Antimicrobial susceptibility testing of a clinical isolate of vancomycin-dependent enterococcus using D-alanine-D-alanine as a growth supplement.

Bacteremia due to a vancomycin-dependent enterococcus (VDE) occurred during long-term vancomycin therapy in a renal transplant recipient with underlying pancreatitis and a vancomycin-resistant enterococcal (VRE) wound infection and bacteremia. The VDE was isolated from blood during vancomycin therapy and grew only in the presence of vancomycin and D-alanine-D-alanine (DADA), a substance required for cell-wall synthesis. Colonies beyond the periphery of growth of the VDE around a vancomycin disk contained vancomycin-independent revertant mutants after 48 hours of incubation. Pulsed-field gel electrophoresis of the VDE, revertant mutant, the initial blood culture isolate of VRE, and an autopsy isolate showed that the four strains were identical. Antimicrobial susceptibility testing was performed using standard macrobroth and microbroth dilution methods. DADA was used as a growth supplement for macrobroth dilution susceptibility testing of the VDE isolate. Minimum inhibitory concentrations (MICs) were similar for the VRE isolate and the VDE revertant, which were both resistant to ampicillin, high-level gentamicin, ciprofloxacin, imipenem, vancomycin, and daptomycin, and were susceptible to fusidic acid, high-level streptomycin, rifampin, and a quinupristin-dalfopristin combination. The MICs of teicoplanin were 2 microg/mL or less and 16 microg/mL for the clinical VRE isolate and the VDE revertant, respectively. The autopsy isolate was resistant to all antimicrobials tested and showed a fourfold increase in MICs for quinupristin-dalfopristin compared with that of the original blood isolate. The VDE was susceptible to all drugs tested except vancomycin.

Imipenem and meropenem activity against mecA-positive homogeneously and heterogeneously oxacillin-resistant and mecA-negative oxacillin-borderline-susceptible staphylococci.

Microbroth dilution and disk-diffusion testing of imipenem and meropenem was performed at 35 and 30 degrees C against 61 phenotypic expression class 3,4 and 9 phenotypic expression class 1,2 oxacillin-resistant isolates of Staphylococcus aureus (ORSA), 51 oxacillin-borderline-susceptible isolates of S. aureus (BORSA), and 37 phenotypic expression class 3,4 and 9 phenotypic expression class 1,2 isolates of Staphylococcus epidermidis (ORSE). Imipenem MIC ranges at 35 degree C were 0.6 to > 64 micrograms/ml for class 3,4 ORSA, 0.03 to 0.25 micrograms/ml for class 1,2 ORSA, 0.015 to 0.12 micrograms/ml for BORSA, 0.03 to 64 micrograms/ml for class 3,4 ORSE, and 0.12 to 8 micrograms/ml for class 1,2 ORSE. Corresponding values for meropenem were 0.5 to 64 micrograms/ml, 0.12 to 4 micrograms/ml, 0.06 to 1 microgram/ml, 0.5 to 64 micrograms/ml, and 1 to 8 microgram/ml. MIC ranges at 30 degrees C did not differ by more than 1 log2 dilution from those at 35 degrees C. After 24 h incubation of disk-diffusion tests at 35 degrees C, 44% of class 3,4 and 100% of class 1,2 ORSA isolates were imipenem-susceptible; after an additional 24 h at 25 degrees C, 39 and 100% of these isolates, respectively, remained susceptible to imipenem. Similar values were obtained with 24 h incubation at 30 degrees C followed by 24 h at 25 degrees C. All BORSA isolates were susceptible to imipenem. Of the ORSE isolates, 22 and 78% of isolates in classes 3,4 and 1,2, respectively, were susceptible at 24 h with little change after an additional 24 h at 25 degrees C. Similar trends were observed with meropenem. In parallel disk-diffusion studies with oxacillin, false-susceptibility rates of 5% of class 3,4 and 44% class 1,2 ORSA isolates after 24 h of incubation at 35 degrees C were reduced to 3 and 0%, respectively, after an additional 24 h of incubation at 25 degrees C. Imipenem- and meropenem-resistant subpopulations of oxacillin-resistant staphylococci did not seem to be detected by altered susceptibility testing conditions.

Timed killing kinetic studies of the interaction between ciprofloxacin and beta-lactams against gram-negative bacilli.

Timed killing kinetic studies were performed with ciprofloxacin in combination with aztreonam, ceftazidime, piperacillin/tazobactam, and ticarcillin/clavulanic acid against three isolates each of Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa. Each antimicrobial agent in the combination was tested at its MIC and at one-half and one-quarter of its MIC. Colony counts were determined at 0, 3, 5, and 7 hours. Synergy occurred most frequently at 7 hours and, when present, was most likely to occur when ciprofloxacin and the beta-lactam were tested at concentrations equal to their respective MICs. Synergy after 3 hours of incubation was not predictive of a synergestic interaction at 5 or 7 hours. Antagonism was noted in several instances, particularly when ciprofloxacin and the beta-lactam were combined at one-quarter of their respective MICs.

Evaluation of Vitek GPS-SA card for testing of oxacillin against borderline-susceptible staphylococci that lack mec.

Fifty-one Staphylococcus aureus strains lacking mec for which oxacillin MICs were 1 to 8 micrograms/ml were tested against oxacillin and the combination of oxacillin and clavulanic acid with the Vitek GPS-SA card, the reference broth microdilution method, and the oxacillin agar screen plate. Of the 51 strains, 44 (86%) did not grow on the oxacillin agar screen plate, broth microdilution MICs were 1 to 2 micrograms/ml, and GPS-SA card MICs were < or = 2 micrograms/ml, with the exception of 3 strains that failed to grow in the card on repeated attempts. Another seven strains did grow on the oxacillin agar screen plate. For four of the latter group of strains, oxacillin broth microdilution MICs were > 4 micrograms/ml and GPS-SA card MICs were > or = 4 micrograms/ml; for the other 3 strains, corresponding MICs were 4 and < or = 2 micrograms/ml, respectively. The GPS-SA card classified 86% of strains as oxacillin susceptible.

Activities of three investigational fluoroquinolones (BAY y 3118, DU-6859a, and clinafloxacin) against Neisseria gonorrhoeae isolates with diminished susceptibilities to ciprofloxacin and ofloxacin.

Between 1 January 1992 and 31 December 1993, our laboratory, as part of the Gonococcal Isolate Surveillance Project, found that 39 of 673 isolates of Neisseria gonorrhoeae from one local sexually transmitted diseases clinic demonstrated decreased susceptibilities to both ciprofloxacin and ofloxacin. The MICs of BAY y 3118, DU-6859a, and clinafloxacin at which 90% of the gonococci with decreased susceptibility to ciprofloxacin and ofloxacin were inhibited were all 0.016 microgram/ml, which was eightfold higher than those for ciprofloxacin-susceptible gonococci. Our report substantiates prior observations that diminished susceptibility to one quinolone is often associated with diminished susceptibility to other quinolones.

Evaluation of BBL crystal MRSA ID system.

The BBL Crystal system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) was evaluated for its accuracy in identifying oxacillin resistance in Staphylococcus aureus by testing of mec-specific-gene-positive and -negative isolates. Although the manufacturer makes no claim for use of the product for testing of staphylococci other than S. aureus, the product's potential utility in detecting oxacillin resistance in isolates of mec gene-positive and -negative Staphylococcus epidermidis was also explored. All mec gene-negative staphylococci yielded a negative MRSA ID test reaction. There was a close correlation between mec gene positivity and a positive reaction in the methicillin-resistant S. aureus identification system with 63 of 69 (91%) stock isolates of S. aureus yielding a positive result in 4 h, 66 of 69 (95%) yielding a positive result in 5 h, and 68 of 69 (99%) yielding a positive result in 6 h. The corresponding percentage agreements at 4, 5, and 6 h for mec gene-positive stock isolates of S. epidermidis were 87, 91, and 96%, respectively.

Evaluation of differential inoculum disk diffusion method and Vitek GPS-SA card for detection of oxacillin-resistant staphylococci.

This study was conducted in order to compare the accuracy of detection of oxacillin-resistant staphylococci, defined by microdilution MICs, population analyses, and mec gene hybridization, with the Vitek GPS-SA Susceptibility Card with that of the standard inoculum (10(7) CFU) and high-inoculum (10(9) CFU) disk diffusion tests. By the standard inoculum disk diffusion test, 10 of 67 (15%) isolates of oxacillin-resistant Staphylococcus aureus and 3 of 47 (6%) isolates of Staphylococcus epidermidis were falsely susceptible after 24 h of incubation at 35 degrees C. By the high-inoculum disk diffusion test (10(9) CFU), 4 of the 10 isolates of S. aureus remained falsely susceptible, whereas none of the isolates of S. epidermidis was falsely susceptible. Of the 10 isolates of S. aureus falsely susceptible by the standard disk test, only one remained falsely susceptible after an additional 24 h of incubation at 22 degrees C. All four isolates of S. aureus that were falsely susceptible by the high-inoculum disk diffusion test after 24 h of incubation at 35 degrees C became resistant after an additional 24 h of incubation at 22 degrees C. Thus, extended incubation of both the standard and high-inoculum disk diffusion tests increased their accuracy in detecting oxacillin resistance. All isolates of oxacillin-resistant staphylococci were accurately detected with the Vitek software upgrades (6.1 and 7.1) of the GPS-SA card.

Comparison of vidas Clostridium difficile toxin-A assay and premier C. difficile toxin-A assay to cytotoxin-B tissue culture assay for the detection of toxins of C. difficile.

Damage to the intestinal mucosa by Clostridium difficile (CD) is toxin mediated. Two enzyme immunoassays (EIAs) for toxin-A detection, the automated Vitek immunodiagnostic assay system CDA (Vidas CDA), and the Premier toxin A (Premier) were tested for their ability to detect toxin A in 301 stool samples and compared with an in-house tissue culture assay for toxin B (TCA). Of these 301 samples, 49 were TCA positive and 252 were TCA negative. Agreement between Vidas CDA and TCA on the initial run was 85% (255 of 301) and increased to 94% (278 of 296) when discordant samples were retested from available frozen specimens. Corresponding levels of agreement for Premier were 91% (272 of 301) and 98% (284 of 288), respectively. If tissue culture positivity at any titer was used as the sole criterion for positivity of the specimen, agreement with positive TCA before and after repeat testing was 57% (26 of 49) and 74% (34 of 46) for Vidas CDA and 65% (32 of 49) and 95% (36 of 38) for Premier. Agreement with negative TCA titers was good: 90% for Vidas CDA and 95% for Premier, and 98% for Vidas CDA and 99% for Premier after repeat testing. Predictive values positive and negative after repeat testing were, respectively, 88% and 96% for Vidas CDA, and 95% and 99% for Premier. Results for the automated and manual EIA methods for detection of C. difficile toxin A were obtained in 2.5 h as compared with 36-48 h for tissue culture.

In vitro activity of metronidazole against Helicobacter pylori as determined by agar dilution and agar diffusion.

Metronidazole activity against 25 clinical isolates of Helicobacter pylori was evaluated by agar dilution, epsilometer (E-test; AB Biodisk, Solna, Sweden), and disk diffusion methods after 3 and 5 days of incubation in a microaerophilic atmosphere. Agar dilution, performed in duplicate, provided reproducible results with MICs for 50% of the isolates of less than or equal to 0.12 microgram/ml after 3 and 5 days of incubation and MICs for 90% of the isolates of 2 and 4 micrograms/ml after 3 and 5 days of incubation, respectively. Reproducibility of MICs was slightly better after 5 days than after 3 days of incubation. MICs obtained with the E-test were higher, with 76 and 68% of isolates inhibited by less than or equal to 16 micrograms of metronidazole per ml after 3 and 5 days, respectively, in contrast with corresponding values of 92 and 88% for agar dilution. Zone diameters obtained with the commercially available 80-micrograms metronidazole elution disk were too large (greater than or equal to 41 mm) to allow discrimination between susceptible and resistant isolates, although resistant subpopulations were detected by the appearance of inner colonies in four isolates. In conclusion, the E-test was easy to perform and interpret, and it appeared to be more likely than agar dilution to detect metronidazole resistance in vitro in H. pylori.