The antibacterial activity of the new quinolone compounds enoxacin, norfloxacin, ofloxacin and ciprofloxacin was evaluated in 300 Enterobacteriaceae, 50 Pseu-domonasaeruginosa, 30 Acinetobacter spp., 15 Haemophilus influenzae, 50 Streptococcus faecalis, and 70 Staphylococcus aureus isolates and compared to that of nalidixic acid, gentamicin and various β-lactam compounds. Moreover, the rate of spontaneous mutants resistant to quinolone compounds was evaluated. In concentrations only insignificantly exceeding the minimal inhibitory concentrations (MIC), mutants could be isolated rather frequently (approx. 10^-6 fold); in concentrations of at least 10 times the MIC resistant mutants were barely detectable. In general, the mutants exhibited a 4- to 8-fold increase of the MIC as compared to the wild strain. In S. faecalis mutants were not detectable, whereas they occurred in low frequency ( < 10^-8 fold) in S. aureus strains. In all mutants there was almost, but not entirely, complete cross-resistance between the quinolone derivatives.

Of 104 genotypically diverse methicillin-resistant Staphylococcus aureus (MRSA) isolates tested with the MicroScan WalkAway (Pos MIC 24 panel) and Vitek 2 (AST-P549 card) systems, 7 and 6 isolates, respectively, showed an oxacillin MIC of <2mg/liter. Most of these MRSA isolates were community acquired. However, if the cefoxitin screen of AST-P549 was also considered, MRSA detection failed for only one isolate.The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has increased over the last years. Reliable detection of MRSA is important since a false report of a patient's isolate as methicillin susceptible would result in inadequate therapy with probably fatal consequences (2). Whereas MRSA infections formerly occurred almost exclusively in hospitalized patients, community-acquired MRSA (cMRSA) isolates have been reported recently in patients without any previous contact with the health care system (7).Many laboratories rely on automatic susceptibility testing methods that use oxacillin MIC testing, oxacillin breakpoint detection in the presence of salt, or cefoxitin MIC testing as markers for the presence of methicillin resistance. Many studies have investigated the detection of MRSA by the Vitek 2 system (3,4,8,11,12,13,15,17); however, data for the performance of the MicroScan WalkAway system in MRSA detection are scarce (17).Most studies evaluating the performance of Vitek 2 used consecutive clinical strains (3,8,11,12,15), but this approach may be biased by the overrepresentation of locally predominant clones and may not predict performance in other geographical areas. We therefore used a collection of MRSA strains with distinct pulsed-field gel electrophoresis (PFGE) patterns to study MRSA detection using the MicroScan WalkAway and Vitek 2 systems.From 1998 to 2006, noncopy MRSA isolates (n ϭ 1,516), initially identified by oxacillin screening agar or Vitek, from four hospitals in the Bochum area were collected and typed by PFGE as described previously (5). Of these, 120 isolates with different PFGE patterns were chosen. The patterns were interpreted according to the criteria of Tenover et al. (18), and isolates grouped into PFGE types and subtypes.For susceptibility tests, isolates from frozen storage were subcultured twice on Columbia blood agar at 37°C in 5% CO 2 before being tested with the Vitek 2 system using the AST-P549 card and the MicroScan WalkAway system using the Pos MIC 24 panel.Whenever results for oxacillin in the Vitek 2 or MicroScan WalkAway system or for the cefoxitin screen in the Vitek 2 system were not indicative of MRSA, a mecA PCR was performed from colonies growing on purity control plates of both automatic systems and a S. aureus-specific PCR for SA442 (16) was used as an internal positive control. In addition, the Panton-Valentine leukocidin (PVL)-coding genes lukS-PVL and lukF-PVL were detected by PCR (9). SCCmec typing (10) and spa typing (6) were performed as described previously.Loss of mecA during storage of isolates could be demonstrated in 16 of 120 isolates ...

BackgroundClarithromycin (CLR) is the most commonly recommended antibiotic in Helicobacter pylori eradication regimens, but the prevalence of CLR-resistant H. pylori is increasing. CLR resistance is associated with mutations in the 23S rRNA gene. However, H. pylori eradication can still be achieved with triple therapy, and an additive effect may occur with multiple antibiotics.MethodsTwenty-six CLR-resistant strains were examined. The MIC of clarithromycin was determined by agar-dilution-testing on Columbia agar, as described elsewhere. The conserved region of the H. pylori 23S rRNA gene between nucleotide positions 1445 and 2846 [GenBank: U27270] was amplified. RFLP and sequence analysis were performed with the 1402-bp PCR product. Synergy between clarithromycin and amoxicillin was assessed using the agar dilution checkerboard technique. To confirm the correlation between mutation and synergistic effect with subinhibitory concentrations of AMX, site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.ResultsTwenty-six clarithromycin-resistant strains were examined. The conserved region of the H. pylori 23S rRNA gene was amplified, and the purified PCR product was checked for mutations by restriction fragment length polymorphism (RFLP) analysis and sequencing. A synergistic effect was found in only three of the 12 H. pylori strains (25%) with the A2142G mutation and five of the 10 H. pylori strains (50%) with the A2143G mutation (fractional inhibitory concentration: FIC < 0.5, minimal inhibitory concentration: MIC<2 mg/L) was found. Site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.Three of these isolates harboring a mutation in position A2143G grew under selection with CLR (MIC >16 mg/L), and all three strains showed the synergistic effect (FIC<0.5). In contrast, three of the same four strains transformed with DNA fragments with a mutation in position A2142G were resistant to CLR (MIC>16 mg/L) and showed no synergism with amoxicillin (FIC>2).ConclusionsHere we demonstrate that in 100% of the in vitro transformed strains, a mutation at position A2143G leads to a synergistic effect between clarithromycin and amoxicillin, whereas a mutation at position at A2142G had no discernible effect.

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