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Antimicrobial Agents and Chemotherapy, July 2005, p. 3073-3079, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3073-3079.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Glycopeptide-Resistant Enterococcus faecium Isolates from Portuguese Hospitals

Carla Novais,1 João C. Sousa,2 Teresa M. Coque,3 Luísa V. Peixe,1* and The Portuguese Resistance Study Group

REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal,1 Microbiologia, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal,2 Servicio de Microbiologia, Hospital Ramón y Cajal, Madrid, Spain3

Received 22 December 2004/ Returned for modification 29 January 2005/ Accepted 19 March 2005


   ABSTRACT
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Fifty-one pulsed-field gel electrophoresis types and 17 Tn1546 variants were identified among 101 Enterococcus faecium isolates recovered in three distant Portuguese hospitals. Intra- and interhospital dissemination of specific strains and Tn1546 types was detected, which might largely contribute to the endemicity of vancomycin-resistant E. faecium in Portuguese hospitals, as happened previously in other geographical locations.


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Vancomycin-resistant enterococci (VRE) have been increasingly reported worldwide since first described in 1987, although the epidemiology of these microorganisms varies widely in different geographical areas (1, 18, 23). In the United States, VRE have become established nosocomial pathogens in intensive care units and increasingly in many hospital wards (1, 5, 15, 23, 27). In Europe, they have been mainly recovered from the community setting (1, 32), with sporadic cases of nosocomial outbreaks involving different epidemiological situations (1, 13, 14). Recent studies showing rates of VRE above 10% in six countries (Annual Report of the European Antibiotic Resistance Surveillance System, 2002 [http//:www.earss.rivm.nl]) and intrahospital dissemination of particular strains in some institutions suggest a change in the epidemiology of VRE in Europe (8). The objective of the study was to collect updated data on the clonality, antibiotic resistance genotype, and diversity of Tn1546 of vancomycin-resistant Enterococcus faecium (VREF) from Portuguese hospitals.

One hundred one VREF clinical isolates were studied among those isolated during 1996 to 2003 in three Portuguese hospitals (University Hospital in Coimbra [HUC], 54 isolates; Santo António Hospital [HSA] in Porto, 36 isolates; and São Teotónio Hospital in Viseu [HST], 11 isolates). The sample included all VRE detected in HUC, HSA, and HST isolated from January 2001 to April 2003 and a few VRE saved by microbiology laboratories in HUC from 1996 to 2000. Transfer of patients from HST to HUC occurs when specialized treatment is required. Sites of isolation of the 101 clinical isolates were urine (36%), blood (16%), wound (12%), abdominal (9%), catheter (8%), and respiratory tract (3%), and 15% were from unknown sources. Distribution of the E. faecium clinical isolates in hospital units is shown in Table 1. Of the 101 patients, 64% were in medical wards, 14% in surgical wards, and 16% in intensive care facilities, and 6% could not be classified.


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  		TABLE 1. Features of VRE Enterococcus faecium clones isolated in hospitals located in three different Portuguese areasa

 
Susceptibility testing was performed according to NCCLS guidelines, using the recommended breakpoints to define resistance (19). A multiplex PCR assay was used for species identification and vancomycin resistance gene detection (6). Genes coding for resistance to aminoglycosides and macrolides, the backbone structure of Tn1546 harbored by VREF, and virulence factors (cytolysin [Cyl], gelatinase [Gel], aggregation substance [Agg], hyaluronidase [Hyl], and enterococcal surface protein [Esp]) were investigated by PCR as described previously (7, 17, 24, 29, 30, 32). Conjugation experiments were performed using E. faecium GE1 as the recipient with isolates representing each pulsed-field gel electrophoresis (PFGE) type and subtype (10). Strains were typed by PFGE using SmaI and I-CeuI as restriction enzymes (3, 12). The location of vanA was determined by hybridization of I-CeuI-digested genomic DNA with probes labeled with the ECL kit (Amersham Life Sciences, Uppsala, Sweden) for vanA and 23S rRNA genes as described previously (3). Clonal relationships were established according to standard criteria (26).

Fifty-one PFGE types were identified among the 101 VREF isolates studied, with types 70 (14/101; 14%) and 78 (17/101; 17%) being the more commonly found. Interhospital dissemination of strain 70, 76, 78, 88, or 100 (found in two hospitals) or strain 71 (detected in three hospitals) was observed. Most VREF isolates studied were also resistant to teicoplanin (96/101; 95%), ampicillin (100/101; 99%), erythromycin (99/101; 98%), and ciprofloxacin (96/101; 95%). A high level of resistance to kanamycin, gentamicin, or streptomycin was detected in 67% (68/101), 34% (34/101), and 28% (28/101) of isolates, respectively. Rates of resistance to tetracycline, nitrofurantoin, and chloramphenicol were 34%, 7%, and 8%, respectively. All isolates were susceptible to linezolid and daptomycin.

The vanA gene was identified in all except one (vanB2) VREF isolate. A high level of resistance to gentamicin was due to aac(6')-aph(2"), and resistance to erythromycin was associated with erm(B). The simultaneous presence of aac(6')-aph(2") and aph(3')-IIIa was detected in 10 isolates. vanC1, vanC2, aph(2")I-Ib, aph(2")-Ic, aph(2")-Id, erm(A), erm(C) or mef(A) was not found in any case. vanA transference to the recipient E. faecium strain GE1 by filter mating was achieved for 36 of the 82 selected isolates (44%). Conjugation frequency ranged from 10–1 to 10–8. erm(B) was cotransferred in all except one erythromycin-resistant isolates. A high level of resistance to kanamycin encoded by aph(3')-IIIa was cotransferred only in six isolates.

A subset of 54 E. faecium isolates was selected for Tn1546 typing and included isolates within a particular PFGE cluster recovered from different hospitals or in the same hospital over time and also isolates representing unique PFGE types showing different virulence and/or antibiotic susceptibility profiles. Seventeen different variants of Tn1546 were found among 54 VREF isolates screened (Table 2). Some Tn1546 types were detected in different hospitals (PP-2 and PP-5), among isolates corresponding to different PFGE types (A, PP-2, PP-4, PP-5, PP-9, and PP-13), and during long time periods (PP-4 and PP-5). Interestingly, most Tn1546 variants (13/17) showed alterations downstream of vanA (X, PP-2, PP-3, PP-4, PP-5; PP-9, PP-10, PP-13, PP-20, PP-23, PP-24, PP-25, and PP-27). The more prevalent, disseminated, and long-term-persistent Tn1546-variants, PP-4 and PP-5, contained an ISEf1 insertion located in the vanX-vanY region. They were frequently transferred by conjugation to different E. faecium PFGE types (Table 2), and they also have been found among Enterococcus faecalis clinical isolates from the same institutions (20). Hybridization of I-CeuI-digested genomic DNA from representative isolates containing PP-4 or PP-5 corresponding to distinct PFGE types with the vanA probe was mainly associated with a band around 97 kb, suggesting the presence of a common plasmid. For representative isolates harboring other Tn1546 variants, hybridization of I-CeuI-digested genomic DNA with the vanA probe was associated with bands of high and/or low molecular weight of different lengths, suggesting the location of the vanA gene in different plasmids and/or chromosomal sites.


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  		TABLE 2. Tn1546 types found among E. faecium clinical isolates recovered at three different Portuguese hospitals (1996 to 2003)a

 
Gene coding for Esp, Gel, and Hyl was detected in 33%, 7%, and 4% of the isolates, respectively. These isolates belonged to several PFGE types and were collected in HUC and HSA between 1997 and 2003. Different combinations of putative virulence factors were detected: asa1 positive, gel positive (n = 2), asa1 positive, hyl positive (n = 1), esp positive, hyl positive (n = 2), asa1 positive, gel positive, esp positive (n = 1), hyl positive (n = 4), and esp positive (n = 30). Isolates corresponding to the same clonal type (clones 70, 71, 76, 88, 99, and 100) contained variable virulence patterns as previously described (2). None of these putative virulence trait genes was cotransferred with genetic determinants coding for glycopeptide resistance.

Our study shows a genetically diverse VREF population from Portuguese hospitals with the occurrence of intra- and interhospital dissemination of specific VREF strains and Tn1546 types/plasmids. These results suggest a wide dissemination of VREF colonizing humans but also a successful spread of particular strains which might largely contribute to the endemicity of VREF in Portuguese hospitals (15, 27). Acquisition of Tn1546 by a few widely disseminated ampicillin-resistant E. faecium clones and further spread of specific vancomycin resistance genetic elements to multiple strains led to the increase of VRE in the United States in hospitals during the last two decades (5, 9, 15, 16, 28, 31). Nosocomial VRE outbreaks in European countries have been associated with very diverse epidemiological situations involving spread of specific strains, transposons, and/or plasmids (1, 8, 13, 14, 16, 25, 33). Intrahospital dissemination of particular strains generally has been successfully controlled (1), and to our knowledge, interhospital dissemination of VRE strains has been described only rarely in Europe (4, 20, 33). However, transfer of mobile elements has caused larger outbreaks in different European countries (13, 14, 25). The recent description of specific genetic elements carrying glycopeptide resistance able to persist with or without apparent selection is of concern, since they may locally and in the long term maintain antibiotic resistance and they can be efficiently transferred to multiple genetic backgrounds (11, 28). The predominance of the Tn1546 types PP-5 and PP-4 (48% of the Tn1546 types studied) among epidemic and nonepidemic VREF clones and among E. faecalis strains from the same institutions (20) indicates that horizontal gene transfer also plays a relevant role in the dissemination of glycopeptide resistance in Portuguese hospitals (13, 14, 25). The evidence of a common plasmid band in several PFGE types isolates carrying PP-4 and PP-5 supports this hypothesis. Additionally, the presence of these Tn1546 types on distinct plasmids and chromosomal locations of E. faecalis strains from the same hospitals (20) and also differences in the transferability of specific Tn1546 among VREF isolates suggest a complex epidemiology involving different genetic elements and dissemination mechanisms. Wide dissemination of both particular clones, plasmid and/or Tn1546, might amplify the spread of VRE, as previously shown in American and certain European institutions (13, 14, 15, 25, 28).

Geographical variations in the occurrence of putative virulence traits, such as those encoded by esp or hyl, have been reported and associated with the epidemicity of VREF (22). Our data show a lower prevalence of esp-positive isolates than of VREF strains from the United States or the United Kingdom (33% versus 65% and 61%, respectively) (22, 34). However, the absence of esp among most of the epidemic VREF isolates indicates that other factors are important in the dissemination of antibiotic-resistant E. faecium, as previously suggested (2, 31). Acquisition of esp among isolates of E. faecium in the nosocomial setting (2, 21) might increase the fitness of already-widespread E. faecium clones.

In summary, our study documents a high level of genetic diversity of VREF populations from Portuguese hospitals with the presence of intra- and interhospital dissemination of specific strains and Tn1546 types. These results suggest both a wide dissemination of human-colonizing VREF and a successful spread of particular strains and Tn1546 types/plasmids which might largely contribute to the endemicity of VREF Portuguese hospitals, as previously has happened in another geographical locations.


   ACKNOWLEDGMENTS
 
Carla Novais was supported by a fellowship from Fundação para a Ciência e Tecnologia (SFRH/BD/3372/2000).

Members of the Portuguese Resistance Study Group are Graça Ribeiro, Clementina Vital (Coimbra University Hospital), Isabel Marques, Ana M. Queirós (São Teotónio Hospital, Viseu), and José Amorim and Helena Ramos (Santo António Hospital, Oporto).


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4050-030 Porto, Portugal. Phone: 351-2-22078946. Fax: 351-2-22003977. E-mail: lpeixe{at}ff.up.pt. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Bonten, M. J. M., R. J. Willems, and R. A. Weinstein. 2001. Vancomycin resistant enterococci: why are they here, and where do they come from? Lancet Infect. Dis. 1:314-325.[CrossRef][Medline]
  2. Coque, T. M., R. J. L. Willems, J. Fortún, J. Top, S. Diz, E. Loza, R. Cantón, and F. Baquero. 2005. Population structure of Enterococcus faecium causing bacteremia in a Spanish university hospital: setting the scene for a future increase in vancomycin resistance? Antimicrob. Agents Chemother. 49:2693-2700.[Abstract/Free Full Text]
  3. Dahl, K. H., and A. Sundsfjord. 2003. Transferable vanB2 Tn5382-containing elements in fecal streptococcal strains from veal calves. Antimicrob. Agents Chemother. 47:2579-2583.[Abstract/Free Full Text]
  4. Del Campo, R., C. Tenorio, M. Zarazaga, R. Gomez-Lus, F. Baquero, and C. Torres. 2001. Detection of a single vanA-containing Enterococcus faecalis clone in hospitals in different regions in Spain. J. Antimicrob. Chemother. 48:746-747.[Free Full Text]
  5. De Lencastre, H., A. E. Brown, M. Chung, D. Armstrong, and A. Tomasz. 1999. Role of the transposon Tn5482 in the epidemiology of vancomycin resistant Enterococcus faecium in the pediatric oncology unit of a New York City hospital. Microb. Drug Resist. 5:113-129.[Medline]
  6. 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]
  7. 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.
  8. Goossens, H., D. Habes, R. Rossi, C. Lammens, G. Privitera, and P. Courvalin. 2003. European survey of vancomycin resistant enterococci in at-risk hospital wards and in vitro susceptibility testing of ramoplanin against these isolates. J. Antimicrob. Chemother. 51(Suppl. S3):iii5-iii12.[Abstract]
  9. Hanrahan, J., C. Hoyen, and L. B. Rice. 2000. Geographic distribution of a large mobile element that transfers ampicillin and vancomycin resístance between Enterococcus faecium strains. Antimicrob. Agents Chemother. 44:1349-1351.[Abstract/Free Full Text]
  10. Heaton, M. P., and S. Handwerger. 1995. Conjugative mobilization of a vancomycin resístance plasmid by a putative Enterococcus faecium sex pheromone responsive plasmid. Microb. Drug Resist. 2:177-183.
  11. Johnsen, P. J., J. I. Østerhus, H. Sletvold, M. Sørum, H. Kruse, K. Nielsen, G. S. Simonsen, and A. Sundsfjord. 2005. Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal Enterococcus faecium population. Appl. Environ. Microbiol. 71:159-168.[Abstract/Free Full Text]
  12. 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. Molecular bacteriology: protocols and clinical applications. Humana Press, Inc., Totowa, N.J.
  13. Kawalec, M., M. Gniadkowski, and W. Hryniewicz. 2000. Outbreak of vancomycin-resistant enterococci in a hospital in Gdansk, Poland, due to horizontal transfer of different Tn1546-like transposons variants and clonal spread of several strains. J. Clin. Microbiol. 38:3317-3322.[Abstract/Free Full Text]
  14. Kawalec, M., M. Gniadkowski, M. Zaleska, T. Ozorowski, L. Konopka, and W. Hryniewicz. 2001. Outbreak of vancomycin-resistant Enterococcus faecium of the phenotype VanB in a hospital in Warsaw, Poland: probable transmission of the resistance determinants into an endemic vancomycin-susceptible strain. J. Clin. Microbiol. 39:1781-1787.[Abstract/Free Full Text]
  15. Kim, W., R. Weinstein, and M. Hayden. 1999. The changing molecular epidemiology and establishment of endemicity of vancomycin resistance in enterococci at one hospital over a 6-year period. J. Infect. Diseas. 179:163-171.
  16. Leavis, H. L., R. J. L. Willems, J. Top, E. Spalburg, E. M. Mascini, A. C. Fluit, A. Hoepelman, A. J. de Neeling, and M. J. Bonten. 2003. Epidemic and nonepidemic multidrug-resistant Enterococcus faecium. Emerg. Infect. Dis. 9:1108-1112.[Medline]
  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. Murray, B. E. 2000. Vancomycin-resistant enterococcal infections. N. Engl. J. Med. 342:710-721.[Free Full Text]
  19. 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.
  20. 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]
  21. Oancea, C., I. Klare, W. Witte, and G. Werner. 2004. Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis. J. Antimicrob. Chemother. 54:232-235.[Abstract/Free Full Text]
  22. Rice, L. B., L. Carias, S. Rudin, C. Vael, H. Goossens, C. Konstabel, I. Klare, S. R. Nallapareddy, W. Huang, and B. E. Murray. 2003. A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. J. Infect. Dis. 187:508-512.[CrossRef][Medline]
  23. Shepard, B. D., and M. S. Gilmore. 2002. Antibiotic-resistant enterococci: the mechanisms and dynamics of drug introduction and resistance. Microbes Infect. 4:215-224.[CrossRef][Medline]
  24. 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]
  25. Suppola, J. H., E. Jolho, S. Salmenlinna, E. Tarkka, J. Vuopio-Varkila, and M. Vaara. 1999. vanA and vanB incorporate into an endemic ampicillin-resistant vancomycin-sensitive Enterococcus faecium strain: effect on interpretation of clonality. J. Clin. Microbiol. 37:3934-3939.[Abstract/Free Full Text]
  26. 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]
  27. Thal, L., S. Donabedian, B. Robinson-Dunn, J. W. Chow, L. Dembry, D. B. Clewell, D. Alshab, and M. J. Zervos. 1998. Molecular analysis of glycopeptide-resistant Enterococcus faecium isolates collected from Michigan hospitals over a 6-year period. J. Clin. Microbiol. 36:3303-3308.[Abstract/Free Full Text]
  28. 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]
  29. 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]
  30. 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]
  31. Willems, R. J. L., J. Top, M. van Santen, A. R. Robinson, T. M. Coque, F. Baquero, H. Grundmann, and M. Bonten. 2005. Global epidemic of vancomycin resistant Enterococcus faecium is caused by the spread of a distinct nosocomially adapted genetic complex. Emerg Infect. Dis. 11:821-828.
  32. 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]
  33. Woodford, N., D. Morrison, A. P. Johnson, V. Briant, R. C. George, and B. Cookson. 1993. Application of DNA probes for rRNA and vanA genes to investigation of a nosocomial cluster of vancomycin-resistant enterococci. J. Clin. Microbiol. 31:653-658.[Abstract/Free Full Text]
  34. Woodford, N., M. Soltani, and K. J. Hardy. 2001. Frequency of esp in Enterococcus faecium isolates. Lancet 358:584.

Antimicrobial Agents and Chemotherapy, July 2005, p. 3073-3079, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3073-3079.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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