Advertisement

Antibiotic Resistance Genes and Antibiotic Susceptibility of Oral Enterococcus faecalis Isolates Compared to Isolates from Hospitalized Patients and Food

  • Annette Carola Anderson
  • Huria Andisha
  • Elmar Hellwig
  • Daniel Jonas
  • Kirstin Vach
  • Ali Al-Ahmad
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1057)

Abstract

Enterococcus faecalis, a commensal of the intestinal tract of humans and animals is of great significance as leading opportunistic pathogen, and also prevalent in oral diseases, such as endodontic infections, as well as the healthy oral cavity. To investigate the potential of oral E. faecalis to constitute a reservoir of antibiotic resistance, isolates from supragingival plaque/saliva and from endodontic infections were screened regarding their resistance to selected antibiotics in comparison to nosocomial and food isolates.

70 E. faecalis isolates were analyzed with PCR regarding their equipment with the resistance genes tetM, tetO, ermB, ermC, vanA, vanB and blaTEM. Additionally, they were tested for their phenotypic resistance to doxycycline, azithromycin, rifampicin, amoxicillin and streptomycin using the Etest.

High percentages of the plaque/saliva, nosocomial and food isolates were resistant to doxycycline and azithromycin, particularly plaque/saliva isolates (81%) and nosocomial isolates (73.3%) showed resistance to doxycycline, significantly more than among the food and endodontic isolates. Rifampicin resistance was widespread among isolates from plaque/saliva (52.4%), endodontic infections (50%) and nosocomial infections (40%); all isolates were susceptible to amoxicillin and all oral isolates to high-level streptomycin. TetM genes were detected in the majority of all isolates and ermB genes were present in many nosocomial and plaque/saliva isolates. Thirty percent of the endodontic isolates and 53% of the nosocomial isolates were equipped with blaTEM genes.

The results suggest that the oral cavity can harbor E. faecalis strains with multiple resistances against different antibiotics and thus be regarded as a potential source of resistance traits.

Keywords

Antibiotic resistance Endodontic infections Enterococci Food Nosocomial infections Oral cavity Plaque 

Notes

Acknowledgements

The authors thank Bettina Spitzmüller, Kristina Kollmar and Annette Wittmer for excellent technical assistance and Nicole Arweiler, Daniel Jonas and Ingrid Huber for providing part of the isolates, as well as Grant Anderson for English language correction.

Conflicts of Interest

The authors deny any conflicts of interest related to this study.

Ethical Statement

All endodontic and clinical isolates were obtained after approval by the Ethics Committee (no. 140/09, University of Freiburg).

References

  1. Al-Ahmad A, Maier J, Follo M, Spitzmuller B, Wittmer A, Hellwig E et al (2010) Food-borne enterococci integrate into oral biofilm: an in vivo study. J Endod 36(11):1812–1819. doi: 10.1016/j.joen.2010.08.011CrossRefPubMedGoogle Scholar
  2. Al-Ahmad A, Ameen H, Pelz K, Karygianni L, Wittmer A, Anderson AC et al (2014) Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth. J Endod 40(2):223–230. doi: 10.1016/j.joen.2013.07.023CrossRefPubMedGoogle Scholar
  3. Amsler K, Santoro C, Foleno B, Bush K, Flamm R (2010) Comparison of broth microdilution, agar dilution, and Etest for susceptibility testing of doripenem against gram-negative and gram-positive pathogens. J Clin Microbiol 48(9):3353–3357. doi: 10.1128/jcm.00494-10CrossRefPubMedPubMedCentralGoogle Scholar
  4. Anderson AC, Al-Ahmad A, Elamin F, Jonas D, Mirghani Y, Schilhabel M et al (2013) Comparison of the bacterial composition and structure in symptomatic and asymptomatic endodontic infections associated with root-filled teeth using pyrosequencing. PLoS One 8(12):e84960. doi: 10.1371/journal.pone.0084960CrossRefPubMedPubMedCentralGoogle Scholar
  5. Anderson AC, Jonas D, Huber I, Karygianni L, Wolber J, Hellwig E et al (2015) Enterococcus faecalis from food, clinical specimens, and oral sites: prevalence of virulence factors in association with biofilm formation. Front Microbiol 6:1534. doi: 10.3389/fmicb.2015.01534CrossRefPubMedGoogle Scholar
  6. Arias CA, Murray BE (2012) The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol 10(4):266–278. doi: 10.1038/nrmicro2761CrossRefPubMedPubMedCentralGoogle Scholar
  7. Barbosa-Ribeiro M, De-Jesus-Soares A, Zaia AA, Ferraz CC, Almeida JF, Gomes BP (2016) Antimicrobial susceptibility and characterization of virulence genes of Enterococcus faecalis isolates from teeth with failure of the endodontic treatment. J Endod. doi: 10.1016/j.joen.2016.03.015CrossRefPubMedGoogle Scholar
  8. Call DR, Bakko MK, Krug MJ, Roberts MC (2003) Identifying antimicrobial resistance genes with DNA microarrays. Antimicrob Agents Chemother 47(10):3290–3295CrossRefGoogle Scholar
  9. CLSI (2013) CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. CLSI document M100-S23. Clinical and Laboratory Standards Institute, Wayne, PAGoogle Scholar
  10. Dahlen G, Samuelsson W, Molander A, Reit C (2000) Identification and antimicrobial susceptibility of enterococci isolated from the root canal. Oral Microbiol Immunol 15(5):309–312CrossRefGoogle Scholar
  11. Dahlen G, Blomqvist S, Almstahl A, Carlen A (2012) Virulence factors and antibiotic susceptibility in enterococci isolated from oral mucosal and deep infections. J Oral Microbiol:4. doi: 10.3402/jom.v4i0.10855CrossRefGoogle Scholar
  12. Depardieu F, Kolbert M, Pruul H, Bell J, Courvalin P (2004) VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis. Antimicrob Agents Chemother 48(10):3892–3904. doi: 10.1128/aac.48.10.3892-3904.2004CrossRefPubMedPubMedCentralGoogle Scholar
  13. Duggan JM, Sedgley CM (2007) Biofilm formation of oral and endodontic Enterococcus faecalis. J Endod 33(7):815–818. doi: 10.1016/j.joen.2007.02.016CrossRefPubMedGoogle Scholar
  14. Dutka-Malen S, Evers S, Courvalin P (1995) Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33(1):24–27PubMedPubMedCentralGoogle Scholar
  15. EUCAST (2016) The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 6.0, 2016 http://www.eucast.org. http://www.eucast.org/clinical_breakpoints/
  16. Fass RJ (1993) Erythromycin, clarithromycin, and azithromycin: use of frequency distribution curves, scattergrams, and regression analyses to compare in vitro activities and describe cross-resistance. Antimicrob Agents Chemother 37(10):2080–2086CrossRefGoogle Scholar
  17. Fisher K, Phillips C (2009) The ecology, epidemiology and virulence of Enterococcus. Microbiology 155(Pt 6):1749–1757. doi: 10.1099/mic.0.026385-0CrossRefPubMedGoogle Scholar
  18. Franz CM, Huch M, Abriouel H, Holzapfel W, Galvez A (2011) Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151(2):125–140. doi: 10.1016/j.ijfoodmicro.2011.08.014CrossRefPubMedGoogle Scholar
  19. Gaetti-Jardim EC, Marqueti AC, Faverani LP, Gaetti-Jardim E Jr (2010) Antimicrobial resistance of aerobes and facultative anaerobes isolated from the oral cavity. J Appl Oral Sci 18(6):551–559CrossRefGoogle Scholar
  20. Hammerum AM (2012) Enterococci of animal origin and their significance for public health. Clin Microbiol Infect 18(7):619–625. doi: 10.1111/j.1469-0691.2012.03829.xCrossRefPubMedGoogle Scholar
  21. Jacinto RC, Gomes BP, Ferraz CC, Zaia AA, Filho FJ (2003) Microbiological analysis of infected root canals from symptomatic and asymptomatic teeth with periapical periodontitis and the antimicrobial susceptibility of some isolated anaerobic bacteria. Oral Microbiol Immunol 18(5):285–292CrossRefGoogle Scholar
  22. Jamet E, Akary E, Poisson MA, Chamba JF, Bertrand X, Serror P (2012) Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses. Food Microbiol 31(2):191–198. doi: 10.1016/j.fm.2012.03.009CrossRefPubMedGoogle Scholar
  23. Jorgensen JH, Ferraro MJ (2009) Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis 49(11):1749–1755. doi: 10.1086/647952CrossRefPubMedGoogle Scholar
  24. Jungermann GB, Burns K, Nandakumar R, Tolba M, Venezia RA, Fouad AF (2011) Antibiotic resistance in primary and persistent endodontic infections. J Endod 37(10):1337–1344. doi: 10.1016/j.joen.2011.06.028CrossRefPubMedPubMedCentralGoogle Scholar
  25. Komiyama EY, Lepesqueur LS, Yassuda CG, Samaranayake LP, Parahitiyawa NB, Balducci I et al (2016) Enterococcus species in the oral cavity: prevalence, virulence factors and antimicrobial susceptibility. PLoS One 11(9):e0163001. doi: 10.1371/journal.pone.0163001CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kouidhi B, Zmantar T, Mahdouani K, Hentati H, Bakhrouf A (2011) Antibiotic resistance and adhesion properties of oral Enterococci associated to dental caries. BMC Microbiol 11:155. doi: 10.1186/1471-2180-11-155CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kristich CJ, Rice LB, Arias CA (2014) Enterococcal infection-treatment and antibiotic resistance. In: Gilmore MS, Clewell DB, Ike Y, Shankar N (eds) Enterococci: from commensals to leading causes of drug resistant infection. Massachusetts Eye and Ear Infirmary, BostonGoogle Scholar
  28. Kuch A, Willems RJ, Werner G, Coque TM, Hammerum AM, Sundsfjord A et al (2012) Insight into antimicrobial susceptibility and population structure of contemporary human Enterococcus faecalis isolates from Europe. J Antimicrob Chemother 67(3):551–558. doi: 10.1093/jac/dkr544CrossRefPubMedGoogle Scholar
  29. Lietzau S, Hoewner M, von Baum H, Marre R, Brenner H (2006) Antibiotic resistant fecal isolates of Enterococci among unselected patients outside the clinical sector: an epidemiological study from Southern Germany. Pharmacoepidemiol Drug Saf 15(4):275–277. doi: 10.1002/pds.1167CrossRefPubMedGoogle Scholar
  30. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG et al (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18(3):268–281. doi: 10.1111/j.1469-0691.2011.03570.xCrossRefPubMedGoogle Scholar
  31. Manson JM, Hancock LE, Gilmore MS (2010) Mechanism of chromosomal transfer of Enterococcus faecalis pathogenicity island, capsule, antimicrobial resistance, and other traits. Proc Natl Acad Sci U S A 107(27):12269–12274. doi: 10.1073/pnas.1000139107CrossRefPubMedPubMedCentralGoogle Scholar
  32. McBride SM, Fischetti VA, Leblanc DJ, Moellering RC Jr, Gilmore MS (2007) Genetic diversity among Enterococcus faecalis. PLoS One 2(7):e582. doi: 10.1371/journal.pone.0000582CrossRefPubMedPubMedCentralGoogle Scholar
  33. Murray BE (1990) The life and times of the Enterococcus. Clin Microbiol Rev 3(1):46–65CrossRefGoogle Scholar
  34. Paganelli FL, Willems RJ, Leavis HL (2012) Optimizing future treatment of enterococcal infections: attacking the biofilm? Trends Microbiol 20(1):40–49. doi: 10.1016/j.tim.2011.11.001CrossRefPubMedGoogle Scholar
  35. Palmer KL, Kos VN, Gilmore MS (2010) Horizontal gene transfer and the genomics of enterococcal antibiotic resistance. Curr Opin Microbiol 13(5):632–639. doi: 10.1016/j.mib.2010.08.004CrossRefPubMedPubMedCentralGoogle Scholar
  36. Paulsen IT, Banerjei L, Myers GS, Nelson KE, Seshadri R, Read TD et al (2003) Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299(5615):2071–2074. doi: 10.1126/science.1080613CrossRefPubMedGoogle Scholar
  37. Perez-Trallero E, Montes M, Orden B, Tamayo E, Garcia-Arenzana JM, Marimon JM (2007) Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005. Antimicrob Agents Chemother 51(4):1228–1233. doi: 10.1128/aac.01054-06CrossRefPubMedPubMedCentralGoogle Scholar
  38. Perreten V, Vorlet-Fawer L, Slickers P, Ehricht R, Kuhnert P, Frey J (2005) Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J Clin Microbiol 43(5):2291–2302. doi: 10.1128/jcm.43.5.2291-2302.2005CrossRefPubMedPubMedCentralGoogle Scholar
  39. Pinheiro ET, Gomes BP, Drucker DB, Zaia AA, Ferraz CC, Souza-Filho FJ (2004) Antimicrobial susceptibility of Enterococcus faecalis isolated from canals of root filled teeth with periapical lesions. Int Endod J 37(11):756–763. doi: 10.1111/j.1365-2591.2004.00865.xCrossRefPubMedGoogle Scholar
  40. Poeta P, Igrejas G, Goncalves A, Martins E, Araujo C, Carvalho C et al (2009) Influence of oral hygiene in patients with fixed appliances in the oral carriage of antimicrobial-resistant Escherichia coli and Enterococcus isolates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108(4):557–564. doi: 10.1016/j.tripleo.2009.06.002CrossRefPubMedGoogle Scholar
  41. Portenier I, Waltimo TMT, Haapasalo M (2003) Enterococcus faecalis– the root canal survivor and ‘star’ in post-treatment disease. Endod Top 6(1):135–159. doi: 10.1111/j.1601-1546.2003.00040.xCrossRefGoogle Scholar
  42. Poveda Roda R, Bagan JV, Sanchis Bielsa JM, Carbonell Pastor E (2007) Antibiotic use in dental practice: a review. Med Oral Patol Oral Cir Bucal 12(3):E186–E192PubMedGoogle Scholar
  43. Preshaw PM, Hefti AF, Jepsen S, Etienne D, Walker C, Bradshaw MH (2004) Subantimicrobial dose doxycycline as adjunctive treatment for periodontitis: a review. J Clin Periodontol 31(9):697–707. doi: 10.1111/j.1600-051X.2004.00558.xCrossRefPubMedGoogle Scholar
  44. Rams TE, Feik D, Mortensen JE, Degener JE, van Winkelhoff AJ (2013) Antibiotic susceptibility of periodontal Enterococcus faecalis. J Periodontol 84(7):1026–1033. doi: 10.1902/jop.2012.120050CrossRefPubMedGoogle Scholar
  45. Reinert RR, Filimonova OY, Al-Lahham A, Grudinina SA, Ilina EN, Weigel LM et al (2008) Mechanisms of macrolide resistance among Streptococcus pneumoniae isolates from Russia. Antimicrob Agents Chemother 52(6):2260–2262. doi: 10.1128/aac.01270-07CrossRefPubMedPubMedCentralGoogle Scholar
  46. Roberts AP, Mullany P (2010) Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance. Expert Rev Anti-Infect Ther 8(12):1441–1450. doi: 10.1586/eri.10.106CrossRefPubMedGoogle Scholar
  47. Roberts AP, Mullany P (2011) Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance. FEMS Microbiol Rev 35(5):856–871. doi: 10.1111/j.1574-6976.2011.00283.xCrossRefPubMedGoogle Scholar
  48. Roberts AP, Cheah G, Ready D, Pratten J, Wilson M, Mullany P (2001) Transfer of TN916-like elements in microcosm dental plaques. Antimicrob Agents Chemother 45(10):2943–2946. doi: 10.1128/aac.45.10.2943-2946.2001CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rocas IN, Siqueira JF Jr (2013) Detection of antibiotic resistance genes in samples from acute and chronic endodontic infections and after treatment. Arch Oral Biol 58(9):1123–1128. doi: 10.1016/j.archoralbio.2013.03.010CrossRefPubMedGoogle Scholar
  50. Ruiz-Garbajosa P, Canton R, Pintado V, Coque TM, Willems R, Baquero F et al (2006) Genetic and phenotypic differences among Enterococcus faecalis clones from intestinal colonisation and invasive disease. Clin Microbiol Infect 12(12):1193–1198. doi: 10.1111/j.1469-0691.2006.01533.xCrossRefPubMedGoogle Scholar
  51. Schirrmeister JF, Liebenow AL, Braun G, Wittmer A, Hellwig E, Al-Ahmad A (2007) Detection and eradication of microorganisms in root-filled teeth associated with periradicular lesions: an in vivo study. J Endod 33(5):536–540. doi: 10.1016/j.joen.2007.01.012CrossRefPubMedGoogle Scholar
  52. Schlegelova J, Babak V, Klimova E, Lukasova J, Navratilova P, Sustackova A et al (2002) Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples. J Vet Med B Infect Dis Vet Public Health 49(5):216–225CrossRefGoogle Scholar
  53. Sedgley CM, Molander A, Flannagan SE, Nagel AC, Appelbe OK, Clewell DB et al (2005a) Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp. Oral Microbiol Immunol 20(1):10–19. doi: 10.1111/j.1399-302X.2004.00180.xCrossRefPubMedGoogle Scholar
  54. Sedgley CM, Nagel AC, Shelburne CE, Clewell DB, Appelbe O, Molander A (2005b) Quantitative real-time PCR detection of oral Enterococcus faecalis in humans. Arch Oral Biol 50(6):575–583. doi: 10.1016/j.archoralbio.2004.10.017CrossRefPubMedGoogle Scholar
  55. Sedgley C, Buck G, Appelbe O (2006) Prevalence of Enterococcus faecalis at multiple oral sites in endodontic patients using culture and PCR. J Endod 32(2):104–109. doi: 10.1016/j.joen.2005.10.022CrossRefPubMedGoogle Scholar
  56. Sedgley CM, Lee EH, Martin MJ, Flannagan SE (2008) Antibiotic resistance gene transfer between Streptococcus gordonii and Enterococcus faecalis in root canals of teeth ex vivo. J Endod 34(5):570–574. doi: 10.1016/j.joen.2008.02.014CrossRefPubMedGoogle Scholar
  57. Siqueira JF Jr, Rocas IN (2004) Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 97(1):85–94. doi: 10.1016/s1079210403003536CrossRefPubMedGoogle Scholar
  58. Souto R, Colombo AP (2008) Prevalence of Enterococcus faecalis in subgingival biofilm and saliva of subjects with chronic periodontal infection. Arch Oral Biol 53(2):155–160. doi: 10.1016/j.archoralbio.2007.08.004CrossRefPubMedGoogle Scholar
  59. Sun J, Song X, Kristiansen BE, Kjaereng A, Willems RJ, Eriksen HM et al (2009) Occurrence, population structure, and antimicrobial resistance of enterococci in marginal and apical periodontitis. J Clin Microbiol 47(7):2218–2225. doi: 10.1128/jcm.00388-09CrossRefPubMedPubMedCentralGoogle Scholar
  60. Templer SP, Baumgartner A (2007) Enterococci from Appenzeller and Schabziger raw milk cheese: antibiotic resistance, virulence factors, and persistence of particular strains in the products. J Food Prot 70(2):450–455CrossRefGoogle Scholar
  61. Tenover FC, Baker CN, Swenson JM (1996) Evaluation of commercial methods for determining antimicrobial susceptibility of Streptococcus Pneumoniae. J Clin Microbiol 34(1):10–14PubMedPubMedCentralGoogle Scholar
  62. Thurnheer T, Belibasakis GN (2015) Integration of non-oral bacteria into in vitro oral biofilms. Virulence 6(3):258–264. doi: 10.4161/21505594.2014.967608CrossRefPubMedGoogle Scholar
  63. Van Tyne D, Gilmore MS (2014) Friend turned foe: evolution of enterococcal virulence and antibiotic resistance. Annu Rev Microbiol 68:337–356. doi: 10.1146/annurev-micro-091213-113003CrossRefPubMedPubMedCentralGoogle Scholar
  64. Wenzler S, Schmidt-Eisenlohr E, Daschner F (2004) Comparative in vitro activities of three new quinolones and azithromycin against aerobic pathogens causing respiratory tract and abdominal wound infections. Chemotherapy 50(1):40–42. doi: 10.1159/000077284CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Annette Carola Anderson
    • 1
  • Huria Andisha
    • 1
  • Elmar Hellwig
    • 1
  • Daniel Jonas
    • 2
  • Kirstin Vach
    • 3
  • Ali Al-Ahmad
    • 1
  1. 1.Department of Operative Dentistry and Periodontology, Medical Center - University of Freiburg, Faculty of MedicineUniversity of FreiburgFreiburgGermany
  2. 2.Institute for Environmental Health Sciences and Hospital Infection ControlMedical Center - University of Freiburg, Faculty of Medicine, University of FreiburgFreiburgGermany
  3. 3.Center for Medical Biometry and Medical Informatics, Institute for Medical Biometry and Statistics, Medical Center - University of Freiburg, Faculty of MedicineUniversity of FreiburgFreiburgGermany

Personalised recommendations