Antianaerobic antimicrobials: spectrum and susceptibility testing

Abstract

Susceptibility testing of anaerobic bacteria recovered from selected cases can influence the choice of antimicrobial therapy. The Clinical and Laboratory Standards Institute (CLSI) has standardized many laboratory procedures, including anaerobic susceptibility testing (AST), and has published documents for AST. The standardization of testing methods by the CLSI allows comparisons of resistance trends among various laboratories. Susceptibility testing should be performed on organisms recovered from sterile body sites, those that are isolated in pure culture, or those that are clinically important and have variable or unique susceptibility patterns. Organisms that should be considered for individual isolate testing include highly virulent pathogens for which susceptibility cannot be predicted, such as Bacteroides, Prevotella, Fusobacterium, and Clostridium spp.; Bilophila wadsworthia; and Sutterella wadsworthensis. This review describes the current methods for AST in research and reference laboratories. These methods include the use of agar dilution, broth microdilution, Etest, and the spiral gradient endpoint system. The antimicrobials potentially effective against anaerobic bacteria include beta-lactams, combinations of beta-lactams and beta-lactamase inhibitors, metronidazole, chloramphenicol, clindamycin, macrolides, tetracyclines, and fluoroquinolones. The spectrum of efficacy, antimicrobial resistance mechanisms, and resistance patterns against these agents are described.

Fig 1

Agar dilution technique. (Left) A Steers replicator is used to apply inocula onto a agar plate. (Right) Series of agar dilution plates. Each spot represents a different strain. The arrow indicates a spot of dye, which is generally added to orient the plate. The plate for which the growth is no longer present should be considered the MIC.

Fig 2

Broth microdilution. (Left) Sensititre pipette for filling microdilution plate. (Middle) Plate being inoculated with strains. (Right) Plate after growth of strains. The MIC is read as the lowest dilution of antimicrobial resulting in no growth. (Left and right panels courtesy of Trek Diagnostics Systems, Inc., reproduced with permission.)

Fig 3

Etest (AB Biodisk). (Courtesy of bioMérieux, reproduced with permission.)

Fig 4

Spiral gradient endpoint technique. (Left) Dye representing the gradient application of antimicrobial stock solution, decreasing from the center of the plate. (Middle) Growth of bacterial strains inoculated in a radial manner onto a plate. The radius from the center of the plate to end of growth is measured and translated into an MIC by a software program. (Right) Detail of the endpoint and observation of resistant colonies past the endpoint.

Fig 5

Multiplex PCR assay to detect common resistance determinants in B. fragilis. Amplification was done with a set of primers designed for detecting five resistance genes, including carbapenems (cfiA), cephalosporins (cepA), clindamycin (ermF), metronidazole (nimA-F), and tetracycline (tetQ), plus a set of primers for the B. fragilis 16S rRNA gene (positive control). Lane 0, negative control; lane M, DNA standards. Lanes 1 to 11 were either single or multiple B. fragilis clinical isolates with previously determined resistance determinants. The multiplex PCR assay was able to determine all resistance determinants present in either single- or multiple-strain samples.