Microbial Ecology

, Volume 79, Issue 1, pp 30–37 | Cite as

First Report of KPC-2 and KPC-3-Producing Enterobacteriaceae in Wild Birds in Africa

  • Houssem Ben Yahia
  • Sarra Chairat
  • Haythem Gharsa
  • Carla Andrea Alonso
  • Rym Ben Sallem
  • Nerea Porres-Osante
  • Nabil Hamdi
  • Carmen TorresEmail author
  • Karim Ben SlamaEmail author
Environmental Microbiology


The increased incidence of antibiotic-resistant Enterobacteriaceae is a public health problem worldwide. The aim of this study was to analyze the potential role of wild birds, given their capacity of migrating over long distances, in the spreading of carbapenemase, extended-spectrum β-lactamase (ESBL), and acquired-AmpC beta-lactamase-producing Enterobacteriaceae in the environment. Fecal and pellet samples were recovered from 150 wild birds in seven Tunisian regions and were inoculated in MacConkey-agar plates for Enterobacteriaceae recovery (one isolate/animal). Ninety-nine isolates were obtained and acquired resistance mechanisms were characterized in the five detected imipenem-resistant and/or cefotaxime-resistant isolates, by PCR and sequencing. The following ESBL, carbapenemase, and acquired-AmpC beta-lactamase genes were detected: blaCTX-M-15 (two Escherichia fergusonii and one Klebsiella oxytoca isolates), blaKPC-2 (one K. oxytoca), blaKPC-3 (one E. fergusonii), blaACT-36, and blaACC-2 (two K. oxytoca, four E. fergusonii, and two E. coli). The IncFIIs, IncF, IncFIB, IncK, IncP, and IncX replicons were detected among these beta-lactamase Enterobacteriaceae producers. The blaKPC-2, tetA, sul3, qnrB, and cmlA determinants were co-transferred by conjugation from K. oxytoca strain to E. coli J153, in association with IncK and IncF replicons. Our results support the implication of wild birds as a biological vector for carbapenemase, ESBL, and acquired-AmpC-producing Enterobacteriaceae.


Carbapenemase Wild birds Tunisia Enterobacteriaceae 



We extend our sincere thanks to Dr. Nabil Hamdi and Mr. Ridha Ouni for their active participation during sample collection.


Our work was partly supported by the Ministère de l’enseignement supérieur et de la recherche scientifique, Tunisie (Ref. LR03ES03). The work performed in Spain was financed by project SAF2016-76571-R of the Agencia Estatal de Investigación (AEI) of Spain and the Fondo Europeo de Desarrollo Regional (FEDER). Carla Andrea Alonso had a pre-doctoral fellowship FPI from MINECO.

Compliance with Ethical Standards

The protocol was approved by the local Research Ethic Committee.

Conflicts of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Guenther S, Ewers C, Wieler LH (2011) Extended-spectrum beta-lactamases producing E. coli in wildlife, yet another form of environmental pollution? Front Microbiol 2:246. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ruppe E, Woerther PL, Barbier F (2015) Mechanisms of antimicrobial resistance in Gram-negative bacilli. Ann Intensive Care 5:61. CrossRefPubMedGoogle Scholar
  3. 3.
    Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL (2012) Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev 25:682–707. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Nordmann P, Poirel L (2014) The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clinical microbiology and infection: the official publication of the European Society of Clinical. Microbiol Infect Dis 20:821–830. CrossRefGoogle Scholar
  5. 5.
    Dolejska M, Masarikova M, Dobiasova H, Jamborova I, Karpiskova R, Havlicek M, Carlile N, Priddel D, Cizek A, Literak I (2016) High prevalence of Salmonella and IMP-4-producing Enterobacteriaceae in the silver gull on Five Islands, Australia. J Antimicrob Chemother 71:63–70. CrossRefPubMedGoogle Scholar
  6. 6.
    Baez J, Hernandez-Garcia M, Guamparito C, Diaz S, Olave A, Guerrero K, Canton R, Baquero F, Gahona J, Valenzuela N, Del Campo R, Silva J (2015) Molecular characterization and genetic diversity of ESBL-producing Escherichia coli colonizing the migratory Franklin’s gulls (Leucophaeus pipixcan) in Antofagasta, North of Chile. Microb Drug Resist 21:111–116. CrossRefPubMedGoogle Scholar
  7. 7.
    Veldman K, van Tulden P, Kant A, Testerink J, Mevius D (2013) Characteristics of cefotaxime-resistant Escherichia coli from wild birds in the Netherlands. Appl Environ Microbiol 79:7556–7561. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cuzon G, Naas T, Lesenne A, Benhamou M, Nordmann P (2010) Plasmid-mediated carbapenem-hydrolysing OXA-48 beta-lactamase in Klebsiella pneumoniae from Tunisia. Int J Antimicrob Agents 36:91–93. CrossRefPubMedGoogle Scholar
  9. 9.
    Saidani M, Hammami S, Kammoun A, Slim A, Boutiba-Ben Boubaker I (2012) Emergence of carbapenem-resistant OXA-48 carbapenemase-producing Enterobacteriaceae in Tunisia. J Med Microbiol 61:1746–1749. CrossRefPubMedGoogle Scholar
  10. 10.
    Ben Sallem R, Ben Slama K, Rojo-Bezares B, Porres-Osante N, Jouini A, Klibi N, Boudabous A, Saenz Y, Torres C (2014) IncI1 plasmids carrying bla(CTX-M-1) or bla(CMY-2) genes in Escherichia coli from healthy humans and animals in Tunisia. Microb Drug Resist 20:495–500. CrossRefPubMedGoogle Scholar
  11. 11.
    Ben Sallem R, Ben Slama K, Saenz Y, Rojo-Bezares B, Estepa V, Jouini A, Gharsa H, Klibi N, Boudabous A, 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. CrossRefPubMedGoogle Scholar
  12. 12.
    Kumsa B, Socolovschi C, Parola P, Rolain JM, Raoult D (2012) Molecular detection of Acinetobacter species in lice and keds of domestic animals in Oromia Regional State, Ethiopia. PLoS One 7:e52377. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Brahmi S, Touati A, Cadiere A, Djahmi N, Pantel A, Sotto A, Lavigne JP, Dunyach-Remy C (2016) First description of two sequence type 2 Acinetobacter baumannii isolates carrying OXA-23 carbapenemase in Pagellus acarne fished from the Mediterranean Sea near Bejaia, Algeria. Antimicrob Agents Chemother 60:2513–2515. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    CLSI. Twenty-Fifth Informational Supplement (2015) Performance Standards for Antimicrobial Susceptibility Testing. Clinical and Laboratory Standards Institute, Wayne, PA M100-S25Google Scholar
  15. 15.
    Birgy A, Bidet P, Genel N, Doit C, Decre D, Arlet G, Bingen E (2012) Phenotypic screening of carbapenemases and associated beta-lactamases in carbapenem-resistant Enterobacteriaceae. J Clin Microbiol 50:1295–1302. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197CrossRefGoogle Scholar
  17. 17.
    Poirel L, Pitout JD, Nordmann P (2007) Carbapenemases: molecular diversity and clinical consequences. Future Microbiol 2:501–512. CrossRefPubMedGoogle Scholar
  18. 18.
    Ben Slama K, Ben Sallem R, Jouini A, Rachid S, Moussa L, Saenz Y, Estepa V, Somalo S, Boudabous A, Torres C (2011) Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital. Curr Microbiol 62:1794–1801. CrossRefPubMedGoogle Scholar
  19. 19.
    Perez-Perez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153–2162CrossRefGoogle Scholar
  20. 20.
    Eckert C, Gautier V, Arlet G (2006) DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 57:14–23. CrossRefPubMedGoogle Scholar
  21. 21.
    Husickova V, Cekanova L, Chroma M, Htoutou-Sedlakova M, Hricova K, Kolar M (2012) Carriage of ESBL- and AmpC-positive Enterobacteriaceae in the gastrointestinal tract of community subjects and hospitalized patients in the Czech Republic. Biomed Pap Med Fac Univ Palacky Olomouc Czechoslovakia 156:348–353. CrossRefGoogle Scholar
  22. 22.
    Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239CrossRefGoogle Scholar
  23. 23.
    Timofte D, Maciuca IE, Evans NJ, Williams H, Wattret A, Fick JC, Williams NJ (2014) Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 beta-lactamases from bovine mastitis isolates in the United Kingdom. Antimicrob Agents Chemother 58:789–794. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ (2005) Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63:219–228. CrossRefPubMedGoogle Scholar
  25. 25.
    Couturier M, Bex F, Bergquist PL, Maas WK (1988) Identification and classification of bacterial plasmids. Microbiol Rev 52:375–395PubMedPubMedCentralGoogle Scholar
  26. 26.
    Porres-Osante N, Azcona-Gutierrez JM, Rojo-Bezares B, Undabeitia E, Torres C, Saenz Y (2014) Emergence of a multiresistant KPC-3 and VIM-1 carbapenemase-producing Escherichia coli strain in Spain. J Antimicrob Chemother 69:1792–1795. CrossRefPubMedGoogle Scholar
  27. 27.
    Ruiz E, Ocampo-Sosa AA, Rezusta A, Revillo MJ, Roman E, Torres C, Martinez-Martinez L (2012) Acquisition of carbapenem resistance in multiresistant Klebsiella pneumoniae strains harbouring blaCTX-M-15, qnrS1 and aac(6′)-Ib-cr genes. J Med Microbiol 61:672–677. CrossRefPubMedGoogle Scholar
  28. 28.
    Bouaziz A, Loucif L, Ayachi A, Guehaz K, Bendjama E, Rolain JM (2017) Migratory white stork (Ciconia ciconia): a potential vector of the OXA-48-producing Escherichia coli ST38 clone in Algeria. Microb Drug Resist 24:461–468. CrossRefPubMedGoogle Scholar
  29. 29.
    Woodford N, Wareham DW, Guerra B, Teale C (2014) Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: an emerging public health risk of our own making? J Antimicrob Chemother 69:287–291. CrossRefPubMedGoogle Scholar
  30. 30.
    Guerra B, Fischer J, Helmuth R (2014) An emerging public health problem: acquired carbapenemase-producing microorganisms are present in food-producing animals, their environment, companion animals and wild birds. Vet Microbiol 171:290–297. CrossRefPubMedGoogle Scholar
  31. 31.
    Abdallah HM, Reuland EA, Wintermans BB, Al Naiemi N, Koek A, Abdelwahab AM, Ammar AM, Mohamed AA, Vandenbroucke-Grauls CM (2015) Extended-Spectrum beta-lactamases and/or carbapenemases-producing Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt. PLoS One 10:e0136052. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Lahlaoui H, Poirel L, Barguellil F, Moussa MB, Nordmann P (2012) Carbapenem-hydrolyzing class D beta-lactamase OXA-48 in Klebsiella pneumoniae isolates from Tunisia. Eur J Clin Microbiol Infect Dis 31:937–939. CrossRefPubMedGoogle Scholar
  33. 33.
    Ben Tanfous F, Alonso CA, Achour W, Ruiz-Ripa L, Torres C, Ben Hassen A (2016) First description of KPC-2-producing Escherichia coli and ST15 OXA-48-positive Klebsiella pneumoniae in Tunisia. Microb Drug Resist 23:365–375. CrossRefPubMedGoogle Scholar
  34. 34.
    Tavares PC, Monteiro LR, Lopes RJ, Correia Santos MM, Furness RW (2004) Intraspecific variation of mercury contamination in chicks of black-winged stilt (Himantopus himantopus) in coastal wetlands from southwestern Europe. Bull Environ Contam Toxicol 72:437–444. CrossRefPubMedGoogle Scholar
  35. 35.
    Hamza E, Dorgham SM, Hamza DA (2016) Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt. J Glob Antimicrob Resist 7:8–10. CrossRefPubMedGoogle Scholar
  36. 36.
    Wang Y, Zhang R, Li J, Wu Z, Yin W, Schwarz S, Tyrrell JM, Zheng Y, Wang S, Shen Z, Liu Z, Liu J, Lei L, Li M, Zhang Q, Wu C, Zhang Q, Wu Y, Walsh TR, Shen J (2017) Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production. Nat Microbiol 2:16260. CrossRefPubMedGoogle Scholar
  37. 37.
    K. Seedikkoya PAAeEAAS (2007) “Cattle egret as a biocontrol agent” zoos’. Print J 22:2864–2866CrossRefGoogle Scholar
  38. 38.
    Mani Y, Mansour W, Mammeri H, Denamur E, Saras E, Boujaafar N, Bouallegue O, Madec JY, Haenni M (2017) KPC-3-producing ST167 Escherichia coli from mussels bought at a retail market in Tunisia. J Antimicrob Chemother 72:2403–2404. CrossRefPubMedGoogle Scholar
  39. 39.
    Caltagirone M, Nucleo E, Spalla M, Zara F, Novazzi F, Marchetti VM, Piazza A, Bitar I, De Cicco M, Paolucci S, Pilla G, Migliavacca R, Pagani L (2017) Occurrence of extended spectrum beta-lactamases, KPC-type, and MCR-1.2-producing Enterobacteriaceae from Wells, river water, and wastewater treatment plants in Oltrepo Pavese area, Northern Italy. Front Microbiol 8:2232. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Atterby C, Ramey AM, Hall GG, Jarhult J, Borjesson S, Bonnedahl J (2016) Increased prevalence of antibiotic-resistant E. coli in gulls sampled in Southcentral Alaska is associated with urban environments. Infect Ecol Epidemiol 6:32334. CrossRefPubMedGoogle Scholar
  41. 41.
    Alcala L, Alonso CA, Simon C, Gonzalez-Esteban C, Oros J, Rezusta A, Ortega C, Torres C (2015) Wild birds, frequent carriers of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli of CTX-M and SHV-12 types. Microb Ecol 72:861–869. CrossRefPubMedGoogle Scholar
  42. 42.
    Raza S, Mohsin M, Madni WA, Sarwar F, Saqib M, Aslam B (2017) First report of bla CTX-M-15-type ESBL-producing Klebsiella pneumoniae in wild migratory birds in Pakistan. EcoHealth. 14:182–186. CrossRefPubMedGoogle Scholar
  43. 43.
    Zong Z, Partridge SR, Iredell JR (2010) ISEcp1-mediated transposition and homologous recombination can explain the context of bla(CTX-M-62) linked to qnrB2. Antimicrob Agents Chemother 54:3039–3042. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Poirel L, Decousser JW, Nordmann P (2003) Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene. Antimicrob Agents Chemother 47:2938–2945CrossRefGoogle Scholar
  45. 45.
    Singh NS, Singhal N, Virdi JS (2018) Genetic environment of blaTEM-1, blaCTX-M-15, blaCMY-42 and characterization of integrons of Escherichia coli isolated from an Indian urban aquatic environment. Front Microbiol 9:382. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Ben Sallem R, Ben Slama K, Estepa V, Jouini A, Gharsa H, Klibi N, Saenz Y, Ruiz-Larrea F, Boudabous A, Torres C (2012) Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia. Eur J Clin Microbiol Infect Dis 31:1511–1516. CrossRefPubMedGoogle Scholar
  47. 47.
    Carattoli A (2009) Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother 53:2227–2238. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Guenther S, Grobbel M, Lubke-Becker A, Goedecke A, Friedrich ND, Wieler LH, Ewers C (2010) Antimicrobial resistance profiles of Escherichia coli from common European wild bird species. Vet Microbiol 144:219–225. CrossRefPubMedGoogle Scholar
  49. 49.
    Giacopello C, Foti M, Mascetti A, Grosso F, Ricciardi D, Fisichella V, Lo Piccolo F (2016) Antimicrobial resistance patterns of Enterobacteriaceae in European wild bird species admitted in a wildlife rescue centre. Vet Ital 52:139–144. CrossRefPubMedGoogle Scholar
  50. 50.
    Pinto A, Simoes R, Oliveira M, Vaz-Pires P, Brandao R, da Costa PM (2015) Multidrug resistance in wild bird populations: importance of the food chain. J Zoo Wildl Med 46:723–731. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de TunisUniversité de Tunis El ManarTunisTunisia
  2. 2.Institut Supérieur des Sciences Biologiques Appliquées de TunisUniversité de Tunis El ManarTunisTunisia
  3. 3.Area de Bioquímica y Biología MolecularUniversidad de La RiojaLogroñoSpain
  4. 4.U/R de Bio-Écologie et Systématique Évolutive; Faculté des Sciences de TunisTunisTunisia

Personalised recommendations