Evaluation of methods for distinguishing gram-positive from gram-negative anaerobic bacteria

Several species of anaerobic bacteria display variable Gram stain reactions which often make identification difficult. We have evaluated the Gram stain, the potassium hydroxide test (3% KOH), an antibiotic susceptibility test (10 mg/L vancomycin), and the L-alanine aminopeptidase test (utilising fluorogenic L-alanine-4-methoxy-beta-naphthylamide [LAMNA] and chromogenic L-alanine-4-nitroanilide [LANA] substrates) for the preliminary grouping of Gram-positive and Gram-negative anaerobes. By testing 88 clinical isolates, 35 Gram-positive and 53 Gram-negative, we obtained the following results: sensitivity, 49, 94, 21 and 23%; specificity, 100, 98, 91 and 91%; positive predictive value, 100, 97, 79 and 80%; negative predictive value, 56, 96, 43 and 44%, for the KOH test, the vancomycin susceptibility test, L-alanine-4-methoxy-beta-naphthylamide test and L-alanine-4-nitroanilide test, respectively. The KOH test correctly grouped all the Gram-positive strains but incorrectly grouped 27 of 53 Gram-negative strains (e.g. Prevotella spp., Fusobacterium spp., Veillonella spp, and Leptotrichia spp.). The vancomycin test correctly identified 33 of 35 Gram-positive strains (resistance with Clostridium innocuum and Actinomyces odontolyticus) and 52 of 53 Gram-negative strains (susceptible with Prevotella melaninogenica). Both LAMNA and LANA tests correctly grouped 32 of 35 Gram-positive strains but the LAMNA test incorrectly grouped 42 of 53 Gram-negative strains whilst the LANA test incorrectly grouped 41. From the methods examined we conclude that the vancomycin susceptibility test is the most useful for the preliminary classification of anaerobes in the clinical laboratory. (C) 1996 Academic Press