This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Novais, C.
Right arrow Articles by Peixe, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Novais, C.
Right arrow Articles by Peixe, L.
Agricola
Right arrow Articles by Novais, C.
Right arrow Articles by Peixe, L.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2005, p. 3364-3368, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3364-3368.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Environmental Contamination with Vancomycin-Resistant Enterococci from Hospital Sewage in Portugal

Carla Novais,1 Teresa M. Coque,2 Helena Ferreira,1 João Carlos Sousa,3 and Luisa Peixe1*

REQUIMTE Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal,1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain,2 Microbiologia, Faculdade Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal3

Received 27 September 2004/ Accepted 20 December 2004


arrow
ABSTRACT
 
Vancomycin-resistant enterococci (VRE) were detected in samples of sewage obtained downstream of hospitals of the Porto area in Portugal, and in samples from the Douro Estuary. Clonal analysis, Tn1546 typing, and presence of putative virulence traits indicate the clinical origin of these isolates. This observation highlights the importance of hospital sewage in the VRE contamination of the environment.


arrow
INTRODUCTION
 
Enterococci have been traditionally considered as indicators of fecal contamination of drinking and recreational waters (1), although they are usually recovered at high concentrations from natural environments lacking exposure to heavy fecal contamination, such as rivers, seawater, and nonagricultural soils (2, 11). Release of antibiotic-resistant bacteria to the community is therefore of particular concern since they might proliferate in soil and surface waters, persist and spread in different environments (11, 14, 15, 25, 26), and transfer antibiotic resistance genes among different species (3, 12). The enterococcal population in wastewater from different European countries and the United States has been previously evaluated (4, 11, 12), but knowledge of the relationship between enterococcal populations from sewage or surface waters and those recovered from hospitalized patients is scarce (11).

Vancomycin-resistant enterococci (VRE) are one of the most worrisome pathogens in hospitals in the United States, and they are starting to increase in European health institutions, Portugal being the area with the currently highest VRE prevalence (Annual Report of European Antibiotic Resistance Surveillance System, 2002 [http//:www.earss.rivm]). We screened hospital sewage and river water samples for the presence of antibiotic-resistant enterococci, characterizing clonal relatedness and genes coding for antibiotic resistance and putative virulence traits.

Twenty-six sewage samples (SW) were collected in an urban sewer from January 2001 to May 2002 in an area near four Porto hospitals. Twelve samples were collected from sewage upstream of the hospitals (SWU), flowing from residential neighborhoods. Fourteen samples were collected from sewage downstream of health institutions (SWD). Porto wastewaters are drained to the Douro river estuary. Samples were obtained from the shore of this estuary (n = 3) and from the Coura and Sousa Rivers (n = 2), tributaries of the Douro before the estuary.

Samples were collected in sterile bottles and kept at 4°C until processing (2 to 8 h). Slanetz-Bartley agar plates with and without vancomycin (6 mg/liter) were used to incubate membranes with filtered aliquots (0.45-mm-pore-size membranes; Millipore Corporation) of 100-ml river samples and 0.1-ml aliquots of SWU and SWD that were directly spread on plates. Susceptibility testing was performed following CLSI (formerly NCCLS) guidelines and using the recommended breakpoints to define resistance (18). A multiplex PCR assay was used for species identification and vancomycin resistance gene detection (8). VRE isolates were typed by pulsed-field gel electrophoresis (PFGE) (16, 21). Genes coding for resistance to aminoglycosides, streptogramins, and macrolides, the backbone structure of the Tn1546 harbored by VRE isolates, and the presence of some putative enterococcal virulence factors (cytolysin [Cyl], gelatinase [Gel], aggregation substance [Agg], hyaluronidase [Hyl], and enterococcal surface protein [Esp]) were investigated as described previously (10, 17, 20, 23, 24, 27). Conjugation experiments were performed using Enterococcus faecalis strain JH2-2 or E. faecium GE1 as recipients (19). Amplification and sequencing of the purK allele from vancomycin-resistant Enterococcus faecium clones were performed as previously reported (13). Statistical significance for comparison proportions was calculated by the chi-square test (P < 0.05 was considered to be statistically significant).

Enterococci were isolated in 25 out of 26 (96%) of the sewage samples and 5 out of 5 (100%) of the river samples. VRE were isolated from 11/14 (79%) samples collected from SWD and in 2/3 samples from the Douro Estuary, being better recovered from plates containing vancomycin (21/31 isolates; 68%) than from those supplemented with ampicillin (6/31; 19%) or gentamicin (3/31; 10%) or from Slanetz plates without antibiotic (1/31; 3%). VRE were not detected in SWU or in the rivers before the Douro Estuary. Rates of resistance to ampicillin, ciprofloxacin, and erythromycin and of high-level resistance (HLR) to streptomycin, gentamicin, or kanamycin were higher among isolates from SWD samples than among those from SWU samples. Similar rates of resistance to tetracycline and chloramphenicol were found among SWU and SWD (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. MIC distribution and percentage of resistance to different antibiotics tested in enterococci isolated from Portuguese sewage and rivers

vanA, vanB, and vanC1 genes were detected in 27, 4, and 1 VRE isolates, respectively. Among these strains, HLR to gentamicin and erythromycin was always due to aac(6')-aph(2''), and erm(B). Transference of vanA was achieved for 13/24 (54%) isolates. vanA and erm(B) were observed in 8/14 (57%) cases, and cotransference of vanA and aph(3')-IIIa in 13/14 (93%) cases. Four transconjugants harbored vanA, erm(B), and aph(3')-IIIa (29%) (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Features of VRE clones isolated Portuguese environment: PFGE, antibiotic resistance, and virulence profiles; Tn1546 types; and frequency of transfer of the studied traitsa

Nine PFGE types (8 E. faecium and 1 E. faecalis) were identified among VRE from sewage, and two E. faecium PFGE types were found among VRE from the Douro Estuary (Table 2). Clones B, C, and L were observed on at least two different occasions over 1 year, type B being recovered from sewage of hospitals I and II. Some of the PFGE types (B, C, D, F, N, and P) were also observed in the hospital setting (data not shown). Six different variants of Tn1546 (PP3, PP5, PP-16, PP-17, PP-20, and X) were identified (Table 3). Four variants (PP-16, PP-17, PP-20, and X) lack amplicons with primers corresponding to regions associated with transposition functions, and five Tn1546 types (PP-3, PP5, PP-17, PP-20, and X) showed alterations in regions downstream of vanA. Tn1546 variants PP5 and PP3, found in E. faecium isolates from the Douro Estuary, contained an ISEf1 insertion, which has been described only among Tn1546 from clinical enterococcal isolates from Portugal (19). Most vancomycin-resistant E. faecium isolates (n = 22/31; 71%) contained at least one trait associated with virulence. Different combinations of putative virulence factors were detected: esp+ (n = 6), hyl+ (n = 5), asa1+ (n = 3), asa1+ gel+ (n = 1), asa1+ gel+ cyl+ (n = 3), and asa1+ gel+ esp+ (n = 3). Isolates within clonal types B, C, and L contained variable virulence patterns. The allele purK-1, associated with epidemic E. faecium clinical isolates (13), was detected in river and sewage clones.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Tn1546 types found among VRE clinical isolates recovered in environmental samplesa

Antibiotic resistance, virulence profiles, and the presence of the allele purK-1 in enterococcal isolates from SWD and river estuary, but not in those from SWU samples, suggest a nosocomial origin of these strains and thus a poor efficiency of wastewater management, which is often observed in European and American wastewater treatment plants (7, 11, 12, 25). The persistence of particular VRE PFGE types in wastewater for at least a 1-year period might reflect a better fitness of specific clones able to survive under harmful conditions or a continuous leaking of particular strains from health institutions, both able to contribute to the spread of hospital-specific enterococcal ecovars to the environment (13). The presence of E. faecalis clone B in hospitals of three different Portuguese cities for a 7-year period (19) suggests a wide dissemination of particular strains in the community which might be occasionally reintroduced in nosocomial institutions (14). In addition, the presence of antibiotic-resistant enterococci, including VRE, in the estuary area of the Douro River is indicative of fecal human contamination from both community and hospitals. Since this zone is used for bathing, recreation, and sport fishing and since enterococci can survive for long periods in the algal mat (26), shellfish (11, 28), and seagulls (5), the risk of acquisition by humans of enterococcal genogroups of hospital origin from the environment cannot be discarded (14).

The variable antibiotic and virulence profiles among isolates of PFGE types B, L, and C suggest multiple horizontal gene transfer events in sewage or release of particular clones carrying different genetic elements available from local metagenomes (3, 19). A great heterogeneity of Tn1546 has been observed among enterococcal isolates from different ecological origins (27, 29), with some types being associated with specific hosts or settings (19, 27, 29) while virulence traits are scarcely found outside the nosocomial environment (6, 9, 10, 13). On the other hand, differences in the transferability of specific Tn1546 among vancomycin-resistant E. faecium isolates belonging to the same clone suggest that different transfer mechanisms may play a role in the dissemination of VRE. The presence of mobile elements containing particular antibiotic resistance genes or virulence determinants in the community could trigger the dissemination of enterococcal isolates carrying elements coding resistance and/or virulence determinants (22) and enhance the risk to transmission to other environmental bacterial as already reported (3, 22).

Our data suggest that both particular clones and mobile elements carrying antibiotic resistance or virulence associated to the clinical setting might be continuously contaminating the community environment through wastewater. Reducing the release of bacteria or genetic elements from the clinical setting to the community is becoming a critical issue to avoid the buildup of environmental reservoirs of antibiotic resistance.


arrow
ACKNOWLEDGMENTS
 
We are very grateful to Fernando Baquero for critical reading of the manuscript and to SMAS Porto, Divisão de Controlo de Qualidade and Departamento de Águas Residuais, for the collection of samples.

Carla Novais was supported by a fellowship from Fundação para a Ciência e Tecnologia (SFRH/BD/3372/2000), and the present work was partially supported by FSE/FEDER (POCTI/EFP/39885/2001).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 4050 Porto, Portugal. Phone: 351-2-22078946. Fax: 351-2-22003977. E-mail: lpeixe{at}ff.up.pt. Back


arrow
REFERENCES
 
    1
  1. Anonymous. 1999. Standard methods for examination of water and wastewater, 20th ed., p. 1220. American Public Health Association and Water Environment Federation, Washington, D.C.
    2. 2
  2. Ator, L. L., and M. J. Starzyk. 1976. Distribution of group D streptococci in rivers and streams. Microbios 16:91-104.[Medline]
    3. 3
  3. Baquero, F. 2004. From pieces to patterns: evolutionary engineering in bacterial pathogens. Nat. Rev. Microbiol. 2:510-518.[CrossRef][Medline]
    4. 4
  4. Blanch, A. R., J. L. Caplin, A. Iversen, I. Kühn, A. Manero, H. D. Taylor, and X. Vilanova. 2003. Comparison of enterococcal populations related to urban and hospital wastewater in various climatic and geographic European regions. J. Appl. Microbiol. 94:994-1002.[CrossRef][Medline]
    5. 5
  5. Choi, S., W. Chu, J. Brown, S. J. Becker, V. J. Harwood, and S. C. Jiang. 2003. Application of enterococci antibiotic resistance patterns for contamination source identification at Huntington Beach, California. Mar. Pollut. Bull. 46:748-755.[CrossRef][Medline]
    6. 6
  6. Coque, T. M., J. M. Steckelberg, J. E. Patterson, and B. E. Murray. 1995. Incidence of hemolysin, gelatinase and aggregation substance among enterococci isolated from patients with endocarditis, other infections and from feces of hospitalized and community based persons. J. Infect. Dis. 171:1223-1229.[Medline]
    7. 7
  7. Dias Miguel, J. 2003. Poluiçao águas perigosas. Visão 2003:112-114.
    8. 8
  8. Dukta-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33:24-27.[Abstract]
    9. 9
  9. Eaton, T. J., and M. J. Gasson. 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl. Environ. Microbiol. 67:1628-1635.[Abstract/Free Full Text]
    10. 10
  10. Gilmore, M. S., P. S. Coburn, S. R. Nallapareddy, and B. E. Murray. 2002. Enterococcal virulence, p. 301-354. In M. S. Gilmore (ed.), The enterococci: pathogenesis, molecular biology, and antibiotic resistance. American Society for Microbiology, Washington, D.C.
    11. 11
  11. Guardabassi, L., and A. Dalsgaard. 2004. Occurrence, structure, and mobility of Tn1546-like elements in environmental isolates of vancomycin-resistant enterococci. Appl. Environ. Microbiol. 70:984-990.[Abstract/Free Full Text]
    12. 12
  12. Harwood, V. J., M. Brownell, W. Perusek, and J. E. Whitlock. 2001. Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States. Appl. Environ. Microbiol. 67:4930-4933.[Abstract/Free Full Text]
    13. 13
  13. Homan, W. L. D., S. Tribe, M. Poznanski, G. Li, E. Hogg, J. D. A. Spalburg, R. L. J. van Embden, and Willems. 2002. Multilocus sequence typing scheme for Enterococcus faecium. J. Clin. Microbiol. 40:1963-1971.[Abstract/Free Full Text]
    14. 14
  14. Iversen, A., I. Kühn, M. Rahman, A. Franklin, L. G. Burman, B. Olsson-Liljequist, E. Torell, and R. Möllby. 2004. Evidence for transmission between humans and the environment of a nosocomial strain of Enterococcus faecium. Environ. Microbiol. 6:55-59.[CrossRef][Medline]
    15. 15
  15. Iversen, A., I. Kühn, A. Franklin, and R. Möllby. 2002. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl. Environ. Microbiol. 68:2838-2842.[Abstract/Free Full Text]
    16. 16
  16. Kaufmann, M. E. 1998. Pulsed-field gel electrophoresis, p. 17-31. In N. Woodford and A. P. Johnson (ed.), Methods in molecular medicine, vol. 15. Humana Press Inc., Totowa, N.J.
    17. 17
  17. Lim, J. A., A. R. Kwon, S. K. Kim, Y. Chong, K. Lee, and E. C. Choi. 2002. Prevalence of resistance to macrolide, lincosamide and streptogramin antibiotics in gram-positive cocci isolated in a Korean hospital. J. Antimicrob. Chemother. 49:489-495.[Abstract/Free Full Text]
    18. 18
  18. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5. National Committee for Clinical Laboratory Standards. Wayne, Pa.
    19. 19
  19. Novais, C., T. M. Coque, J. C. Sousa, F. Baquero, and L. Peixe. 2004. Local genetic patterns within a vancomycin-resistant Enterococcus faecalis clone isolated in three hospitals in Portugal. Antimicrob. Agents Chemother. 48:3613-3617.[Abstract/Free Full Text]
    20. 20
  20. Soltani, M., D. Beighton, J. Howard, and N. Woodford. 2000. Mechanisms of resistance to quinupristin-dalfopristin among isolates of Enterococcus faecium from animals, raw meat, and hospital patients in Western Europe. Antimicrob. Agents Chemother. 44:433-436.[Abstract/Free Full Text]
    21. 21
  21. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
    22. 22
  22. Tomita, H., C. Pierson, S. K. Lim, D. B. Clewell, and Y. Ike. 2002. Possible connection between a widely disseminated conjugative gentamicin resistance (pMG1-like) plasmid and the emergence of vancomycin resistance in Enterococcus faecium. J. Clin. Microbiol. 40:3326-3333.[Abstract/Free Full Text]
    23. 23
  23. Vakulenko, S. B., S. M. Donabedian, A. M. Voskresenskiy, M. J. Zervos, S. A. Lerner, and J. W. Chow. 2003. Multiplex PCR for detection of aminoglycoside resistance genes in enterococci. Antimicrob. Agents Chemother. 47:1423-1426.[Abstract/Free Full Text]
    24. 24
  24. Vankerckhoven, V., T. Van Autgaerden, C. Vael, C. Lammens, S. Chapelle, R. Rossi, D. Jabes, and H. Goossens. 2004. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J. Clin. Microbiol. 42:4473-4479.[Abstract/Free Full Text]
    25. 25
  25. Vilanova, X., A. Manero, M. Cerda-Cuellar, and A. R. Blanch. 2004. The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants. J. Appl. Microbiol. 96:279-288.[CrossRef][Medline]
    26. 26
  26. Whitman, R. L., D. A. Shively, H. Pawlik, M. B. Nevers, and M. N. Byappanahalli. 2003. Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan. Appl. Environ. Microbiol. 69:4714-4719.[Abstract/Free Full Text]
    27. 27
  27. Willems, R. J. L., J. Top, N. van der Braak, A. van Belkum, D. J. Mevius, G. Hendriks, M. van Santen-Verheuvel, and J. D. van Embden. 1999. Molecular diversity of Tn1546-like elements in enterococci from humans and animals. Antimicrob. Agents Chemother. 43:483-491.[Abstract/Free Full Text]
    28. 28
  28. Wilson, I. G., and G. G. McAfee. 2002. Vancomycin-resistant enterococci in shellfish, unchlorinated waters, and chicken. Int. J. Food Microbiol. 79:143-151.[Medline]
    29. 29
  29. Woodford, N., A. M. A. Adebiyi, M. F. I. Palepou, and B. Cookson. 1998. Diversity of VanA glycopeptide resistance elements in enterococci from humans and animals. Antimicrob. Agents Chemother. 42:502-508.[Abstract/Free Full Text]

Applied and Environmental Microbiology, June 2005, p. 3364-3368, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3364-3368.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Freitas, A. R., Novais, C., Ruiz-Garbajosa, P., Coque, T. M., Peixe, L. (2009). Dispersion of Multidrug-Resistant Enterococcus faecium Isolates Belonging to Major Clonal Complexes in Different Portuguese Settings. Appl. Environ. Microbiol. 75: 4904-4908 [Abstract] [Full Text]  
  • Freitas, A. R., Novais, C., Ruiz-Garbajosa, P., Coque, T. M., Peixe, L. (2009). Clonal expansion within clonal complex 2 and spread of vancomycin-resistant plasmids among different genetic lineages of Enterococcus faecalis from Portugal. J Antimicrob Chemother 63: 1104-1111 [Abstract] [Full Text]  
  • Novais, C., Freitas, A. R., Sousa, J. C., Baquero, F., Coque, T. M., Peixe, L. V. (2008). Diversity of Tn1546 and Its Role in the Dissemination of Vancomycin-Resistant Enterococci in Portugal. Antimicrob. Agents Chemother. 52: 1001-1008 [Abstract] [Full Text]  
  • Quinteira, S., Peixe, L. (2006). Multiniche Screening Reveals the Clinically Relevant Metallo-{beta}-Lactamase VIM-2 in Pseudomonas aeruginosa Far from the Hospital Setting: an Ongoing Dispersion Process?. Appl. Environ. Microbiol. 72: 3743-3745 [Abstract] [Full Text]  
  • Novais, C., Coque, T. M., Costa, M. J., Sousa, J. C., Baquero, F., Peixe, L. V. (2005). High occurrence and persistence of antibiotic-resistant enterococci in poultry food samples in Portugal. J Antimicrob Chemother 56: 1139-1143 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Novais, C.
Right arrow Articles by Peixe, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Novais, C.
Right arrow Articles by Peixe, L.
Agricola
Right arrow Articles by Novais, C.
Right arrow Articles by Peixe, L.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»