Molecular analysis of vancomycin-resistant Enterococcus faecalis from Michigan hospitals during a 10 year period

Preeti N. Malani1,4, LeeAnn Thal1,2,5, Susan M. Donabedian1,2,5, Barbara Robinson-Dunn6,7, Carol A. Kauffman1,4, Joseph W. Chow1,2,6, Ellie Hershberger1,2,5 and Marcus J. Zervos1,2,5,6,*

1Departments of Internal Medicine; 2Division of Infectious Disease, 3Veterans Affairs Healthcare Center, Ann Arbor, MI; 4University of Michigan Medical School, Ann Arbor, MI; 5William Beaumont Hospital, Royal Oak, MI; 6Wayne State University, Detroit, MI; 7Michigan Department of Community Health, Lansing, MI, USA

Received 28 August 2001; returned 25 November 2001; revised 30 January 2002; accepted 5 February 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We evaluated the molecular relatedness of 47 clinical isolates of vancomycin-resistant Enterococcus faecalis collected from 15 Michigan hospitals from 1991 to 2000. There were 17 PFGE strain types for the 47 isolates. Ten of 15 hospitals demonstrated interhospital, and three of 15 intrahospital, dissemination of some isolates. Forty-two isolates (89.4%) demonstrated vanB resistance. All vanA isolates comprised unique PFGE groups, suggesting transposon dissemination or the presence of a similar plasmid. The results of this study suggest inter- and intrahospital dissemination of strains of vancomycin-resistant E. faecalis during a 10 year period in Michigan.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, enterococci have emerged as significant pathogens in an increasing number of serious nosocomial infections.1,2 Of particular concern are the increased numbers of isolates resistant to multiple antimicrobial agents.1 The spread of these resistant enterococcal isolates has occurred not only within individual hospitals, but also between hospitals of varied geographical location. Although the epidemiology of vancomycin-resistant Enterococcus faecium is well described,3 less is known about vancomycin-resistant isolates of Enterococcus faecalis. Such knowledge may facilitate effective infection control efforts.

In this study, we compared the molecular relatedness of vancomycin-resistant E. faecalis isolates from Michigan hospitals using pulsed field-gel electrophoresis (PFGE) and PCR analysis. Our purpose was to obtain information about the geographical dispersion of isolates and genes responsible for vancomycin resistance. Such analysis is helpful in determining the mechanisms responsible for resistance—whether clonal, plasmid- or transposon-mediated, or by a combination of mechanisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty-seven vancomycin-resistant E. faecalis isolates were collected over a 10 year period from 1991 to 2000, from a total of 33 participant institutions as part of a Michigan Department of Community Health surveillance project of the epidemiology of vancomycin-resistant Enterococcus spp. in Michigan. All isolates of vancomycin-resistant E. faecalis identified at participant hospitals during the study period are included. Individual hospitals submitted 0–23 isolates.

Isolates were from urine (21), blood (11), wounds (six), intra-abdominal sources (five), stool (two) and a catheter tip (one). The source of one isolate was unspecified. Duplicate isolates from the same patient were excluded. Isolates were identified as E. faecalis using standard biochemical reactions. Susceptibility of isolates to vancomycin (Eli Lilly and Co., Indianapolis, IN, USA) was determined by a microdilution assay using National Committee for Clinical Laboratory Standards recommendations.4

Genomic DNA was prepared in agarose plugs, digested with SmaI (New England BioLabs, Beverly, MA, USA) and electrophoresed on a CHEF-DRIII (Bio-Rad Laboratories, Richmond, CA, USA) as described previously.5 Total numbers of visible bands were counted for each isolate, and patterns were compared visually. Isolates that were indistinguishable (no band differences) were placed in the same strain group using published consensus criteria for strain delineation.6

PCR hybridization experiments for detection of vancomycin resistance determinants for vanA and vanB were performed on all isolates. Genomic DNA, prepared by a method described previously,7 was amplified by PCR using a Perkin-Elmer 480 thermal cycler and the PCR Reagent System (Gibco-BRL, Gaithersburg, MD, USA). The vanA gene probe was amplified from E. faecium SF6460 using a 698 bp BamHI fragment as the probe. The 433 bp vanB probe was amplified from E. faecium SF6621.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the study period, 2% of E. faecalis and 37% of E. faecium isolates were resistant to vancomycin. Among the 33 hospitals studied, 15 had isolates of E. faecalis that were resistant to vancomycin and 28 had isolates of E. faecium that were vancomycin resistant. Analysis of the 47 vancomycin-resistant E. faecalis isolates indicated 17 PFGE strain types. The majority of study isolates, 34/47 (72%), were from south-eastern Michigan hospitals. Table 1 describes PFGE strain types by mode of dissemination, resistance, year(s) isolated and total number of isolates. In general, there was an increasing number of resistant E. faecalis isolates over time.


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Table 1. Summary of PFGE strain types, dissemination mode, type of resistance, year(s) isolated and total number of isolates
 
Strain type M4 was obtained from 11 patients in two hospitals from 1994 to 1999. Strain type M12 was obtained from seven patients in five hospitals from 1996 to 1999. Nine isolates represented unique strains. Five strain types contained more than one isolate but were only detected in one hospital (intrahospital spread). There was evidence for intrahospital spread of vancomycin-resistant E. faecalis at 10 of 15 hospitals. Three PFGE strain types (M4, M9 and M12) contained 21/47 isolates; each of these strains was found at two or more hospitals. M9 and M12 had the widest ranges of dissemination, isolated from three and five hospitals, respectively.

Five (10.6%) isolates demonstrated vanA resistance and 42 (89.4%) vanB resistance. The five vanA isolates comprised five unique PFGE groups (M10, M13, M14, M15 and M16) from four hospitals, whereas the 42 vanB isolates constituted 12 different PFGE types. Vancomycin MICs were >256 mg/L for vanA isolates and 16–512 mg/L for vanB isolates.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Shortly after Leclercq et al.8 reported the first observed inducible, plasmid-mediated, high-level resistance to both vancomycin and teicoplanin in E. faecium, other reports of vancomycin resistance in E. faecium and E. faecalis followed.9,10 Although resistance was rare in the USA before 1989, by 1999 25% of enterococcal isolates causing infection in USA intensive care units demonstrated resistance to vancomycin.1

Vancomycin-resistant E. faecalis strains are less common than E. faecium and comprised only 2% of total E. faecalis isolates surveyed in this state-wide study. In contrast with E. faecium, there is little information on the epidemiology of vancomycin resistance in E. faecalis. Our data demonstrated multiple strain types of vancomycin-resistant E. faecalis from Michigan hospitals over a 10 year period. Both vanA and vanB resistance was observed. Among the 17 PFGE strain types, nine were unique, indicating that some strain types appear once or infrequently but have not yet been involved in spread to other patients or hospitals. Five strain types contained more than one isolate but were detected in only one hospital, with dissemination limited to two to six patients. The remaining three strain types were found at multiple hospitals.

Ten hospitals shared PFGE strain types. Four hospitals contained only unique PFGE strain types (not associated with any dissemination). The E. faecalis isolates demonstrated predominately vanB (89.4%) resistance. In contrast, earlier analysis of vancomycin-resistant E. faecium from the same hospitals revealed vanA resistance in the majority of isolates (92%) tested.1,5

The results of this study provide further evidence for intra- and interhospital spread of resistant enterococcal isolates. Evaluation of isolates from a single institution showed epi-demiologically related vanB strains with different PFGE types, indicating the possibility of transposon dissemination or the presence of a similar plasmid among these isolates.


    Acknowledgements
 
Supported in part by the William Beaumont Hospital Research Institute and by Public Health Service Grant H50/CCH513220-01 from the Centers for Disease Control and Prevention.


    Footnotes
 
* Correspondence address. William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, MI 48073, USA. Tel: +1-248-551-0419; Fax: +1-248-551-5069; E-mail: mzervos{at}Beaumont.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . National Nosocomial Infections Surveillance System Report. (1999). Data summary from January 1990–May 1999. American Journal of Infection Control 27, 520–32.

2 . Centers for Disease Control and Prevention. (1993). Nosocomial enterococci resistant to vancomycin—United States, 1989–1993. Morbidity and Mortality Weekly Report 42, 597–9.

3 . Hayden, M. K. (2000). Insights into the epidemiology and control of infection with vancomycin-resistant enterococci. Clinical Infectious Diseases 31, 1058–65.[ISI][Medline]

4 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

5 . Donabedian, S., Chow, J. W., Shlaes, D. M., Green, M. & Zervos, M. J. (1995). DNA probes and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level. Journal of Clinical Microbiology 33, 141–5.[Abstract]

6 . 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, 2233–9.[Free Full Text]

7 . Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith J. A. et al., Eds. (1993). Current Protocols in Molecular Biology, Vol. 1, Unit 2.4. John Wiley and Sons, New York.

8 . Leclercq, R., Derlot, E., Duval, J. & Courvalin, P. (1988). Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. New England Journal of Medicine 319, 157–61.[ISI][Medline]

9 . Kim, W. J., Weinstein, R. A. & Hayden, M. K. (1999). The changing epidemiology and establishment of endemicity of vancomycin resistance in enterococci at one hospital over a 6-year period. Journal of Infectious Diseases 179, 163–71.[ISI][Medline]

10 . Sahm, D. F., Kissinger, J., Gilmore, M. S., Murray, P. R., Mulder, R., Solliday, J. et al. (1989). In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 33, 1588–91.[ISI][Medline]