World Journal of Microbiology and Biotechnology

, Volume 27, Issue 8, pp 1775–1785 | Cite as

In vitro transference and molecular characterization of bla TEM genes in bacteria isolated from Portuguese ready-to-eat foods

  • P. Amador
  • R. Fernandes
  • I. Duarte
  • L. Brito
  • C. Prudêncio
Original Paper


The principal aim of this study was to investigate the possibility of transference to Escherichia coli of β-lactam resistance genes found in bacteria isolated from ready-to-eat (RTE) Portuguese traditional food. From previous screenings, 128 β-lactam resistant isolates (from different types of cheese and of delicatessen meats), largely from the Enterobacteriaceae family were selected and 31.3% of them proved to transfer resistance determinants in transconjugation assays. Multiplex PCR in donor and transconjugant isolates did not detect bla CTX, bla SHV and bla OXY, but bla TEM was present in 85% of them, while two new TEMs (TEM-179 and TEM-180) were identified in two isolates. The sequencing of these amplicons showed identity between donor and transconjugant genes indicating in vitro plasmid DNA transfer. These results suggest that if there is an exchange of genes in natural conditions, the consumption of RTE foods, particularly with high levels of Enterobacteriaceae, can contribute to the spread of antibiotic resistance.


Ready-to-eat (RTE) food β-lactam-resistant Enterobacteriaceae blaTEM-1 gene Transconjugants 



The authors thank Dr. Maria Tärnberg (Clinical Microbiology, Linköping University, Sweden) for providing the strains E. coli HY0401091, E. coli UB0402407, K. pneumoniae HY0301692, K. pneumoniae AA0404346, K. pneumoniae MR050068, E. coli UB0402407 and K.pneumonia HY0301692 used as positive controls in multiplex PCR. The authors also thank Dr. Maximiliano Alvarez for strain E. coli J53 azide-resistant (AzR) used in this study as recipient cells.


  1. Amador P, Fernandes R, Prudêncio C, Brito L (2009) Resistance to β-lactams in bacteria isolated from different types of Portuguese Cheese. Int J Mol Sci 10:1538–1551CrossRefGoogle Scholar
  2. Amador P, Fernandes R, Brito L, Prudêncio C (2010) Antibiotic resistance in Enterobacteriaceae isolated from portuguese delicatessen meats. J Food Saf. doi: 10.1111/j.1745-4565.2010.00258.x
  3. Ambler RP, Coulson AF, Frère JM, Ghuysen JM, Joris B, Forsman M, Levesque RC, Tiraby G, Waley SG (1991) A standard numbering scheme for the class A β-lactamases. Biochem J 276:269–272Google Scholar
  4. Angulo FJ, Nargund VN, Chiller TC (2004) Evidence of an association between use of antimicrobial agents in food animals and antimicrobial resistance among bacteria isolated from humans and the human health consequences of such resistance. J Vet Med B 51:374–379CrossRefGoogle Scholar
  5. Briñas L, Zarazaga M, Sáenz Y, Ruiz-Larrea F, Torres C (2002) β-lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob Agents Chemother 46:3156–3163CrossRefGoogle Scholar
  6. Caniça MM, Deloménie C, Labia R, Krishnamoorthy R, Paul G (1996) Characterization of the mutant produced by site-directed mutagenesis generating the substitution Asn-2763Asp in the critical proximity of Arg-244 in TEM-1 β-lactamase. Abstract presented at: the 16th Interdisciplinary Meeting on Anti-Infectious Chemotherapy; Abstract no. 24/C3, p. 100; ParisGoogle Scholar
  7. Catry B, Laevens H, Devriese LA, Opsomer G, de Kruif A (2003) Antimicrobial resistance in livestock. J Vet Pharmacol Ther 26:81–93CrossRefGoogle Scholar
  8. Cavaco LM, Abatih E, Aarestrup FM, Guardabassi L (2008) Selection and persistence of CTX-M-producing Escherichia coli in the intestinal flora of pigs treated with amoxicillin, ceftiofur, or cefquinome. Antimicrob Agents Chemother 52:3612–3616CrossRefGoogle Scholar
  9. Chang F-Y, Siu LK, Fung C-P, Huang M-H, Ho M (2001) Diversity of SHV and TEM β-lactamases in Klebsiella pneumoniae: gene evolution in Northern Taiwan and two novel β-lactamases, SHV-25 and SHV-26. Antimicrob Agents Chemother 45:2407–2413CrossRefGoogle Scholar
  10. CLSI (Clinical Laboratory Standards Institute) (2007) Performance standards for antimicrobial susceptibility testing; 17th informational supplement. CLSI document M100–S17. Clinical and Laboratory Standards Institute, Wayne, PAGoogle Scholar
  11. Coque TM, Baquero F, Canton R (2008) Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill 13:1–11Google Scholar
  12. Courvalin P (1994) Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Antimicrob Agents Chemother 38:1447–1451Google Scholar
  13. Decré D, Burghoffer B, Gautier V, Petit J-C, Arlet G (2004) Outbreak of multi-resistant Klebsiella oxytoca involving strains with extended-spectrum β-lactamases and strains with extended-spectrum activity of the chromosomal β-lactamase. J Antimicrob Chemother 54:881–888CrossRefGoogle Scholar
  14. Fevre C, Jbel M, Passet V, Weill FX, Grimont PAD, Brisse S (2005) Six groups of OXY β-lactamase evolved over millions of years in Klebsiella oxytoca. Antimicrob Agents Chemother 49:3453–3462CrossRefGoogle Scholar
  15. Fournier B, Lu CY, Lagrange PH, Krishnamoorthy R, Philippon A (1995) Point mutation in the pribnow box, the molecular basis of beta-lactamase overproduction in Klebsiella oxytoca. Antimicrob Agents Chemother 39:1365–1368Google Scholar
  16. Gniadkowski M (2008) Evolution of extended-spectrum β-lactamases by mutation. Clin Microbiol Infect 14:11–32CrossRefGoogle Scholar
  17. Gupta V (2007) An update on newer β-lactamases. Indian J Med Res 126:417–427Google Scholar
  18. Hammad AM, Ishida Y, Shimamoto T (2009) Prevalence and molecular characterization of ampicillin-resistant Enterobacteriaceae isolated from traditional Egyptian Domiati cheese. J Food Prot 72:624–630Google Scholar
  19. Ho P-L, Wong RC-W, Chow K-H, Yip K, Wong SS-Y, Que T-L (2008) CTX-M type β-lactamases among fecal Escherichia coli and Klebsiella pneumoniae isolates in non-hospitalized children and adults. J Microbiol Immunol Infect 41:428–432Google Scholar
  20. IFT (Institute of Food Ttechnologists) (2006) Antimicrobial resistance: implications for the food system. Compr Rev Food Sci Food Saf 5:71–137CrossRefGoogle Scholar
  21. Jacoby GA, Munoz-Price LS (2005) The new β-lactamases. N Engl J Med 352:380–391CrossRefGoogle Scholar
  22. Johnson JR, Kuskowski MA, Smith K, O’Bryan TT, Tatini S (2005) Antimicrobial-resistant and extraintestinal pathogenic Escherichia coli in retail foods. J Infect Dis 191:1040–1049CrossRefGoogle Scholar
  23. LACED (2010) The lactamase engineering database. Institute of Technical Biochemistry at the University of Stuttgart. Accessed 18 July 2010
  24. Lahey Clinic (2010) β-lactamase classification and amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant β-lactamases. Accessed 18 July 2010
  25. Livermore DM (1995) β-lactamases in laboratory and clinical resistance. Clin Microbiol Rev 8:557–584Google Scholar
  26. Macovei L, Zurek L (2007) Influx of enterococci and associated antibiotic resistance and virulence genes from ready-to-eat food to the human digestive tract. Appl Environ Microbiol 73:6740–6747CrossRefGoogle Scholar
  27. Monstein HJ, Östholm-Balkhed Å, Nilsson NV, Nilsson M, Dornbusch K, Nilsson LE (2007) Multiplex PCR amplification assay for the detection of bla SHV, bla TEM and bla CTX-M genes in Enterobacteriaceae. APMIS 115:1400–1408CrossRefGoogle Scholar
  28. Monstein HJ, Tärnberg M, Nilsson LE (2009) Molecular identification of CTX-M and bla OXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons. BMC Infect Dis. doi: 10.1186/1471-2334-9-7
  29. Moubareck C, Bourgeois N, Courvalin P, Doucet-Populaire F (2003) Multiple antibiotic resistance gene transfer from animal to human enterococci in the digestive tract of gnotobiotic mice. Antimicrob Agents Chemother 47:2993–2996CrossRefGoogle Scholar
  30. Novais A, Comas I, Baquero F, Cantón R, Coque TM, Moya A, González-Candelas F, Galán JC (2010) Evolutionary trajectories of β-lactamase CTX-M-1 cluster enzymes: predicting antibiotic resistance. PloS Pathog 6:e1000735. doi: 10.1371/journal.ppat.1000735
  31. Olesen I, Hasman H, Aarestrup FM (2004) Prevalence of β-lactamase among ampicillin-resistant Escherichia coli and Salmonella isolated from food animal in Denmark. Microb Drug Resist 10:334–340CrossRefGoogle Scholar
  32. Phillips I, Casewell M, Cox T, De Groot B, Friis C, Jones R, Nightingale C, Preston R, Waddell J (2004) Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemother 53:28–52CrossRefGoogle Scholar
  33. Schmitt J, Jacobs E, Schmidt H (2007) Molecular characterization of extended-spectrum β-lactamases in Enterobacteriaceae from patients of two hospitals in Saxony, Germany. J Med Microbiol 56:241–249CrossRefGoogle Scholar
  34. Schwartz S, Chaslus-Dancla E (2001) Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet. Res 32:201–225Google Scholar
  35. Shoemaker NB, Vlamakis H, Hayes K, Salyers AA (2001) Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl Environ Microbiol 67:561–568CrossRefGoogle Scholar
  36. Silbergeld EK, Graham J, Price LB (2008) Industrial food animal production, antimicrobial resistance, and human health. Annu Rev Public Health 29:151–169CrossRefGoogle Scholar
  37. Simeoni D, Rizzotti L, Cocconcelli P, Gazzola S, Dellaglio F, Torriani S (2008) Antibiotic resistance genes and identification of staphylococci collected from the production chain of swine meat commodities. Food Microbiol 25:196–201CrossRefGoogle Scholar
  38. Smet A, Martel A, Persoons D, Dewulf J, Heyndrickx M, Catry B, Herman L, Haesebrouck F, Butaye P (2008) Diversity of extended-spectrum β-lactamases and class c β-lactamases among cloacal Escherichia coli isolates in Belgian broiler farms. Antimicrob Agents Chemother 52:1238–1243CrossRefGoogle Scholar
  39. Sow AG, Wane AA, Diallo MH, Boye CSB, Aïdara-Kane A (2007) Genotypic characterization of antibiotic-resistant Salmonella Enteritidis isolates in Dakar, Senegal. J Infect Dev Ctries. 1:284–288Google Scholar
  40. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X, windows interface, flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefGoogle Scholar
  41. Van TTH, Moutafis G, Tran LT, Coloe PJ (2007) Antibiotic resistance in food-borne bacterial contaminants in Vietnam. Appl Environ Microbiol 73:7906–7911CrossRefGoogle Scholar
  42. Vo AT, van Duijkeren E, Fluit AC, Gaastra W (2007) Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses. Vet Microbiol 124:248–255CrossRefGoogle Scholar
  43. Weill FX, Lailler R, Praud K, Kérouanton A, Fabre L, Brisabois A, Grimont PAD, Cloeckaert A (2004) Emergence of extended-spectrum-β-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France. J Clin Microbiol 42:5767–5773CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • P. Amador
    • 1
    • 4
  • R. Fernandes
    • 2
    • 3
  • I. Duarte
    • 4
  • L. Brito
    • 1
  • C. Prudêncio
    • 2
    • 3
  1. 1.Laboratório de Microbiologia, CBAA/DRAT, Instituto Superior de AgronomiaTechnical University of LisbonLisbonPortugal
  2. 2.Ciências Químicas e das Biomoléculas, Escola Superior de Tecnologia de Saúde do PortoInstituto Politécnico do PortoVila Nova de GaiaPortugal
  3. 3.Centro de Farmacologia e Biopatologia QuímicaFaculdade de Medicina da Universidade do PortoAlameda Hernâni MonteiroPortoPortugal
  4. 4.Ciências Exactas e do Ambiente, Sector de Biologia e Ecologia, Escola Superior AgráriaInstituto Politécnico de CoimbraCoimbraPortugal

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