Bacteria
The 52 S. maltophilia strains (one per patient) that were isolated from nosocomial infections between 1998 and 2002 in the Hospital of Trakya University were included in the study. The strains were identified by conventional bacteriological methods and were stored at -70°C in skim-milk media (Becton Dickinson, USA). Before the study, they were twice passaged onto 5% sheep blood agar and the identification was confirmed by Crystal ID Enteric-nonfermenter (Becton Dickson, USA).
Antimicrobial susceptibility tests
The drug powders for the agar dilution test were obtained from the following suppliers: Ceftazidime pentahydrate (Glaxo-Welcome, UK), CPM (Bristol-Myers Squibb, USA), PIP and tazobactam (Lederle, USA), ticarcillin disodium and clavulanate lithium (GlaxoSmithKline, UK), CIP (Bayer, Turkey), trimethoprim and sulfamethoxazole (Roche, Turkey). Standard antimicrobial discs (Oxoid, UK) were used for the disc diffusion tests and E-test strips were supplied by AB Biodisk, Sweden.
Antimicrobial susceptibility tests were carried out using the disc diffusion and agar dilution techniques as described by NCCLS [7, 8]. The Agar dilution and E-test results were interpreted using the NCCLS criteria established for non-enterobacteriaceae, and the disc diffusion test was interpreted using the criteria established for P. aeruginosa [7–9]. The E-test technique was carried out according to the manifacturer's instructions. The tests were evaluated after 20–24 hours incubation at 35°C and were repeated if they were found to be discordant.
Escherichia coli the American Type Culture Collection (ATCC) 25922 and P. aeruginosa ATCC 27853 were used as quality control strains.
Definitions
The agar dilution method was accepted as the reference method. Categorical agreement was defined if the tests results were within the same susceptibility category, and errors of disc diffusion and E-test methods were determined as follows: Very major error; (resistant by reference method, susceptible by test method); major error; (susceptible by reference method, resistant by test method); and minor error; (intermediate result was obtained by one method but not the other) [10]. Percentage errors were calculated based on the total number of isolates which were tested. A good agreement was defined as complete category agreement over 90% and the total of very major and major errors below 5% [11].
Arbitrarily primed PCR
The method of vanCouwenberghe et al. [12] was used for the preparation of the DNA and AP-PCR, with minor modifications. Briefly, after an overnight culture at 37°C in 5% sheep blood agar, the bacteria were suspended in 1 ml TE buffer (10 mM Tris, 1 mM EDTA, pH: 8.0) to regulate the density to a 4 McFarland standard. Then, they were heated at 100°C for 10 min. The suspension was centrifuged at 2500 rpm for 10 minutes and the supernatant was used for AP-PCR. DNA in the supernatant was quantitated by spectrophotometry at an optical density of 260 nm. PCR mixtures were prepared in 100 μl of 1X buffer (10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2) and contained 1 μg DNA, 0.1 mM each dNTP, 2.5 U Taq polymerase and 30 pmol of pBR322 Sal I primer (AGTCATGCCCCGCGC). PCR was initiated with five cycles of low stringency, which included a denaturing step at 95°C for 1 min, annealing of the primer at 28°C for 1 min, and 2 min of extension at 72°C. After the initial 5 cycles, 55 additional cycles were conducted with annealing of the primer at 50°C. The reaction was terminated with a final extension cycle at 72°C for 10 min.
Samples were electrophoresed in a 1.5% agarose gel (Sigma, Germany) in 1X Tris-borate-EDTA buffer for 90 min at 100 V and visualized under UV light after staining with ethidium bromide. To ensure reproducibility, all amplifications were done in duplicate and were also repeated using DNA extracted on different days. Dice coefficients of similarity were calculated for every pair of isolates by visual comparison of restriction patterns. If DNA profiles of isolates were indistinguishable or differing by only three or fewer DNA band shifts, then, the isolates were deemed same or related and included in the same pattern [13].
Statistical analysis
Chi-square test (Fisher's exact test when necessary) was used.
