The spread of antimicrobial-resistant bacteria in wildlife must be viewed as a major concern with serious implications for human and animal health. Escherichia coli and enterococcal isolates were recovered from faecal samples of 49 wild rabbits (Oryctolagus cuniculus) on specific media and were characterised using biochemical and molecular tests. For all isolates, antimicrobial susceptibility testing was performed, and resistance genes were detected by PCR. Molecular typing of isolates was carried out by pulsed-field gel-electrophoresis, and E. coli strains were also tested for the presence of intimin (eae) gene characteristic of rabbit enteropathogenic E. coli. A total of 34 E. coli and 36 enterococci [E. hirae (52.8%) and E. faecalis (47.2%)] were obtained. For E. coli, resistance to tetracycline (94%), streptomycin (62%), ciprofloxacin (47%), trimethoprim-sulphamethoxazole (35%) and chloramphenicol (6%) was observed. Resistance to third-generation cephalosporins was detected in one E. coli strain that carried the blaCMY-2 and blaTEM-1 genes. Class 1 integrons were detected in eight isolates. For enterococci, resistance to tetracycline (63.9%), erythromycin (30.5%), streptomycin (18.2%), and chloramphenicol (5.5%) was detected. The tet(M)+tet(L), erm(B) and ant (6)-Ia genes were identified in thirteen, seven and three resistant Enterococcus strains, respectively. Molecular typing showed a high diversity among our strains. Wild rabbits could represent a reservoir of E. coli, and enterococci carrying antimicrobial resistance genes and E. coli additionally carrying the eae gene of enteropathogenic pathotypes could both contaminate the environment. our finding seems to represent the first report of eae-positive E. coli in wild rabbits.
Allen, H. K., Donato, J., Wang, H. H., Cloud-Hansen, K. A., Davies, J. and Handelsman, J. (2010): Call of the wild: antibiotic resistance genes in natural environments. Nat. Rev. Microbiol. 8, 251–259.
Alonso, C. A., Mora, A., Díaz, D., Blanco, M., González-Barrio, D., Ruiz-Fons, F., Simón, C., Blanco, J. and Torres, C. (2017): Occurrence and characterization of stx and/or eae-positive Escherichia coli isolated from wildlife, including a typical EPEC strain from a wild boar. Vet. Microbiol. 207, 69–73.
Ben Said, L., Jouini, A., Alonso, C. A., Klibi, N., Dziri, R., Boudabous, A., Ben Slama, K. and Torres, C. (2016): Characteristics of extended-spectrum β-lactamase (ESBL) and pAmpC beta-lactamase-producing Enterobacteriaceae of water samples in Tunisia. Sci. Total Environ. 550, 1103–1109.
Ben Said, L., Klibi, N., Dziri, R., Borgo, F., Boudabous, A., Ben Slama, K. and Torres, C. (2015): Prevalence, antimicrobial resistance and genetic lineages of Enterococcus spp. from vegetable food, soil and irrigation water in farm environments in Tunisia. J. Sci. Food Agric. 96, 1627–1633.
Ben Sallem, R., Ben Slama, K., Sáenz, Y., Rojo-Bezares, B., Estepa, V., Jouini, A., Gharsa, H., Klibi, N., Boudabous, A. and Torres, C. (2012): Prevalence and characterization of extended-spectrum beta-lactamase (ESBL)- and CMY-2-producing Escherichia coli isolates from healthy food-producing animals in Tunisia. Foodborne Pathog. Dis. 9, 1137–1142.
Ben Slama, K., Jouini, A., Ben Sallem, R., Somalo, S., Sáenz, Y., Estepa, V., Boudabous, A. and Torres, C. (2010): Prevalence of broad-spectrum cephalosporin-resistant Escherichia coli isolates in food samples in Tunisia and characterization of integrons and antimicrobial resistance mechanisms implicated. Int. J. Food. Microbiol. 137, 281–286.
Butaye, P., Devriese, L. A. and Haesebrouck, F. (2001): Differences in antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium strains isolated from farm and pet animals. Antimicrob. Agents Chemother. 45, 1374–1378.
CLSI (2017): Performance Standards for Antimicrobial Susceptibility Testing. Twenty-fifth Informational Supplement. CLSI Document, M100–S25.
Cole, D., Drum, D. J. V., Stallknecht, D. E., White, D. G., Lee, M. D., Ayers, S., Sobsey, M. and Maurer, J. J. (2005): Free-living Canada geese and antimicrobial resistance. Emerg. Infect. Dis. 11, 935–938.
Costa, D., Poeta, P., Sáenz, Y., Vinué, L., Coelho, A., Matos, M., Rojo-Bezares, B., Rodrigues, J. and Torres, C. (2008): Mechanisms of antibiotic resistance in Escherichia coli isolates recovered from wild animals. Microb. Drug Resist. 14, 71–77.
Da Costa, P. M., Loureiro, L. and Matos, A. J. F. (2013): Transfer of multidrug resistant bacteria between intermingled ecological niches: the interface between humans, animals and the environment. Int. J. Environ. Res. Public Health 10, 278–294.
Dotto, G., Giacomelli, M., Grilli, G., Ferrazzi, V., Carattoli, A., Fortini, D. and Piccirillo, A. (2014): High prevalence of oqxAB in Escherichia coli isolates from domestic and wild lagomorphs in Italy. Microb. Drug Resist. 20, 118–123.
Elhani, D., Klibi, N., Dziri, R., Ben Hassan, M., Asli, M. S., Ben Said, L., Mahjoub, A., Ben Slama, K., Jemli, B., Bellaj, R., Barguellil, F. and Torres, C. (2014): vanA-containing E. faecium isolates of clonal complex CC17 in clinical and environmental samples in a Tunisian hospital. Diagn. Microbiol. Infect. Dis. 79, 60–63.
Figueiredo, N., Radhouani, H., Gonçalves, A., Rodrigues, J., Carvalho, C., Igrejas, G. and Poeta, P. (2009): Genetic characterization of vancomycin-resistant enterococci isolates from wild rabbits. J. Basic. Microbiol. 49, 491–494.
Franz, C. M. A. P., Holzapfel, W. H. and Stiles, M. E. (1999): Enterococci at the crossroads of food safety? Int. J. Food. Microbiol. 47, 1–24.
Guardabassi, L., Schwarz, S. and Lloyd, D. H. (2004): Pet animals as reservoirs of antimicrobial-resistant bacteria. J. Antimicrob. Chemother. 54, 321–332.
Guerrero-Ramos, E., Cordero, J., Molina-González, D., Poeta, P., Igrejas, G., Alonso-Calleja, C. and Capita, R. (2016): Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain. Food. Microbiol. 53, 156–164.
Huys, G., D’Haene, G. K., Collard, J. M. and Swings, J. (2004): Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl. Environ. Microbiol. 70, 1555–1562.
Jackson, C. R., Fedorka-Cray, P. J., Davis, J. A., Barrett, J. B., Brousse, J. H., Gustafson, J. and Kucher, M. (2010): Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States. J. Appl. Microbiol. 108, 2171–2179.
Jouini, A., Ben Slama, K., Saenz, Y., Klibi, N., Costa, D., Vinué, L., Zarazaga, M., Boudabous, A. and Torres, C. (2009): Detection of multiple-antimicrobial resistance and characterization of the implicated genes in Escherichia coli isolates from food of animal origin in Tunis. J. Food Prot. 72, 1082–1088.
Klibi, N., Aouini, R., Borgo, F. C., Ben Said, L., Ferrario, C., Dziri, R., Boudabous, A., Torres, C. and Ben Slama, K. (2015a): Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia. Ann. Microbiol. 65, 1–8.
Klibi, N., Ben Amor, I., Rahmouni, M., Dziri, R., Gtari, D., Ben Said, L., Lozano, C., Boudabous, A., Ben Slama, K., Mansouri, R. and Torres, C. (2015b): Diversity of species and antibiotic resistance among fecal enterococci from wild birds in Tunisia. Detection of vanA-containing Enterococcus faecium isolates. Eur. J. Wildl. Res. 61, 319–323.
Klibi, N., Ben Lagha, A., Ben Slama, K., Boudabous, A. and Torres, C. (2013a): Faecal enterococci from camels in Tunisia: species, antibiotic resistance and virulent genes. Vet. Rec. 172, 213.
Klibi, N., Ben Said, L., Jouini, A., Ben Slama, K., López, M., Ben Sallem, R., Boudabous, A. and Torres, C. (2013b): Species distribution, antibiotic resistance and virulence traits in enterococci from meat in Tunisia. Meat Sci. 93, 675–680.
Klibi, N., Ben Slama, K., Masmoudi, A., Gharbi, S., Ruiz-Larrea, F., Fendri, C., Boudabous, A. and Torres, C. (2006): Diversity of structures carrying the aac(6’)-aph(2”) gene in clinical E. faecalis and E. faecium strains isolated in Tunisia. J. Chemother. 18, 353–359.
Linaje, R., Coloma, M. D., Perez-Martinez, G. and Zuniga, M. (2004): Characterization of faecal enterococci from rabbits for the selection of probiotic strains. J. Appl. Microbiol. 96, 761–771.
López, M., Sáenz, Y., Rojo-Bezares, B., Martínez, S., del Campo, R., Ruiz-Larrea, F., Zarazaga, M. and Torres, C. (2009): Detection of vanA and vanB2-containing enterococci from food samples in Spain, including Enterococcus faecium strains of CC17 and the new singleton ST425. Int. J. Food Microbiol. 133, 172–178.
Marinho, C., Igrejas, G., Gonçalves, A., Silva, N., Santos, T., Monteiro, R., Gonçalves, D., Rodrigues, T. and Poeta, P. (2014): Azorean wild rabbits as reservoirs of antimicrobial resistant Escherichia coli. Anaerobe 30, 116–119.
Poeta, P., Costa, D., Rodrigues, J. and Torres, C. (2006): Antimicrobial resistance and the mechanisms implicated in faecal enterococci from healthy humans, poultry and pets in Portugal. Int. J. Antimicrob. Agents 27, 131–137.
Poeta, P., Costa, D., Saenz, Y., Klibi, N., Ruiz-Larrea, F., Rodrigues, J. and Torres, C. (2005): Characterization of antibiotic resistance genes and virulence factors in faecal enterococci of wild animals in Portugal. J. Vet. Med. 52, 396–402.
Ritchie, J. M., Thorpe, C. M., Rogers, A. B. and Waldor, M. K. (2003): Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits. Infect. Immun. 71, 7129–7139.
Sáenz, Y., Brinăs, L., Domínguez, E., Ruiz, J., Zarazaga, M., Jordi, V. and Torres, C. (2004): Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal and food origins. Antimicrob. Agents Chemother. 48, 3996–1001.
Sáenz, Y., Zarazaga, M., Briňas, L., Lantero, M., Ruiz-Larrea, F. and Torres, C. (2001): Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int. J. Antimicrob. Agents 18, 353–358.
Santos, T., Silva, N., Igrejas, G., Rodrigues, P., Micael, J., Rodrigues, T., Resende, R., Gonçalves, A., Marinho, C., Gonçalves, D., Cunha, R. and Poeta, P. (2013): Dissemination of antibiotic resistant Enterococcus spp. and Escherichia coli from wild birds of Azores Archipelago. Anaerobe 24, 25–31.
Sayah, R. S., Kaneene, J. B., Johnson, Y. and Miller, R. (2005): Patterns of antimicrobial resistance observed in Escherichia coli isolates obtained from domestic- and wild-animal fecal samples, human septage, and surface water. Appl. Environ. Microbiol. 71, 1394–1404.
Silva, N., Igrejas, G., Figueiredo, N., Gonçalves, A., Radhouani, H., Rodrigues, J. and Poeta, P. (2010): Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus). Sci. Total Environ. 408, 4871–4876.
Teshager, T., Herrero, I. A., Porrero, M. C., Garde, J., Moreno, M. A. and Domĕnguez, L. (2000): Surveillance of antimicrobial resistance in Escherichia coli strains isolated from pigs at Spanish slaughterhouses. Int. J. Antimicrob. Agents 15, 137–142.
Torres, C., Tenorio, C., Portillo, A., Garcia, M., Martínez, C., del Campo, R., Ruiz-Larrea, F. and Zarazaga, M. (2003): Intestinal colonization by vanA or vanB2-containing enterococcal isolates of healthy animals in Spain. Microb. Drug Resist. 9, 47–52.
Turabelidze, D., Kotetishvili, M., Kreger, A., Morris, J. G. and Sulakvelidze, A. (2000): Improved pulsed-field gel electrophoresis for typing vancomycin-resistant enterococci. J. Clin. Microbiol. 38, 4242–4245.
Van den Bogaard, A. E. and Stobberingh, E. E. (2000): Epidemiology of resistance to antibiotics. Links between animals and humans. Int. J. Antimicrob. Agents 14, 327–335.