Bioluminescent assays offungal ATP in cultures of Candida albicans exposed to tioconazole and ketoconazole demonstrated that intracellular ATP levels were directly related to cell viability. At high concentrations of drug, a fungicidal effect was indicated by a very rapid and heavy leakage of ATP and a simultaneous, dramatic decrease in intracellular ATP. This leakage was due to direct membrane damage, which was less pronounced after exposure to ketoconazole than to tioconazole. After exposure to lower imidazole concentrations, intracellular ATP indicated growth inhibition without significant leakage of ATP. These findings support the hypothesis that imidazole antifungal agents basically act in the same manner but at different concentrations.Two modes of action have been suggested for the antimycotic imidazoles clotrimazole and miconazole (10, 11), but not for ketoconazole (11 University, Regionsjukhuset, Sweden, 1981). In the present report, we have studied extracellular and intracellular ATP in C. albicans exposed to tioconazole and ketoconazole. The results were correlated with cell viability to further elucidate the mode of action of these drugs. MATERIALS AND METHODSAntifungal agents. Ketoconazole (Leo AB, Helsingborg, Sweden) and tioconazole (Pfizer, Inc., Sandwich, England) were each dissolved in 100% dimethyl sulfoxide (DMSO) to stock solutions of 10 mg/ml. Just before each experiment, working solutions were prepared in the medium to be used. Luciferase assay of fungal ATP. (i) Analytical equipment. Light emission from the bioluminescent assay was measured in a 1250 luminometer (LKB-Wallac, Turku, Finland) and recorded on a 2210 potentiometric recorder (LKB-Products, Bromma, Sweden). The heat extraction of intracellular fungal ATP was performed in an LKB-Biocal 2973 incubator (LKB-Products).(ii) Analytical reagents. ATP-monitoring reagent (LKBWallac) was used in the assay of ATP. Apyrase (purified grade I) (Sigma Chemical Co., St. Louis, Mo.) was used to eliminate extracellular ATP before the extraction of intracellular ATP. Other reagents were of analytical grade.Intracellular ATP levels in cultures of C. albicans. Twofold serial dilutions of ketoconazole and tioconazole were prepared in appropriate test media (0.9 ml) and inoculated with a 10-fold dilution of the overnight culture (0.1 ml) giving a final yeast concentration of ca. 106 CFU/ml. Each experiment included a control culture that was not exposed to antifungal agents. The yeast cultures were incubated at 37°C with shaking, and intracellular ATP was determined by the following procedure.(i) Elimination of extracellular ATP. A 50-,ul sample from the culture was incubated for 10 min at 37°C with 50 ,u of an apyrase solution consisting of 0.04% apyrase made up in YNB.(ii) Extraction of intracellular ATP.

A selective method for distinguishing bacterial and nonbacterial adenosine triphosphate (ATP) in clinical bacteriological specimens was studied. The method involved incubation of samples with the detergent Triton X-100 and the ATP-hydrolyzing enzyme apyrase. The incubation selectively destroyed ATP in suspensions of various human cells while not affecting the ATP content in microbial cells. ATP remaining in the sample after incubation was extracted in boiling buffer and assayed by the firefly luciferase assay. Application of the method to 469 clinical urine specimens showed that the ATP level after treatment with Triton/apyrase was correlated to bacterial counts and that the sensitivity of the assay was sufficient for the detection of 105 bacteria/ml. The ATP levels per bacterial cell remaining in the urine specimens after treatment with Triton/apyrase were close to values observed in laboratory-grown cultures. The specificity and sensitivity of the luciferase assay for the detection of urinary bacteria and its possible use as a bacteriuria screening method are discussed.

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Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria.Testing of Mycobacterium tuberculosis susceptibility to antimycobacterial agents on the basis of growth on solid media requires 3 to 4 weeks of incubation (2, 3). Among these methods, the resistance ratio method on LowensteinJensen medium (2) is most used in Europe, while the plate proportional method on 7H10 agar (3) is the main method used in the United States. To achieve more rapid susceptibility testing, alternative techniques based on bacterial growth in broth cultures have been adapted. The rapid radiometric system (BACTEC) quantifies 14CO2 produced by mycobacteria growing in broth containing 14C-labeled palmitic acid (4). Results are provided after 1 week of incubation, and susceptibility is defined as a 99% reduction of the metabolic activity of a drug-exposed culture compared with that of an unexposed control culture (9). This technique is widely used, and the results are in good agreement with those of reference methods on solid media (4,(8)(9)(10)(11).The firefly bioluminescence assay of bacterial ATP has been used for studies of effects of antimicrobial agents on various bacteria (6, 7) and for rapid susceptibility testing of M. tuberculosis (1).The aim of this study was to evaluate this technique for rapid susceptibility testing of M. tuberculosis by comparing it with the resistance ratio method on Lowenstein-Jensen medium and with the rapid radiometric system (BACTEC). Antimycobacterial agents. Stock solutions of 10,000 pLg of active drug per ml were prepared from ethambutol (Cyanamid of Great Britain Ltd., Gosport, England), isoniazid (Ferrosan, Malmo, Sweden), rifampin (Ferrosan), and streptomycin (Glaxo Laboratories Ltd., Greenford, England). standard. The same inoculum was added to one drug-free vial (control A), and for determining the 1% proportion of resistance, another drug-free vial (control B) was inoculated with a 100-fold dilut...

In order to compare the clinical and microbiological efficacy and safety of meropenem with imipenem/cilastatin, 249 patients with intra-abdominal infections participated in an open randomised comparative multicentre trial. Seventy-five men and 57 women (mean age 51 years) were enrolled in the meropenem group and 67 men and 50 women (mean age 52 years) in the imipenem/cilastatin group. The patients received either meropenem, 500 mg q 8 h, or imipenem/cilastatin, 500 mg/500 mg q 8 h by intravenous infusion for up to 17 days (mean 5 days). Ninety-seven of 99 patients (98%) receiving meropenem were clinically cured while 86 of 90 patients (96%) in the imipenem/cilastatin group were clinically cured. The microbiological response was satisfactory in 89 of 94 evaluable patients (95%) receiving meropenem and in 78 of 81 evaluable patients (96%) receiving imipenem/cilastatin. There was no significant difference in clinical and microbiological efficacy between the two treatment groups. Adverse reactions were noted in 26 patients receiving meropenem and in 36 patients receiving imipenem/cilastatin. The present study shows that meropenem is effective and well tolerated in the treatment of intra-abdominal infections.