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.
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Abstract |
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Introduction |
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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 resistancewhether clonal, plasmid- or transposon-mediated, or by a combination of mechanisms.
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Materials and methods |
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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.
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Results |
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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 16512 mg/L for vanB isolates.
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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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References |
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