The in vitro activities of four new beta-lactam antimicrobial agents (moxalactam, cefotaxime, cefoperazone, and piperacillin) and the aminoglycosides against 744 recent clinical isolates of facultative gram-negative bacilli were compared simultaneously by the agar dilution method. The major in vitro difference of these newer beta-lactam compounds appeared to be their antipseudomonal activity; cefoperazone was the most active, whereas cefotaxime had the least potency. The aminoglycosides, however, had the most effective in vitro activity on a weight basis against Pseudomonas aeruginosa.Moxalactam, cefotaxime, cefoperazone, and piperacillin are four new beta-lactam antibiotics developed in the past few years with a wide spectrum of activity against gram-negative bacilli. Preliminary testing has suggested that these new beta-lactam antibiotics possess a broad spectrum of in vitro activity against most clinically important gram-negative bacteria, including Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Enterobacter species, and Bacteroides fragilis. Indeed, the potential clinical usefulness of these new betalactam antibiotics appears to be related to a wider spectrum of activity than other beta-lactam antibiotics and lower toxicity than the aminoglycoside drugs. Several previous in vitro studies (1,5,8,10,11,14)

The bioactivity of imipenem at 20 pLg/ml in various agar and broth media which are commonly used in susceptibility test assays was measured at different storage temperatures over time. Imipenem was found to be more stable at 4°C than at -20°C and least stable in all media at 35°C.Imipenem, a beta-lactam antimicrobial agent, has been reported to demonstrate activity against a variety of microorganisms (1, 3). The stability of this agent incorporated into cation-supplemented Mueller-Hinton broth (CSMHB) in microdilution trays is in excess of 1 year when frozen at continuous temperatures of -70°C or lower (MK-0787 susceptibility powder package insert; Merck Sharp & Dohme Research Laboratories, Rahway, N.J.); the bioactivity of imipenem in broth media at other temperatures or in agar media has not been reported. We used a modification of the agar diffusion assay (4) to measure the bioactivity of imipenem in Middlebrook 7H-11 agar, Wilkins-Chalgren agar, and Mueller-Hinton agar at three temperatures. We also assayed the bioactivity of imipenem in CSMHB, CSMHB with 5% Fildes enrichment, Todd-Hewitt broth, and brain heart infusion broth at 35°C, and in CSMHB at the additional temperatures of -20°C in a non-frost-free freezer, 4°C, and 250C.(This work was presented in part at the 23rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Las Vegas, Nev. [E. J. Baron and J. A. Hindler, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 23rd, Las Vegas, Nev., abstr. no. 553, 1983].) Microbiological assay seed agar plates were prepared in a single batch as follows: Micrococcus lysodeikticus (UCLA stock culture, maintained at -70°C) was inoculated into brain heart infusion broth (Difco Laboratories, Detroit, Mich.) and incubated at 36°C until the culture reached the late-logarithmic phase. The suspension was adjusted to match the turbidity of a McFarland 0.5 standard in sterile distilled water, and 1.5 ml was added to a tube of 18 ml of melted 1% modified Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) for each plate prepared. The seeded agar was poured into a polystyrene petri plate (15 by 150 mm) on a level surface, and the agar was allowed to solidify. Those plates not used immediately were sealed in plastic bags and refrigerated. The seed agar plates remained stable for a minimum of 24 days at 4°C (data not shown).Imipenem reconstituted in 0.1 M phosphate buffer (pH 7.0) from laboratory reference standard powder (kindly provided by H. Kropp, Merck Institute for Therapeutic Research) to a final concentration of 20 ,ug/ml was incorporated into the four broth media to be tested, all purchased from Difco Laboratories. These imipenem-containing broth media were dispensed in 1.0-ml volumes into polystyrene * Corresponding author. t Present address: Clinical Microbiology Laboratory, North Shore University Hospital, Manhasset, NY 11030. tubes (12 by 75 mm; BD Labware, Oxnard, Calif.) and stored at the temperatures to be tested. Test solutions held at -20°C were thawed at the time of bi...

A new immunofluorescent method (FIAX) was compared with a radioimmunoassay procedure for the determination of tobramycin serum concentrations. When assaying three tobramycin control sera repeatedly, within-run and run-to-run variations by the FIAX method were all within acceptable limits, and no statistically significant differences were found. Results (3,9,11,14). Numerous methods have been described for assaying tobramycin serum concentrations including: bioassay (8, 10, 12), enzymatic assays (13), gas-liquid chromatography (6, 7), high-pressure liquid chromatography (1), and radioimmunoassay (RIA) (2).In this study, the FIAX immunofluorescent assay method was compared with RIA to determine the following: (i) within-run precision; (ii) run-to-run reproducibility; (iii) accuracy of determinations by comparison with expected values; and (iv) the reliability of the FIAX method in determining tobramycin levels in patient sera. MATERIALS AND METHODSThis study employed 155 selected serum samples from patients who had been under treatment with tobramycin. Specimens that could not be assayed on the day they were obtained were frozen and stored at -20°C until tested.RIA. Tobramycin RIA kits were obtained from Diagnostic Products Corp., Los Angeles, Calif. All samples were assayed according to their recommended protocol.FIAX. All reagents used for the FIAX assay procedure were supplied by International Diagnostic Technology, Santa Clara, Calif.The solid-phase immunofluorescence method primarily involves competition between a fluoresceinlabeled and an unlabeled antigen for a fixed amount of antibody immobilized on a reactive surface. In this case, specific anti-tobramycin antibody was immobilized on a defined polymeric surface such as that which is present on the StiQ sampler. This sampler was immersed sequentially into a mixture of fluorescein-labeled tobramycin and a sample (standards, controls, or unknown) containing unlabeled tobramycin. Thus, the amount of fluorescein-labeled tobramycin that attaches to the specific anti-tobramycin antibody is inversely proportional to the concentration of unlabeled tobramycin in the sample.The following procedure outlines the assay protocol used in this study: 700 gul of fluorescein-labeled tobramycin was dispensed into 12-by 75-mm glass tubes to which 10 ,ul of sample (standard, control, or unknown) was added; each sample was tested in duplicate. All tubes were shaken briefly by hand. StiQ samplers were placed into these tubes and then mixed on a mechanical shaker for 25 min at room temperature. The tubes and StiQ samplers were removed from the shaker and allowed to equilibrate for an additional 5 min before reading. Fluorescence is measured on a fluorometer set at 995 nm (excitation) and 530 nm (emission). The degree of fluorescence was expressed in fluorescence signal units. The fluorometer was adjusted to read 190 fluorescence signal units with a reference solution containing 1 ,ug of tobramycin per ml.A standard curve was constructed with tobramycin standards (included in t...

The AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) was compared with a reference broth microdilution MIC method to determine the accuracy and reproducibility of aminoglycoside susceptibility testing of gram-negative bacilli. Stock clinical isolates (n = 176) which demonstrated resistance to at least one aminoglycoside, extended-spectrum penicillin, or broad-spectrum cephalosporin (or a combination) were selected for this study. Isolates with moderate susceptibility to the aminoglycosides were also included. Of these isolates, 116 were either resistant or moderately susceptible to one or more of amikacin, gentamicin, netilmicin, and tobramycin. When AMS MIC results for 704 antimicrobial agent-organism combinations were compared with parallel microdilution MIC results, exact agreement (AMS MIC = reference MIC) rates were: amikacin, 71.6%; gentamicin, 71.6%; netilmicin, 83.0%; and tobramycin, 69.3%. Agreement rates within ±+1 log2 dilution were: amikacin, 96.0%; gentamicin, 93.8%; netilmicin, 97.2%; and tobramycin, 96.0%. When National Committee for Clinical Laboratory Standards criteria were used to qualitatively evaluate performance, the overall agreement rates were: amikacin, 100.0%; gentamicin, 99.4%; netilmicin, 98.9%; and tobramycin, 99.4%. There were only four very major discrepancies, which represented 0.6% of the tests performed, and there were no major discrepancies. The percentages of minor discrepancies were: amikacin, 9.6%; gentamicin, 14.2%; netilmicin, 11.9%; and tobramycin, 10.8%. Of the overall average of 11.6% minor discrepancies, 9.7% occurred even though the AMS MIC was within ±1 log2 dilution of the reference MIC. The intralaboratory reproducibility ranged from 93.3 to 100% for the four drugs examined. With this challenge group of gram-negative bacilli, the AMS generated aminoglycoside MIC results that were comparable to those obtained by a reference broth microdilution method.