1 Servicio de Microbiología, Hospital Ramón y Cajal, Carretera de Colmenar, km. 9.1, Madrid 28034, Spain; 2 Research Laboratory for Infectious Diseases (LIO), National Institute of Public Health and the Environment (RIVM) Bilthoven, The Netherlands
Received 22 May 2002; returned 24 June 2002; revised 4 September 2002; accepted 5 September 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: esp, Enterococcus faecium, ampicillin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
An enterococcal surface protein, Esp, has recently been identified as a marker of highly prevalent VREF clones among hospitalized patients.1 This protein contributes to colonization and persistence of Enterococcus faecalis in the urinary tract and the ability of this species to produce biofilms.4,5 Several recent European studies have also demonstrated the presence of the esp gene in vancomycin-sensitive E. faecium (VSEF) frequently found in the hospital environment and recovered from urine samples.1,6,7 However, few data are available on the prevalence of esp-positive isolates in bacteraemia, or among VSEF strains.1,6,7 Moreover, data about the prevalence of esp in clones colonizing healthy people are still needed. The aim of this work was to provide such data and to investigate whether specific E. faecium subpopulations carrying a variant of the E. faecalis esp gene are associated with disease or epidemicity in this species in Spain, where the prevalence of VREF in hospitals remains very low (<3%).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We screened 149 E. faecium isolates comprising 65 blood isolates (from 63 patients) recovered in our hospital between 1995 and 2000 (two were kindly provided by Dr Liñares from Hospital Bellvitge, Barcelona); 31 non-blood isolates from consecutive patients (11 isolated from organic fluids, 10 from soft-tissue infections, nine from urine and one from the respiratory tract) collected between 1999 and 2000; 33 faecal isolates from 33 healthy volunteers (HV) living in the same community and without exposure to a hospital environment or antibiotics in the previous 6 months, which were recovered in 2000, and 20 from environmental samples collected in the same area.
Species identification and antibiotic susceptibility
Presumptive species identification was performed by the semi-automated WIDER system (Fco. Soria Melguizo, Madrid, Spain) for clinical isolates, or by standard biochemical procedures for faecal isolates.8 Confirmation of species identification was performed by PCR using oligonucleotide primer sets to amplify genes coding for EfaA (derived from E. faecalis) and aminoglycoside 6'-N-acetyltransferase [AAC(6')-Ii] (derived from E. faecium).8 Antimicrobial susceptibility was determined by the agar dilution method according to the 2000 NCCLS guidelines.
PFGE and analysis of the banding patterns
Chromosomal DNA was prepared as described previously.8 The SmaI-digested genomic DNA banding patterns were analysed by visual examination by two independent investigators. Following the standard criteria given by Tenover et al.9 to establish clonal relationships, isolates were considered to be related if they exhibited differences of up to six bands (if there was good epidemiological evidence to suggest the relatedness among isolates or if they had been isolated over extended periods of time).
Esp detection
The presence of esp was detected by PCR using primers to amplify an 800 bp gene sequence coding for esp from E. faecium (GenBank Accession number AF443999): 5'-GGAACGCCTTGGTATG-3' and 5'-CCGCTTTTGGTGATTC-3'. E. faecium strain E-774 was used as a positive control. Isolates positive for the esp gene were considered to express Esp on their surface.1
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The presence of esp in isolates susceptible and resistant to different antibiotics indicates that this trait probably emerged prior to the acquisition of resistance not only to Van6,7 but also to other antibiotics commonly used in the hospital setting (this study). The significant association between resistance to Amp, Erm or Cip and esp in the E. faecium isolates studied suggests that antibiotic treatment selects particular clones among those that have reached ecological abundance in the nosocomial habitat due to the presence of esp. This may explain why in some hospitals, strains containing esp were frequent among VRE strains.7 The higher occurrence in our study of this trait in AmpR nosocomial hospital-associated versus community isolates supports this hypothesis. Willems et al.1,10 have shown the existence of E. faecium and E. faecalis ecovars (genogroups associated with particular hosts and environments) and have suggested the existence of a specific VREF genogroup possibly adapted to nosocomial transmission by the presence of esp.
In summary, our results show that esp clones are most prevalent among E. faecium isolates from hospitalized patients, and only rarely occur in the community setting. The frequency of the esp gene in hospitals correlates with the occurrence of antibiotic-resistant E. faecium clones. This observation suggests that antibiotic-resistant variants may arise frequently under antibiotic selective pressure among esp-positive clones reaching ecological abundance in the nosocomial habitat.
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Suppola, J. H., Jolho, E., Salmenlinna, S., Tarkka, E., Vuopio-Varkila, J. & Vaara, M. (1999). van A and van B incorporate into endemic ampicillin-resistant vancomycin-sensitive Enterococcus faecium strain: effect on interpretation of clonality. Journal of Clinical Microbiology 37, 39349.
3
.
Day, N. P. J., Moore, C. E., Enright, M. C., Berendt, A. R., Smith, J. M., Murphy M. F. et al. (2001). A link between virulence and ecological abundance in natural populations of Staphylococcus aureus. Science 292, 1146.
4
.
Shankar, N., Lockatell, C. V., Baghdayan, A. S., Drachenberg, C., Gilmore, M. S. & Johnson, D. E. (2001). Role of Enterococcus faecalis surface protein ESP in the pathogenesis of ascending urinary tract infection. Infection and Immunity 69, 436672.
5
.
Toledo-Arana, A., Valle, J., Solano, C., Arrizubieta, M. J., Cucarella, C., Lamata, M. et al. (2001). The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Applied and Environmental Microbiology 67, 453845.
6 . Baldassarri, L., Bertuccini, L., Ammendolla, M. G., Gherardi, G. & Creti, R. (2001). Variant esp gene in vancomycin-sensitive Enterococcus faecium. Lancet 357, 1802.
7 . Woodford, N., Soltani, M. & Hardy, K. J. (2001). Frequency of esp in Enterococcus faecium isolates. Lancet 358, 584.
8 . Coque, T. M., Seetulsingh, P., Singh, K. V. & Murray, B. E. (1998). Application of molecular techniques to the study of nosocomial infections caused by enterococci. In Methods in Molecular Medicine, Vol. 15: Molecular Bacteriology: Protocols and Clinical Applications (Woodford, N. & Johnson, A., Eds), pp. 46993. Humana Press, Totowa, NJ, USA.
9
.
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
10 . Waar, K., Murcholl-Silberhorn, A. B., Willems, R. J. L., Sloof, M. J. H., Harmsen, H. J. M. & Degener, J. E. (2002). Genogrouping and incidence of virulence factors of Enterococcus faecalis in liver transplant patients differ from blood culture and fecal isolates. Journal of Infectious Diseases 185, 11217.[ISI][Medline]