Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Multiple antibiotic resistances of Enterococcus isolates from raw or sand-filtered sewage


Fifty antibiotic-resistant Enterococcus strains were isolated from raw sewage of a wastewater treatment plant and from the same sewage after trickling through a 25-cm sand column, which retained >99% of the initial population. All 50 Enterococcus isolates were resistant against triple sulfa and trimethoprim/sulfamethoxazole and none were resistant against vancomycin. Most of the isolates from raw sewage were resistant to more antibiotics than the isolates from sand column effluent. One Enterococcus isolate from raw sewage (no. 61) and one Enterococcus isolate from sand column effluent (no. 95) had ten antibiotic resistances each. Isolate no. 95 maintained its resistances in the absence of antibiotics during the whole study. It was compared with isolate no. 70, which was one of the isolates, being resistant only against the two sulfonamides. Phenotypically and biochemically, the two organisms were strains of Enterococcus faecalis. Sequence analysis of partical 16S rDNA allowed alignment of isolate no. 95 as a strain of Enterococcus faecium and of isolate no. 70 as a strain of E. faecalis. E. faecium strain no. 95 carried at least six different plasmids, whereas for E. faecalis strain no. 70, no discrete plasmid band was seen on the gels.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2


  1. Al-Doori Z, Hill RLR, Casewell MW (2001) Use of a “Miniprep” for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium. World J Microbiol Biotechnol 17:517–521

  2. An P, Hua J, Franz M, Winter J, Gallert C (2004) Changes of chemical and biological parameters in soil caused by trickling sewage. Acta Hydrochim Hydrobiol 32:296–303

  3. Bale MJ, Day MJ, Fry JC (1988) Novel method for studying plasmid transfer in undisturbed river Epilithon. Appl Environ Microbiol 54:2756–2758

  4. Chesbro WR, Evans JB (1959) Factors affecting the growth of enterococci in highly alkaline media. J Bacteriol 78:858–862

  5. Davies J (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264:375–382

  6. Drolet M, Wu HY, Liu LF (1994) Roles of DNA topoisomerases in transcription. In: August J, Anders M, Murad F, Coyle J, Liu L (eds) DNA topoisomerases: biochemistry and molecular biology, 29A. Academic, New York

  7. Facklam RR (1972) Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl Microbiol 23:1131–1139

  8. Facklam RR (1973) Comparison of several laboratory media for presumptive identification of enterococci and group D streptococci. Appl Microbiol Biotechnol 26:138–145

  9. Fars S, Oufdou K, Nejmeddine A, Hassani L, Melloul AA, Bousselhaj K, Amahmid O, Bouhoum K, Lakmichi H, Mezrioui N (2005) Antibiotic resistance and survival of faecal coliforms in activated sludge system in a semi-arid region (Beni Mellal, Morocco). World J Microbiol Biotechnol 21:493–500

  10. Gallert C, Fund K, Winter J (2005) Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers. Appl Microbiol Biotechnol 69:106–112

  11. Goni-Urriza M, Capdepuy M, Arpin C, Raymond N, Caumette P, Quentin C (2000) Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp. Appl Environ Microbiol 66:125–132

  12. Gross KC, Houghton MP, Senterfit LB (1975) Presumptive speciation of Streptococcus bovis and other group D streptococci from human sources by using arginine and pyruvate tests. J Clin Microbiol 1:54–60

  13. Hancock LE, Gilmore MS (2006) Pathogenicity of Enterococci. In: Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (eds) Gram-positive pathogens. ASM Press, Washington DC

  14. Hannan PCT (2000) Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. Vet Res 31:373–395

  15. Heuer H, Krögerrecklenfort E, Wellington EMH, Egan S, van Elsas JD, van Overbeek L, Collard J-M, Guillaume G, Karagouni A, Nikolakopoulou D, Smalla K (2002) Gentamycin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42:289–302

  16. Horodniceanu T, Bougueleret L, EI-Solh N, Bieth G, Delbos F (1979) High-level, plasmid-borne resistance to gentamicin in Streptococcus faecalis subsp. zymogenes. Antimicrob Agents Chemother 16:686–689

  17. Hua J, An P, Winter J, Gallert C (2003) Elimination of COD, microorganisms and pharmaceuticals from sewage by trickling through sandy soil below leaking sewers. Water Res 37:4395–4404

  18. Huycke MM, Sahm DF, Gilmore MS (1998) Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg Infect Dis 4:239–249

  19. Jorgensen JH, Turnidge JD, Washington JA (1999) Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray PR, Pfaller MA, Tenover FC, Baron EJ, Yolken RH (ed) Manual of clinical microbiology, 7th edn. ASM Press, Washington DC, pp 1526–1543

  20. Kümmerer K (2003) Significance of antibiotics in the environment. J Antimicrob Chemother 52:5–7

  21. Moellering RC Jr (1992) Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 14(6):1173–1176

  22. Moellering RC, Weinberg AN (1971) Studies on the antibiotic synergism against enterococci: II. Effects of various antibiotics on the uptake of 14C-labeled Streptomycin by enterococci. J Clin Invest 50:2580–2584

  23. Mundt JO (1986) Enterococci. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, 1st edn, vol. 2. Williams & Wilkins, Baltimore, pp 1063–1065

  24. Murray BE (1990) The life and times of the Enterococcus. Clin Microbiol Rev 3:46–65

  25. Murray BE (1998) Diversity among multidrug-resistant enterococci. Emerg Infect Dis 4(1):37–47

  26. National Committee for Clinical Laboratory Standards (1999) Performance standards for antimicrobial susceptibility testing; ninth informational supplement M100-S9. NCCLS, Wayne, PA, USA

  27. Neu HC (1992) The crisis in antibiotic resistance. Science 257:1064–1073

  28. Pucci MJ, Monteschio ME, Kemker CL (1988) Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp. Appl Environ Microbiol 54:281–287

  29. Rainey FA, Ward-Rainey N, Kroppenstedt RM, Stackebrandt E (1996) The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage; proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46:1088–1092

  30. Rhodes MW, Kator H (1999) Sorbitol-fermenting bifidobacteria as indicators of diffuse human faecal pollution in estuarine watersheds. J Appl Microbiol 87:528–535

  31. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

  32. Schaberg DR, Culver DH, Gaynes RP (1991) Major trends in the microbial etiology of nosocomial infections. Am J Med 91(3B):72S–75S

  33. Schlüter A, Heuer H, Szczepanowski R, Forney LJ, Thomas CM, Pühler A, Top EM (2003) The 64 508 bp IncP-1b antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1b group. Microbiology 149:3139–3153

  34. Skold O (2000) Sulfonamide resistance: mechanisms and trends. Drug Resist Updat 3(3):155–160

  35. Smalla K, Krögerrecklenfort E, Heuer H, Dejonghe W, Top E, Osborn M, Niewint J, Tebbe C, Barr M, Bailey M, Greated A, Thomas C, Turner S, Young P, Nikolakopoulou D, Karagouni A, Wolters A, van Elsas JD, Dronen K, Sandaa R, Borin S, Brabhu J, Grohmann E, Sobecky P (2000) PCR-based detection of mobile genetic elements in total community DNA. Microbiology 146:1256–1257

  36. Szczepanowski R, Krahn I, Linke B, Goesmann A, Pühler A, Schlüter A (2004) Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system. Microbiology 150:3613–3630

  37. Thorsted P, Macartney DP, Akhtar P, Haines AS, Ali N, Davidson P, Stafford T, Pocklington MJ, Pansegrau W, Wilkins BM, Lanka E, Thomas CM (1998) Complete sequence of the IncPß plasmid R751: implications for evolution and organisation of the IncP backbone. J Mol Biol 282:969–990

  38. Tobian JA, Macrina FL (1982) Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome. J Bacteriol 152:215–222

  39. Tyrrell GJ, Turnbull L, Teixeira LM, Lefebvre J, Carvalho MGS, Facklam RR, Lovgren M (2002) Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov. Isolated from human clinical specimens. J Clin Microbiol 40:1140–1145

  40. Wise R, Hart T, Cars O, Streulens M, Helmuth R, Huovinen P, Sprenger M (1998) Antimicrobial resistance is a major threat to public health. Br Med J 317:609–610

Download references


This work was supported by Bundesministerium für Bildung und Forschung, Bonn with a grant to Junyi Xu (IPS 05/02P) and by Deutsche Forschungsgemeinschaft, Bonn, grant For 350.

Author information

Correspondence to Josef Winter.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xu, J., Gallert, C. & Winter, J. Multiple antibiotic resistances of Enterococcus isolates from raw or sand-filtered sewage. Appl Microbiol Biotechnol 74, 493–500 (2007).

Download citation


  • Enterococcus sp.
  • Sewage
  • Multiple antibiotic resistances
  • Resistance plasmids