European Journal of Wildlife Research

, Volume 58, Issue 4, pp 677–684 | Cite as

Occurrence of virulence genes in multidrug-resistant Escherichia coli isolates from Iberian wolves (Canis lupus signatus) in Portugal

  • Romeu Simões
  • Carla Ferreira
  • José Gonçalves
  • Francisco Álvares
  • Helena Rio-Maior
  • Sara Roque
  • Ricardo Brandão
  • Paulo Martins da CostaEmail author
Original Paper


While much evidence supports the view that the total consumption of antimicrobials is the critical factor in selecting resistance, the possibility of resistant isolates and/or genes encoding resistance being transferred among different living communities has raised serious concerns. In the present study, Escherichia coli isolates recovered from faecal samples (n = 34) of Iberian wolves (Canis lupus signatus) were characterized for their antimicrobial drug susceptibility. Nearly two thirds of the isolates carried resistance to one or more antimicrobial drugs (in a panel of 19 antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. By screening a set of 20 multidrug-resistant E. coli for virulence genes, we found strains positive for cdt, chuA, cvaC, eaeA, paa and bfpA, which was the most common virulence trait. Phylogenetic analyses have shown that the majority of these E. coli strains fall into phylogenetic groups A and B1. In this study, the diversity of extended-spectrum β-lactamase-producing strains was expressed by both polymorphism of the pulsed-field gel electrophoresis patterns and the presence of various resistance and virulence genes profiles. Finding the specific implications of these multi-resistant bacteria (hosting several virulence factors) in wolf conservation is a challenging topic to be addressed in further investigations.


Canis lupus signatus E. coli Antimicrobial resistance Phylogenetic groups Virulence factors 



Faecal samples from wolves were collected and genetically analysed during research projects funded by VentoMinho-Energias Renováveis, S.A. and ACHLI—Associação de conservação do habitat do lobo-ibérico. We thank M. Nakamura, D. Cadete, S. Pinto, A. Pedro and N. Santos for helping in sample collection and R. Godinho and D. Castro for the genetic identification of faecal samples. Wolves were captured under legal permits issued by ICNB/Ministry of Environment.


  1. Aarestrup FM, Wegener HC, Collignon P (2008) Resistance in bacteria of the food chain: epidemiology and control strategies. Expert Rev Anti-Infect Ther 6(5):733–750CrossRefPubMedGoogle Scholar
  2. Álvares F (2004) Status and conservation of Iberian wolf in Portugal. Wolf Print 20:4–6Google Scholar
  3. Aminov RI (2010) A brief history of the antibiotic era: lessons learned and challenges for the future. Front Microbiol 1:134PubMedCentralCrossRefPubMedGoogle Scholar
  4. Baldy-Chudzik K, Mackiewicz P, Stosik M (2008) Phylogenetic background, virulence gene profiles, and genomic diversity in commensal Escherichia coli isolated from ten mammal species living in one zoo. Vet Microbiol 131(1–2):173–184CrossRefPubMedGoogle Scholar
  5. Bessa-Gomes C, Petrucci-Fonseca F (2003) Using artificial neural networks to assess wolf distribution patterns in Portugal. Anim Conserv 6:221–229CrossRefGoogle Scholar
  6. Bonnedahl J, Drobni M, Gauthier-Clerc M, Hernandez J, Granholm S, Kayser Y, Kahlmeter G, Waldenström J, Johansson A, Olsen B (2009) Dissemination of Escherichia coli with CTX-M type ESBL between humans and yellow legged gulls in the south of France. PLoS One 4(6):e5958PubMedCentralCrossRefPubMedGoogle Scholar
  7. Branger C, Zamfir O, Geoffroy S, Laurans G, Arlet G, Vu Thien H, Gouriou S, Picard B, Denamur E (2005) Genetic background of Escherichia coli and extended-spectrum β-lactamase type. Emerg Infect Dis 11(1):54–61PubMedCentralCrossRefPubMedGoogle Scholar
  8. Chapman TA, Wu X-Y, Barchia I, Bettelheim KA, Driesen S, Trott D, Wilson M, Chin JJ-C (2006) Comparison of virulence gene profiles of Escherichia coli strains isolated from healthy and diarrheic swine. Appl Environ Microbiol 72(7):4782–4795PubMedCentralCrossRefPubMedGoogle Scholar
  9. Clermont O, Bonacorsi S, Bingen E (2000) Rapid and simple determination of Escherichia coli phylogenetic group. Appl Environ Microbiol 66(10):4555–4558PubMedCentralCrossRefPubMedGoogle Scholar
  10. CLSI (2007) Performance standards for antimicrobial susceptibility testing. Seventeenth informational supplement M100-S17. Clinical and Laboratory Standards Institute, WayneGoogle Scholar
  11. Costa D, Poeta P, Saenz Y, Vinue L, Rojo-Bezares B, Jouini A, Zarazaga M, Rodrigues J, Torres C (2007) Detection of Escherichia coli harbouring extended-spectrum β-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal. J Antimicrob Chemother 59(6):1311–1312Google Scholar
  12. da Costa PM, Vaz-Pires V, Bernardo F (2008) Antimicrobial resistance in Escherichia coli isolated in wastewater and sludge from poultry slaughterhouses wastewater plants. J Environ Health 70(7):40–45PubMedGoogle Scholar
  13. Dancer SJ (2004) How antibiotics can make us sick: the less obvious adverse effects of antimicrobial chemotherapy. Lancet Infect Dis 4(10):611–619CrossRefPubMedGoogle Scholar
  14. Eggermann J, Ferrão da Costa G, Guerra AM, Kirchner WH, Petrucci-Fonseca F (2010) Presence of Iberian wolf (Canis lupus signatus) in relation to land cover, livestock and human influence in Portugal. Mamm Biol 76(2):217–221Google Scholar
  15. Enne VI, Delsol AA, Davis GR, Hayward SL, Roe JM, Bennett PM (2005) Assessment of the fitness impacts on Escherichia coli of acquisition of antibiotic resistance genes encoded by different types of genetic element. J Antimicrob Chemother 56(3):544–551CrossRefPubMedGoogle Scholar
  16. Girlich D, Poirel L, Carattoli A, Kempf I, Lartigue MF, Bertini A, Nordmann P (2007) Extended-spectrum β-lactamase CTX-M-1 in Escherichia coli isolates from healthy poultry in France. Appl Environ Microbiol 73(14):4681–4685PubMedCentralCrossRefPubMedGoogle Scholar
  17. Grilo C, Roque S, Rio-Maior H, Petrucci-Fonseca F (2004) The isolated wolf population South of Douro River: status and action priorities for its recovery. Wolf Print 20:13–15Google Scholar
  18. Hamelin K, Bruant G, El-Shaarawi A, Hill S, Edge TA, Fairbrother J, Harel J, Maynard C, Masson L, Brouseau R (2007) Occurrence of virulence and antimicrobial resistance genes in Escherichia coli isolates from different aquatic ecosystems within the St. Clair River and Detroit River areas. Appl Environ Microbiol 73(2):477–484PubMedCentralCrossRefPubMedGoogle Scholar
  19. Hammerum AM, Heuer OE (2009) Human health hazards from antimicrobial-resistant Escherichia coli of animal origin. Clin Infect Dis 48(7):916–921CrossRefPubMedGoogle Scholar
  20. Machado E, Coque TM, Canton R, Sousa JC, Peixe L (2008) Antibiotic resistance integrons and extended-spectrum {beta}-lactamases among Enterobacteriaceae isolates recovered from chickens and swine in Portugal. J Antimicrob Chemother 62(2):296–302CrossRefPubMedGoogle Scholar
  21. Martínez JL (2008) Antibiotics and antibiotic resistance genes in natural environments. Science 321(5887):365–367CrossRefPubMedGoogle Scholar
  22. Meriggi A, Lovari S (1996) A review of wolf predation in southern Europe: does the wolf prefer wild prey to livestock? J Appl Ecol 33(6):1561–1571CrossRefGoogle Scholar
  23. Middleton JH, Ambrose A (2005) Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis). J Wildl Dis 41(2):334–341CrossRefPubMedGoogle Scholar
  24. Pimenta V, Barroso I, Álvares F, Correia J, Ferrão da Costa G, Moreira L, Nascimento J, Petrucci-Fonseca F, Roque S, Santos E (2005) Censo Nacional de Lobo 2002/2003. Technical report. Instituto da Conservação da Natureza/Grupo Lobo, Lisbon, p 158Google Scholar
  25. Pitout JD (2010) Infections with extended-spectrum beta-lactamase-producing Enterobacteriaceae: changing epidemiology and drug treatment choices. Drugs 70(3):313–333CrossRefPubMedGoogle Scholar
  26. Poeta P, Radhouani H, Pinto L, Martinho A, Rego V, Rodrigues R, Gonçalves A, Rodrigues J, Estepa V, Torres C, Igrejas G (2009) Wild boars as reservoirs of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli of different phylogenetic groups. J Basic Microbiol 49(6):584–588CrossRefPubMedGoogle Scholar
  27. Rohland N, Hofreiter M (2007) Comparison and optimization of ancient DNA extraction. Biotechniques 42(3):343–352CrossRefPubMedGoogle Scholar
  28. Roque S, Álvares F, Petrucci-Fonseca F (2001) Utilización espacio-temporal y hábitos alimenticios de un grupo reproductor de lobos en el Noroeste de Portugal. Galemys 13:179–198Google Scholar
  29. Russo TA, Johnson JR (2000) Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J Infect Dis 181(5):1753–1754CrossRefPubMedGoogle Scholar
  30. Schierack P, Römer A, Jores J, Kaspar H, Guenther S, Filter M, Eichberg J, Wieler LH (2009) Isolation and characterization of intestinal Escherichia coli clones from wild boars in Germany. Appl Environ Microbiol 75(3):695–702PubMedCentralCrossRefPubMedGoogle Scholar
  31. Simões R, Poirel L, Martins da Costa P, Nordmann P (2010) Seagulls and beaches as a reservoir for emerging extended spectrum beta-lactamase producers in Escherichia coli. Emerg Infect Dis 16(1):110–112PubMedCentralCrossRefPubMedGoogle Scholar
  32. Siqueira AK, Ribeiro MG, DdaS L, Tiba MR, Moura C, Lopes MD, Prestes NC, Salerno T, Silva AV (2009) Virulence factors in Escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs. Res Vet Sci 86(2):206–210CrossRefPubMedGoogle Scholar
  33. Sjolund M, Bonnedahl J, Hernandez J, Bengtsson S, Cederbrant G, Pinhassi J, Kahlmeter G, Olsen B (2008) Dissemination of multidrug-resistant bacteria into the arctic. Emerg Infect Dis 14(1):70–71PubMedCentralCrossRefPubMedGoogle Scholar
  34. van Elsas JD, Semenov AV, Costa R, Trevors JT (2011) Survival of Escherichia coli in the environment: fundamental and public health aspects. ISME J 5(2):173–183PubMedCentralCrossRefPubMedGoogle Scholar
  35. Vilà C, Amorim IR, Leonard JA, Posada D, Castroviejo J, Petrucci-Fonseca F, Crandall KA, Ellegren H, Wayne RK (1999) Mitochondrial DNA phylogeography and population history of the grey wolf Canis lupus. Mol Ecol 8(12):2089–2103CrossRefPubMedGoogle Scholar
  36. Vos J (2000) Food habits and livestock depredation of two Iberian wolf packs (Canis lupus signatus) in the north of Portugal. J Zool 251:457–462CrossRefGoogle Scholar
  37. Woodford N, Fagan EJ, Ellington MJ (2006) Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J Antimicrob Chemother 57(1):154–155CrossRefPubMedGoogle Scholar
  38. Yong D, Park R, Yum JH, Lee K, Choi EC, Chong Y (2002) Further modification of the Hodge test to screen AmpC beta-lactamase (CMY-1)-producing strains of Escherichia coli and Klebsiella pneumoniae. J Microbiol Meth 51(3):407–410CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Romeu Simões
    • 1
  • Carla Ferreira
    • 1
  • José Gonçalves
    • 1
    • 2
  • Francisco Álvares
    • 3
  • Helena Rio-Maior
    • 4
  • Sara Roque
    • 5
  • Ricardo Brandão
    • 6
  • Paulo Martins da Costa
    • 1
    • 2
    • 7
    Email author
  1. 1.ICBAS—Abel Salazar Institute for the Biomedical SciencesUniversity of PortoPortoPortugal
  2. 2.CIIMAR—Interdisciplinary Center for Marine and Environmental ResearchUniversity of PortoPortoPortugal
  3. 3.Instituto de Ciências Agrárias de VairãoCIBIO—Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do PortoVairãoPortugal
  4. 4.VERANDA—Associação para a Conservação e Divulgação do Património de MontanhaCastro LaboreiroPortugal
  5. 5.GRUPO LOBO—Departamento de Biologia AnimalFaculdade de Ciências da Universidade de LisboaLisboaPortugal
  6. 6.CERVAS—Wild Animal EcologyRehabilitation and Surveillance CenterGouveiaPortugal
  7. 7.Departamento de Produção Aquática, Instituto de Ciências Biomédicas de Abel SalazarUniversidade do PortoPortoPortugal

Personalised recommendations