Broth-Dilution Method for Determining the Antibiotic Susceptibility of Anaerobic Bacteria

Abstract

A broth-dilution method for performing antimicrobial susceptibility tests on anaerobic bacteria has been proposed. The medium used in the test was Schaedler broth, with incubation in a glove box with an atmosphere of 5% CO2, 10% H2, and 85% N2, or in the GasPak system. Minimal inhibitory concentrations for selected antibiotics were determined, under these conditions, by using a conventional twofold dilution scheme for the antibiotics and a "categorization three-tube method" in which two or three clinically significant concentrations of each antibiotic were used. Minimal inhibitory concentrations obtained by both methods were very similar. The categorization method could be used routinely to test the antimicrobial susceptibility of anaerobic bacteria.Because anaerobic bacteria are being isolated from clinical material with increasing frequency, the diagnostic microbiology laboratory is more often called upon to test the susceptibility of these organisms to a variety of antimicrobial agents. The determination of the drug susceptibility of anaerobic organisms is generally more complicated than for aerobic organisms, because of the difficulty in isolating and identifying them, the obligation to test them in the anaerobic environment, and the lack of a standard method for testing their susceptibility.The approach of many investigators has been to use conventional techniques for aerobic and facultative organisms to test the drug susceptibility of anaerobes in an oxygenless environment. Therefore, antimicrobial susceptibilities have been determined by disk-diffusion, agardilution, and broth-dilution techniques. Although there are several common sources of variation for these techniques, e.g., inoculum concentrations, pH of the medium, and components of the medium, one major disadvantage of the disk-diffusion and agar-dilution techniques for testing anaerobic bacteria in comparison with the broth-dilution technique is that the organisms are much more likely to be subjected to lethal doses of oxygen when they are spread onto the surface of agar. Furthermore, the addition of agar to a broth medium adds to the complexity of the medium and to the possibility of some effects on the susceptibility test. For these reasons we chose to use a broth-dilution technique as our reference method for determining minimal inhibitory concentrations (MICs) of several antimicrobial agents for various anaerobic bacteria.The initial consideration in attempts to standardize a method for susceptibility testing should be the achievement of optimal growth conditions for the organisms being tested. We have previously shown that Schaedler broth (BBL) in an atmosphere of 5% CO2, 10% H2, and 85% N2 was superior to eight other media and other combinations of gas mixtures for growing anaerobic bacteria likely to be isolated from clinical material (10). A broth-dilution method, with this medium and atmosphere, were used in this study. Susceptibility tests were also performed with a modified broth-dilution technique in which only two or three concentr...

Sonorensin: an Antimicrobial Peptide, Belonging to the Heterocycloanthracin Subfamily of Bacteriocins, from a New Marine Isolate, Bacillus sonorensis MT93

“…The MICs of bacteriocin for different indicator strains were evaluated by using a microtiter broth dilution method as described by Stalons and Thornsberry (29). The lowest concentration that inhibited the growth of test strains and did not show any increase in absorption after 48 h was considered the MIC.…”

Collaborative Evaluation of a Proposed Reference Dilution Method of Susceptibility Testing of Anaerobic Bacteria

“…Individuals within the group then began investigations on various aspects of susceptibility testing of anaerobic bacteria. Studies conceming the development of the medium, inoculum requirements, and culture conditions have been published elsewhere (2)(3)(4)(5)7). Details of an agar dilution method were agreed upon and preliminary trials of the method were reported in 1976 (V. L. Sutter, A.Laboratory Standards under a contract negotiated with the Division of Anti-Infective Drugs of the Food and Drug Administration.…”

In vitro activity of mecillinam against anaerobic bacteria

“…The in vitro activity of mecillinam against anerobic bacteria was investigated by employing a microtiter broth dilution method (8 Roholt and co-workers, in a study of 9 patients, found peak serum levels after intramuscular administration of either 250 or 500 mg of mecillinam to be approximately 8 and 10 lOg/ml, respectively (7). The serum half-life of mecillinam intramuscularly administered was approximately 1 h (7).…”