic and wild pigeons with resistance or reduced susceptibility to each antibiotic tested. Ampicillin (AMP); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); kanamycin (K); streptomycin (STR); erythromycin (E); fosfomycin (FOS); nitrofurantoin (F); rifampicin (RD); teicoplanin (TEC); vancomycin (VA), tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
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Table
Table 1. Culture media, incubation times, temperatures and references used for microbiological analysis.
Table 1. Culture media, incubation times, temperatures and references used for microbiological analysis.
Microbial GroupCulture MediumIncubationReference
T (°C)Time
Aerobic plate count (APC) 1Plate count agar (PCA)3072 h[15]
Psychrotrophs 1Plate count agar (PCA)710 d[16]
Enterobacteriaceae2,3Violet red bile glucose agar (VRBGA)3524 h[17]
Fecal coliforms 2,3Violet red bile agar (VRBA)4424 h[17]
Enterococci 2Kanamycin aesculin azide (KEA) agar4224 h[17]
Micrococcaceae1Mannitol salt agar (MSA)3524–48 h[18]
Lactic acid bacteria 2De Man, Rogosa and Sharpe (MRS) agar3072 h[17]
Brochothrix thermosphacta1Streptomycin thallous acetate actidione (STAA) agar2548 h[18]
1 Spread plate technique (0.1 mL); 2 pour plate technique (1 mL); 3 overlay procedure. All media were purchased from Oxoid Ltd. (Hampshire, UK).
Table
Table 2. Microbial counts (log10 cfu/cm2) on meat from domestic and wild pigeons in Spain.
Table 2. Microbial counts (log10 cfu/cm2) on meat from domestic and wild pigeons in Spain.
Type of Pigeon
Microbial GroupTotalDomesticWild
Aerobic plate count (APC)3.16 ± 0.95 c3.76 ± 0.92 bb2.90 ± 0.84 acd
Psychrotrophs3.68 ± 1.40 d3.84 ± 2.04 ab3.61 ± 1.04 ae
Enterobacteriaceae1.56 ± 1.25 a2.06 ± 1.19 ba1.35 ± 1.23 aab
Fecal coliforms1.40 ± 1.17 a2.11 ± 1.02 ba1.10 ± 1.11 aa
Enterococci1.82 ± 1.12 a2.17 ± 0.85 aa1.67 ± 1.20 ab
Micrococcaceae2.73 ± 1.16 b3.12 ± 1.05 bb2.56 ± 1.17 ac
Lactic acid bacteria (LAB)3.40 ± 1.04 cd3.81 ± 0.93 bb3.23 ± 1.05 ade
Brochothrix thermosphacta1.53 ± 2.08 a1.47 ± 2.17 aa1.56 ± 2.05 aab
Data (average ± STD) in the same row (superscripts) with no letters in common are significantly different (p < 0.05). Data in the same column (subscripts) with no letters in common are significantly different (p < 0.05).
Table
Table 3. Number and percentage of Escherichia coli strains susceptible, intermediate or resistant to each antibiotic tested.
Table 3. Number and percentage of Escherichia coli strains susceptible, intermediate or resistant to each antibiotic tested.
AntibioticStrains from Domestic Pigeons
(n = 32)		Strains from Wild Pigeons (n = 28)All Strains (n = 60)
Number of Isolates% of Resistant Strains% of Resistant and Intermediate StrainsNumber of Isolates% of Resistant Strains% of Resistant and Intermediate StrainsNumber of Isolates% of Resistant Strains% of Resistant and Intermediate Strains
SIRSIRSIR
AMC28400.00%12.50%20627.14%28.57%481023.3320.00%
AMP25700.00%21.88%195414.29%32.14%441246.6726.67%
CAZ31100.00%3.13%26200.00%7.14%57300.005.00%
CTX27413.13%15.63%25300.00%10.71%52711.6713.33%
FOX32000.00%0.00%241310.71%14.29%56135.006.67%
IPM32000.00%0.00%28000.00%0.00%60000.000.00%
ATM28313.13%12.50%231414.29%17.86%51458.3315.00%
NA32000.00%0.00%25213.57%10.71%57211.675.00%
CIP32000.00%0.00%27100.00%3.57%59100.001.67%
C30200.00%6.25%27100.00%3.57%57300.005.00%
CN30200.00%6.25%230517.86%17.86%53258.3311.67%
AK31100.00%3.13%222414.29%21.43%53346.6711.67%
STR151526.25%53.13%121513.57%57.14%273035.0055.00%
TOB27413.13%15.63%214310.71%25.00%48846.6720.00%
TE31013.13%3.13%25213.57%10.71%56223.336.67%
SXT32000.00%0.00%28000.00%0.00%60000.000.00%
Amoxicillin-clavulanic acid (AMC); ampicillin (AMP); ceftazidime (CAZ); cefotaxime (CTX); cefoxitin (FOX); imipenem (IPM); aztreonam (ATM); nalidixic acid (NA); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); amikacin (AK); streptomycin (STR); tobramycin (TOB); tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).
Table
Table 4. Number and percentage of Enterococcus faecalis strains susceptible, intermediate or resistant to each antibiotic tested.
Table 4. Number and percentage of Enterococcus faecalis strains susceptible, intermediate or resistant to each antibiotic tested.
AntibioticStrains from Domestic Pigeons (n = 16)Strains from Wild Pigeons (n = 29)All Strains (n = 45)
Number of Isolates% of Resistant Strains% of Resistant and Intermediate StrainsNumber of Isolates% of Resistant Strains% of Resistant and Intermediate StrainsNumber of Isolates% of Resistant Strains% of Resistant and Intermediate Strains
SIRSIRSIR
AMP410212,50%75.00%138827.59%55.17%17181022.22%62.22%
CIP041275.00%100%3131344.83%89.66%3172555.56%93.33%
C7900.00%56.25%32513.45%89.66%103412.22%77.78%
CN36743.75%81.25%332379.31%89.66%693066.67%86.67%
K021487.50%100%741862.07%75.86%763271.11%84.44%
STR011593.75%100%312586.21%89.66%324088.89%93.33%
E09743.75%100%119931.03%96.55%1281635.56%97.78%
FOS76318.75%56.25%1411413.79%51.72%2117715.56%53.33%
F35850.00%81.25%452068.97%86.21%7102862.22%84.44%
RD73637.50%56.25%362068.97%89.66%1092657.78%77.78%
TEC7900.00%56.25%22613.45%24.14%291512.22%35.56%
VA6916.25%62.50%519517.24%82.76%1128613.33%75.56%
TE051168.75%100%2101758.62%93.10%2152862.22%95.56%
SXT11500.00%31.25%20813.45%31.03%311312.22%31.11%
Ampicillin (AMP); ciprofloxacin (CIP); chloramphenicol (C); gentamicin (CN); kanamycin (K); streptomycin (STR); erythromycin (E); fosfomycin (FOS); nitrofurantoin (F); rifampicin (RD); teicoplanin (TEC); vancomycin (VA), tetracycline (TE); trimethoprim-sulfamethoxazole (SXT).

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Cordero, J.; Alonso-Calleja, C.; García-Fernández, C.; Capita, R. Microbial Load and Antibiotic Resistance Patterns of Escherichia coli and Enterococcus faecalis Isolates from the Meat of Wild and Domestic Pigeons. Foods 2019, 8, 536. https://0-doi-org.brum.beds.ac.uk/10.3390/foods8110536

AMA Style

Cordero J, Alonso-Calleja C, García-Fernández C, Capita R. Microbial Load and Antibiotic Resistance Patterns of Escherichia coli and Enterococcus faecalis Isolates from the Meat of Wild and Domestic Pigeons. Foods. 2019; 8(11):536. https://0-doi-org.brum.beds.ac.uk/10.3390/foods8110536

Chicago/Turabian Style

Cordero, Jorge, Carlos Alonso-Calleja, Camino García-Fernández, and Rosa Capita. 2019. "Microbial Load and Antibiotic Resistance Patterns of Escherichia coli and Enterococcus faecalis Isolates from the Meat of Wild and Domestic Pigeons" Foods 8, no. 11: 536. https://0-doi-org.brum.beds.ac.uk/10.3390/foods8110536

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