First Characterization of Escherichia coli Strains Isolated from Wildlife Griffon Vulture (Gyps fulvus) in the Southeast of Spain


The aim of the present study was to characterize a collection of Escherichia coli strains isolated from asymptomatic griffon vulture (Gyps fulvus) during a reintroduction program in the southeast of Spain, in order to establish if griffon vulture could play a role in the spread of resistant or potentially pathogenic E. coli strains. For this purpose, 14 E. coli strains obtained from 10 griffon vulture were studied to establish their serotypes, phylogroups, virulencegene profiles and antimicrobial resistances. High heterogeneity was observed within the 14 strains isolated which belonged to three phylogroups (A, B1 and D), 8 serogroups (O2, O21, O29, O60, 073, O78, O103 and O141) and 13 different serotypes. Out of 34 genes screened, we have detected eight virulence genes that are typical of extraintestinal pathogenic E. coli (ExPEC) (fimH, fimAvMT78, iroN, iucD, cvaC, iss, traT and tsh); however, none of the studied strains showed the ExPEC status. The 14 strains were also analyzed for the production of extended-spectrum beta-lactamases (ESBLs) and for antimicrobial resistances. None of the 14 strains were ESBL-producing E. coli, but high resistance-prevalences to ampicillin and cotrimoxazole were detected. To our knowledge, this is the first characterization of E. coli strains isolated from griffon vulture and although they did not show high virulencegene scores, they showed cotrimoxazole resistance.

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Mora, A. , Ortega, N. , Garcia, E. , Viso, S. , Candela, M. , Dahbi, G. , Cuello, F. and Caro, M. (2014) First Characterization of Escherichia coli Strains Isolated from Wildlife Griffon Vulture (Gyps fulvus) in the Southeast of Spain. Open Journal of Veterinary Medicine, 4, 329-333. doi: 10.4236/ojvm.2014.412040.

1. Introduction

Griffon vulture (Gyps fulvus) is a large-size bird of prey that greatly relies on food found at muladares and other refuse dumps. Few studies have been carried out on the role of vultures in the spread of disease [1] [2] , and none in relation to Escherichia coli, a ubiquitous microorganism of the Enterobacteriaceae family. E. coli comprises different intestinal and extraintestinal pathogenic (ExPEC) groups for animals and humans [3] . Avian pathogenic E. coli (APEC) is included in the group of extraintestinal pathogenic E. coli (ExPEC) and is considered an outstanding pathogen for the poultry industry [4] . A wide number of serogroups have been identified within APEC, many of which have also been implicated in human extraintestinal infections [4] . Moreover, certain important clonal groups have been reported as responsible for human and animal extraintestinal E. coli infections during the last years [5] .

The emergence of multiresistant bacteria of human and veterinary origin is nowadays a great health concern. Wildlife, normally not exposed to clinically useful antimicrobial agents, can acquire resistant bacteria mainly through water polluted from feces of human and farm activity. Current data reveal that carriage of multiresistant strains is widespread in, at least, some wild populations like waterfowl, birds of prey, and rodents [6] -[8] . The aim of the present study was to characterize the serotypes, phylogroups, virulence-gene profiles and antimicrobial resistances of a group of E. coli strains isolated from asymptomatic griffon vulture, and to establish if griffon vulture could play a role in the spread of resistant or potentially pathogenic E. coli strains.

2. Materials and Methods

The samples were collected in 2011 from a muladar (a place where farm carrion are discarded by scavenger’s species) in Alicante (south-east of Spain). Seventeen healthy griffon vultures (15 immature, 1 sub-adult and 1 adult) were tested for E. coli via cloacae swabs with stuart transport medium. Samples were cultured within 24 h of collection. For isolation, swabs were plated directly onto lactose MacConkey agar (L-MC) (Oxoide Ltd.) and incubated at 37˚C for 18 to 24 h. From each L-MC plate, two colonies with typical E. coli morphology were selected, identified by the API 20E system (bioMèrieux) and further studied.

Determination of O and H antigens was carried out using the method previously described by Guinée et al. [9] , with all available O (O1-O181) and H (H1-H56) antisera. Isolates that did not react with O and H antiserum were classified as non-type able (ONT and HNT, respectively).

The phylogenetic group (A, B1, B2 and D) was established by the multiplex PCR-based method of Clermont et al. [10] .

The presence of 34 virulence genes was analyzed as documented previously [3] [5] , using primers specific for genes and operons that encode extraintestinal virulence factors characteristic of ExPEC (fimH; fimAvMT78; papEF; papG I; papG II; papG III; sfa/focDE; sfaS; focG; afa/draBC; cnf1; cdtB; sat; hlyA; iucD; iron; kpsM II, establishing neuC-K1, -K2 and -K5 variants; kpsM III; cvaC; iss; traT; ibeA; malX; usp; tsh), of verotoxigenic E. coli (VTEC) (stx1; stx2; eae) and enterotoxigenic E. coli (ETEC) (eltA; estA; estB).

Susceptibility to antibiotics was analyzed by disc diffusion. Resistances against ampicillin, amoxicillin/ clavulanate acid, cefazoline, gentamicin, trimethoprim/sulfamethoxazole, nalidixic acid and ciprofloxacine were interpreted based on the recommended breakpoints of the CLSI [11] . Suggestive evidence of ESBL production was defined as synergy between amoxicillin/clavulanate and at least one of cefotaxime, ceftazidime, aztreonam or cefepime. Furthermore, PCR was performed using the TEM, SHV and CTX-M specific primers reported previously [5] .

3. Results and Discussion

Fourteen different E. coli strains were recovered from 10 out of the 17 griffon vultures sampled (4 animals showed E. coli strains belonging to 2 different serotypes) (Table 1).

Few data are available on E. coli serotypes within normal avian microbiota. Serotyping showed that the 14 strains belonged to 8 serogroups, 6 of which (O2, O21, O29, O78, O103 and O141) had already been reported within healthy poultry, and serogroup O60 had also been reported within APEC strains [4] (Table 1). However, we have only detected previously four (O2:H18, O21:H19, ONT:H11 and ONT:H19) of the 13 serotypes established in griffon vulture, among 430 serotypes of 1640 APEC strains characterized in a European study and isolated from chickens, turkeys and ducks (data not published).

The 14 E. coli strains belonged to phylogroups A (7 strains), B1 (5 strains) and D (2 strains). None isolate belonged to phylogroup B2 (Table 1). This is in consistent with the fact that isolates are obtained via cloacae from healthy birds. Several studies suggest that virulent clonal groups are derived primarily from phylogroup B2, and to a lesser extent from phylogroup D [5] [12] .

Eight virulence genes typical of ExPEC (fimH, fimAvMT78, iroN, iucD, cvaC iss, traT and tsh) were detected of 34 genes screened (Table 1). The virulence-gene score (number of virulence genes harbored) ranged from 1 to 7, however none of the 14 strains showed the ExPEC status according to the definition of Johnson et al. [13] . A strain satisfied the criteria for being ExPEC if it carried two or more of the following genes: pap, sfa/focDE, afa/draBC, iucD and kpsM II.

Eleven of the 14 E. coli strains showed resistance to any of the 7 antimicrobials tested, being 9 strains ampicillin-resistant and 8 cotrimoxazole-resistant. Furthermore, 2 strains showed intermediate resistance to nalidixic acid and ciprofloxacin (Table 2). Although none of the 14 strains tested were ESBL-producing E. coli, differently to that reported by some authors in relation with birds of prey different from griffon vulture [6] [7] , high resistance-prevalences to ampicillin and cotrimoxazole were detected. In fact, 6 strains showed co-resistance to both antibiotics. These prevalences detected in griffon vultures are similar to those found among healthy poultry strains [14] .

Table 1. Serotypes, phylogroups and virulence-gene profile of the 14 E. coli strains included in the present study.

Table 2. Antibiotic susceptibility of the 14 E. coli strains included in the present study. AM = ampicillin; AMC = amoxicillin-clavulanic acid; CZ = cephazolin; GM = gentamicin; SXT = trimethoprim-sulfamethoxazole (cotrimoxazole); NA = nalidixic acid; CIP = ciprofloxacin, R = resistant; S = sensitive; I = intermediate resistance.


This work was financially supported by Xunta de Galicia and the European Regional Development Fund [ERDF] (2007/000044-0).


*Corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Loria, G.R., Ferrantelli, E., Giardina, G., Li Vecchi, L., Sparacino, L., Oliveri, F., McAuliffe, L. and Nicholas, R.A. (2008) Isolation and Characterization of Unusual Mycoplasma spp. from Captive Eurasian Griffon (Gyps fulvus) in Sicily. Journal of Wildlife Diseases, 44, 159-163.
[2] Darwich, L., Cabezón, O., Echeverria, I., Pabón, M., Marco, I., Molina-López, R., Alarcia-Alejos, O., López-Gatius, F., Lavín, S. and Almería, S. (2012) Presence of Toxoplasma gondii and Neospora caninum DNA in the Brain of Wild Birds. Veterinary Parasitology, 183, 377-381.
[3] Mora, A., Herrrera, A., López, C., Dahbi, G., Mamani, R., Pita, J.M., Alonso, M.P., Llovo, J., Bernárdez, M.I., Blanco, J.E., Blanco, M. and Blanco, J. (2011) Characteristics of the Shiga-Toxin-Producing Enteroaggregative Escherichia coli O104:H4 German Outbreak Strain and of STEC Strains Isolated in Spain. International Microbiology, 14, 121- 141.
[4] Blanco, J.E., Blanco, M., Mora, A., Jansen, W.H., García, V., Vázquez, M.L. and Blanco, J. (1998) Serotypes of Escherichia coli Isolated from Septicaemic Chickens in Galicia (Northwest Spain). Veterinary Microbiology, 61, 229- 235.
[5] Mora, A., Viso, S., López, C., Alonso, M.P., García-Garrote, F., Dabhi, G., Mamani, R., Herrera, A., Marzoa, J., Blanco, M., Blanco, J.E., Moulin-Schouleur, M., Schouler, C. and Blanco, J. (2013) Poultry as Reservoir for Extrain-testinal Pathogenic Escherichia coli O45:K1:H7-B2-ST95 in Humans. Veterinary Microbiology, 167, 506-512.
[6] Guenther, S., Grobbel, M., Lübke-Becker, A., Goedecke, A., Friedrich, N.D., Wieler, L.H. and Ewers, C. (2010) Antimicrobial Resistance Profiles of Escherichia coli from Common European Wild Bird Species. Veterinary Microbiology, 144, 219-225.
[7] Guenther, S., Ewers, C. and Wieler, L.H. (2011) Extended-Spectrum Beta-Lactamases Producing E. coli in Wildlife, yet Another Form of Environmental Pollution? Frontiers in Microbiology, 2, 246.
[8] Guenther, S., Aschenbrenner, K., Stamm, I., Bethe, A., Semmler, T., Stubbe, A., Stubbe, M., Batsajkhan, N., Glupczynski, Y., Wieler, L.H. and Ewers, C. (2012) Comparable High Rates of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli in Birds of Prey from Germany and Mongolia. PLoS ONE, 7, e53039.
[9] Guinèe, P.A.M., Jansen, H.W., Wadström, T. and Sellwood, R. (1981) Escherichia coli Associated with Neonatal Diarrhoea in Piglets and Calves. In: de Leeww, P.W. and Guinèe, P.A.M., Eds., Laboratory Diagnosis in Neo-Natal Calf and Pig Diarrhoea: Current Topics in Veterinary and Animal Science, Vol. 13, Martinus Nijhoff Pub, The Netherlands, 126-162.
[10] Clermont, O., Bonacorsi, S. and Bingen, E. (2000) Rapid and Simple Determination of the Escherichia coli Phylogenetic Group. Applied and Environmental Microbiology, 66, 4555-4558.
[11] Clinical Laboratory Standards Institute (CLSI) (2011) Performance Standards for Antimicrobial Susceptibility Testing: Twenty-First Informational Supplement. CLSI Document M100-S21. National Committee for Clinical Laboratory Standards, CLSI, Wayne.
[12] Mora, A., López, C., Dabhi, G., Blanco, M., Blanco, J.E., Alonso, M.P., Herrera, A., Mamani, R., Bonacorsi, S., Moulin-Schouleur, M. and Blanco, J. (2009) Extraintestinal Pathogenic Escherichia coli O1:K1:H7/NM from Human and Avian Origin: Detection of Clonal Groups B2 ST95 and D ST59 with Different Host Distribution. BMC Microbiology, 9, 132.
[13] Johnson, J.R., Murray, A.C., Gajewski, A., Sullivan, M., Snippes, P., Kuskowski, M.A. and Smith, K.E. (2003) Isolation and Molecular Characterization of Nalidixic Acid-Resistant Extraintestinal Pathogenic Escherichia coli from Retail Chicken Products. Antimicrobial Agents and Chemotherapy, 47, 2161-2168.
[14] Blanco, J.E., Blanco, M., Mora, A. and Blanco, J. (1997) Prevalence of Bacterial Resistance to Quinolones and Other Antimicrobials among Avian Escherichia coli Strains Isolated from Septicemic and Healthy Chickens in Spain. Journal Clinical Microbiology, 35, 2184-2185.

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