Microdilution Technique for Antimicrobial Susceptibility Testing of Anaerobic Bacteria

Abstract

A microdilution technique using commercially available media and materials was developed and used to determine the minimal inhibitory concentrations (MICs) of clindamycin, chloramphenicol, tetracycline, minocycline, ampicillin, carbenicillin, cephalothin, and gentamicin for 101 anaerobic isolates. Representative strains of Bacteroides, Clostridium, Fusobacterium, Peptococcus , and Peptostreptococcus were tested. The use of Schaedler broth at pH 7.2, an inoculum of 10 5 to 10 7 colony-forming units per ml, and incubation at 35 C in an anaerobic glove box with an atmosphere of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide resulted in good growth and easily interpretable results. After 48 h of incubation, 97% of strains tested were inhibited by 3.1 μg or less of clindamycin per ml and 98% were inhibited by 12.5 μg or less of chloramphenicol per ml. Tetracycline and minocycline inhibited 81 and 88% of strains tested in concentrations of 1.6 μg or less per ml and 1.6 μg or less per ml, respectively. Ampicillin inhibited all strains other than B. fragilis in concentrations of 3.1 μg or less per ml. Excluding certain strains of Bacteroides and Clostridium , carbenicillin in concentrations of 12.5 μg or less per ml and cephalothin in concentrations of 6.2 μg or less per ml inhibited all strains tested. Gentamicin was inactive although some strains of anaerobic cocci and Bacteroides were inhibited by 3.1 μg or less per ml. After 18 to 24 h of incubation, eight of the 101 strains had not grown sufficiently for MICs to be determined; for the 93 strains which had grown sufficiently, 93% of 744 MICs were the same or one concentration lower than the 48-h MICs.