Low prevalence of VRE gastrointestinal colonization: MATERIALS AND METHODS
Enterococcus species isolate collection: Seven Winnipeg hospitals (Health Sciences Centre, St Boniface General Hospital, Misericordia General Hospital, Concordia General Hospital, Victoria General Hospital, Grace General Hospital, and Seven Oaks General Hospital) as well as hospitals in the rural Manitoba communities of Brandon, Thompson and Portage la Prairie participated. The Health Sciences Centre and St Boniface General Hospital are tertiary care teaching hospitals affiliated with the University of Manitoba. The remaining eight hospitals are community hospitals.
The study was divided into six, eight-week intervals, and extended from January 1 to December 31,1997. Each hospital collected stool specimens submitted for Clostridium difficile toxin and/or culture testing in the first two weeks of each eight week interval from patients in their hospitals. One stool specimen per patient was considered. Stool specimens were frozen following collection and shipped to one of two central reference laboratories (Health Sciences Centre and the St Boniface General Hospital) where they were thawed and planted onto blood agar (BA), colistin-nalidixic acid agar (CNA), and colistin- nalidixic acid agar supplemented with 6 g/mL vancomycin (CNAV). All plates were incubated at 35°C for 48 h.
All morphotypes of suspected Enterococcus species were selected from BA, CNA or CNAV plates, stocked in skim milk at -80°C, and subsequently identified to species level using the following tests: Gram stain; motility assessment; catalase production; growth in 6.5% sodium chloride; L-pyrrolidonyl- -naphthalamide hydrolysis; xylose, mannitol, arabinose and sorbitol utilization; bile/esculin growth/hydrolysis; pigment production; leucine aminopeptidase activity; and acidification of methyl- -D-glucopyranoside. The identification of all discrepant organisms was determined using DNA sequencing.
Vancomycin resistance genotyping and epidemiological typing: DNA lysates of all isolated VREF were subjected to multiplex polymerase chain reaction (PCR) to detect the presence of vanA, vanB, vanC-1 and vanC-2/vanC-3 genotypes, and pulse field gel electrophoresis (PFGE), following Smal digestion, for epidemiological tracing purposes. Simi-
lar PFGE patterns were defined as those that differed from one another by no more than one or two clearly visible bands. Antimicrobial agents and susceptibility testing: All isolated VREF were tested for their susceptibility to vancomycin (Eli Lilly Canada Inc, Scarborough, Ontario), teicoplanin (Hoechst Marion Roussel Canada Inc, Montreal, Quebec), ampicillin (Wyeth Ayerst Canada Inc, North York), streptomycin (Pfizer Canada Inc, Kirkland, Quebec), gentamicin (Schering Canada Inc, Pointe-Claire, Quebec), ciprofloxacin (Bayer Inc, Etobi- coke, Ontario), quinuprisitin/dalfopristin (Rhone Poulenc Rorer, Montreal, Quebec) and LY333328 (Lilly Research Laboratories, Indianapolis, Indiana) using the microdilution broth method and Mueller-Hinton broth as described by the National Committee of Clinical Laboratory Standards (Wayne, Pennsylvania. Before susceptibility testing all Enterococ- cus species were subcultured twice onto blood agar.
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