Evaluation of the Modified Broth-Disk Method for Determining Antibiotic Susceptibilities of Anaerobic Bacteria

Abstract

A total of 110 strains of anaerobic bacteria including 9 different genera was tested for antibiotic susceptibility by the modified broth-disk method of Wilkins and Thiel (1973), using prereduced media and the VPI anaerobic culture system. Results were compared with those obtained with an agar dilution technique with incubation in a GasPak jar. There was greater than 95% agreement between the two methods with ampicillin, cephalothin, chloramphenicol, and clindamycin, and 90% or greater agreement with erythromycin, penicillin, and tetracycline.Routine susceptibility testing of anaerobes has been a problem for clinical laboratories because of the lack of a standardized disk diffusion method that is adequate for use with the anaerobic bacteria which show such wide variations in generation time. In 1973, Wilkins and Thiel (9) published a simple method for determining the susceptibility of anaerobic bacteria to antibiotics. Their method is a modified broth dilution technique using only one concentration of antibiotic and commercially available disks as the source of antibiotic. The purpose of the present study was to compare the broth-disk method with the more accepted agar dilution technique in order to evaluate its accuracy. MATERIALS AND METHODSA total of 110 strains of anaerobic bacteria isolated in the years 1971 to 1973 from clinical specimens in the Diagnostic Microbiology Laboratory of the University of Minnesota Hospitals was used. The strains used included 46 Bacteroides fragilis, 3 Bacteroides melaninogenicus, 7 Bacteroides sp., 8 Bifidobacterium sp., 9 Clostridium perfringens, 7 Clostridium sp., 7 Eubacterium sp., 2 Fusobacterium sp., 1 Lactobacillus sp., 10 Peptococcus sp., 7 Peptostreptococcus sp., and 3 Veillonella sp. All organisms were identified by methods and characteristics defined by Holdeman and Moore (3).All prereduced media used were purchased from Scott Laboratories, Fiskeville, R. I. Culture in prereduced media was carried out by using the VPI anaerobic culture system (Bellco) and oxygen-free C02-The modified broth-disk method was performed as described by Wilkins and Thiel (9). One drop of an overnight culture in prereduced chopped meatglucose broth was used as inoculum for the tests. The antibiotic disks used were obtained from Bioquest.For the agar dilution technique, plates were prepared with brain heart infusion agar supplemented with hemin and vitamin K (Scott Laboratories; 1 ml per 100 ml of agar) and containing serial twofold dilutions of each antibiotic. The plates were held overnight at room temperature before inoculation. For the inoculum, a 1:100 dilution in prereduced brain heart infusion broth was made of the same overnight chopped meat-glucose culture used for the broth-disk method. The agar dilution plates were inoculated with the diluted cultures by using the replicator device of Steers et al. (7). Two plates without any antibiotic were also inoculated for a growth control and an aerobic check. The plates were inoculated in room air, and only 20 organisms were replicated at a...

Susceptibility of Anaerobic Bacteria to Metronidazole, Ornidazole, and Tinidazole and Routine Susceptibility Testing by Standardized Methods

“…Throughout the experiments, prereduced, anaerobically sterilized media were used. These were prepared according to methods described by Holdeman and Moore (13 Blazevic (1). For the agar dilution technique, plates were prepared containing serial twofold dilutions of each antimicrobial agent from 100 to 0.1 ,ug/ml.…”

Comparative Growth Rates of Selected Anaerobic Species in Four Commonly Used Broth Media

“…Regardless of the susceptibility testing technique used, it is important to standardize the initial inoculum because the in vitro antimicrobial susceptibility of an organism can be significantly affected by organism concentration (2,7,16 variety of growth media and inoculum standardization methods; some investigators (1, 8,11,15,16) use the no. 1 MacFarland nephelometric standard or a 50% dilution of the same standard (10) as in the aerobic disk diffusion method, whereas other investigators (4,5,12,13,17,18) use various dilutions for 18-to 24-h broth cultures. In using a nephelometric standard to standardize inocula, one must assume that different cultures with the same turbidity have approximately the same number of colony-forming units (CFU) per milliliter.…”

Susceptibility testing of clinically isolated anaerobic bacteria by an agar dilution technique

“…453, 1976). These procedures have been reviewed and evaluated (3,22). The agar dilution procedure has the greatest degree of precision and is recommended as a reference method for anaerobic susceptibility testing (19).…”