Article

Development of a EUCAST disk diffusion method for the susceptibility testing of rapidly growing anaerobic bacteria using Fastidious Anaerobe Agar (FAA) - Development study using Bacteroides species

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Abstract

Objectives Antimicrobial resistance among anaerobic bacteria is increasing, leading to a growing demand for inexpensive and reliable susceptibility testing methods. The aim of this study was to determine the suitability of Fastidious Anaerobe Agar (FAA) as a medium for disk diffusion of rapidly growing anaerobic bacteria. Methods Reproducibility of zone diameters and quality of growth were tested using six quality control (QC) strains. We compared four anaerobic incubation systems, two incubation temperatures (35 and 37°C) and FAA from four manufacturers. The effect of incubation for 16-20 hours instead of 24 hours was tested on 10 randomly selected Bacteroides fragilis-group isolates. The final method was tested on 170 clinical B. fragilis-group isolates- and compared to agar dilution MICs. Results After 24 hours incubation all QC strains demonstrated confluent growth. The different anaerobic incubation systems were equal regarding quality of growth and zone diameters. Incubation at 35°C resulted in slightly larger zones (1-2 mm) compared to 37°C. Except for Acumedia FAA, the different manufacturers showed good agreement in zone diameters. All B. fragilis-group isolates displayed confluent growth after 16-20 hours. Metronidazole inhibition zones correlated well with the reference MICs. There was an area of poorer separation for meropenem and piperacillin-tazobactam between 19-27 and 14-23 mm respectively. Prolonged incubation (40-44 h) of clindamycin resulted in better separation and the area of overlap was reduced from 13 to 8 mm compared with 16-20 hours incubation. Conclusion FAA is a suitable medium for disk diffusion of this collection of rapidly growing anaerobic bacteria.

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... distasonis isolates in Poland (Kierzkowska et al. 2018). Considering the increasing antimicrobial resistance among anaerobic bacteria, Fastidious Anaerobe Agar is a suitable medium for disk diffusion of these rapidly growing anaerobic bacteria (Bavelaar et al. 2021), which may be considered in the study of antimicrobial resistance of Parabacteroides species. Taken together, Pa. ...
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We compared the susceptibility test results of 220 anaerobes against 14 antimicrobials using the Etest (AB Biodisk, Solna, Sweden) with those using the National Committee for Clinical Laboratory Standards (NCCLS) standard agar dilution method (Wadsworth version). The Etest medium was brucella blood (whole) agar and the inoculum size was equivalent to a no. 1 McFarland standard. Thirty-six percent of Etest results were unreadable after 24 h of anaerobic incubation compared to only 5% after 48 h. Also, there were more results with categorical agreement with the NCCLS method after 48 h (97.9%) than at 24 h (89%) and more very major errors (VMEs) at 24 h (22% of resistant organisms) than at 48 h (3.2%). VMEs and major errors occurred most frequently with clindamycin, ceftriaxone, and trospectomycin (which should not be used with the Etest) and involved the Bacteroides fragilis group and/or Clostridium most commonly. The Etest is simple to perform and is a generally reliable method that is optimally read after 48 h of incubation. It should be an acceptable alternative to the agar dilution standard, although results with certain organism-antimicrobial combinations should be read very conservatively because of the frequency of VMEs.
Article
Clin Microbiol Infect 2011; 17: 371–379 The susceptibilities of 824 Bacteroides fragilis group isolates against nine antibiotics were evaluated in a Europe-wide study involving 13 countries. Species determination, by different methods, was carried out on all but one isolate. Resistance rates were evaluated according to species and geographical areas via CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The present data were compared with those obtained 10 and 20 years ago at a European level. High-level resistance (MIC ≥64 mg/L) to ampicillin was observed in 44.5% of the strains, which is a significant increase relative to 20 years ago (16%). Piperacillin/tazobactam was more active than amoxicillin/clavulanic acid (3.1% and 10.4% resistance, respectively), again with a resistance increase relative to earlier studies. Dramatic increases in resistance were observed for cefoxitin, clindamycin and moxifloxacin, with rates of 17.2%, 32.4% and 13.6%, respectively. The lowest resistances were found for imipenem, metronidazole and tigecycline (1.2%, <1% and 1.7%). Nonsusceptible strains to imipenem and metronidazole were more resistant to other anti-anaerobic drugs. Differences were detected between geographical areas, with higher resistance rates for moxifloxacin in Scandinavian countries (21.4%) than in Mediterranean countries (5.4%), whereas, for clindamycin, the resistance rates were higher in Mediterranean (41.8%) and lower in Scandinavian countries (22.5%). Piperacillin/tazobactam resistance was also higher in Scandinavian countries.
Article
The susceptibility of 90 strains of Bacteroides fragilis to 16 antibacterial drugs was determined by the agar plate dilution test and the disc diffusion test. Good correlation of results was obtained with the two methods, and regression line analysis could be performed for beta-lactam antibiotics, erythromycin, tetracyclines, lincomycin and clindamycin. Thus the standardized disc diffusion method can be used for susceptibility testing, but only a clear distinction between sensitive and resistant strains can be seen.
Article
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Article
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Article
Fastidious anaerobe agar supported the growth of 82 strains of fusobacteria better than brain heart infusion agar, brucella agar, and Wilkins-Chalgren agar. Fastidious anaerobe agar showed less hazing and fewer tailing endpoints with beta-lactam antibiotics. Whole-blood supplementation improved the performance of all media. Wilkins-Chalgren agar without blood failed to support the growth of 17% of the strains. All Fusobacterium ulcerans strains were resistant to clindamycin.
Article
A multilaboratory collaborative study was undertaken to determine whether the anaerobic disk diffusion test of Horn et al. could be performed reproducibly and accurately. Tests with nine different antimicrobial disks were evaluated. Reproducibility of the agar diffusion disk method was similar to that of the reference agar dilution test procedure. The anaerobic disk diffusion procedure was found to be a potentially useful method for testing some antimicrobial agents against rapidly growinganaerobes belonging to the Bacteroides fragilis group. These promising results warrant further investigations and validations.
Article
The susceptibilities of 225 isolates of the Bacteroides fragilis group to six antibiotics were determined by a new disk diffusion test in Wilkins-Chalgren agar and by the standard agar dilution method. For disk diffusion, the bacteria were directly suspended in saline and immediately swabbed onto 15-cm agar plates. Disks of cefoxitin (30 micrograms), chloramphenicol (30 micrograms), clindamycin (2 micrograms), moxalactam (30 micrograms), imipenem (10 micrograms), and ticarcillin (75 micrograms) were applied, and the plates were incubated at 37 degrees C in an anaerobic atmosphere. Zone sizes were measured at 24 h. The results of disk diffusion and agar dilution were compared by regression analysis by the method of least squares and by the error rate-bounded method. Zones were easily measured for 216 strains (96%). The correlation between the MICs and diameters of inhibition for cefoxitin, clindamycin, moxalactam, and ticarcillin was generally good. A correlation could not be established for chloramphenicol and imipenem, as there were too few resistant strains. With the recommended resistance breakpoints, the following susceptible and resistant zone diameter breakpoints could be established: cefoxitin, less than or equal to 19 and greater than or equal to 21 mm; clindamycin, less than or equal to 14 and greater than or equal to 18 mm; moxalactam, less than or equal to 21 and greater than or equal to 25 mm; and ticarcillin, less than or equal to 15 and greater than or equal to 16 mm. By applying these zone criteria, the percentage of false-susceptible strains was less than 1% and of false-resistant strains was less than 4% for the drugs tested.
Article
A method was developed for determination of the antibiotic susceptibility of anaerobic bacteria by use of a single-disc diffusion technique and incorporation of the inoculum in pour plates. The method was standardized by correlation of zone diameters with minimal inhibitory concentrations determined in broth. Zone diameters could be used to approximate the minimal inhibitory concentrations of the seven antibiotics tested: ampicillin, bacitracin, carbenicillin, cephalothin, clindamycin, penicillin, and tetracycline.
Methods for antimicrobial susceptibility testing of anaerobic bacteria
  • Carpenter
Disk diffusion susceptibility testing of the 283
  • R Horn
  • A M Bourgault
  • F Lamothe
Horn R, Bourgault AM, Lamothe F. Disk diffusion susceptibility testing of the 283
Antimicrobial susceptibility of Bacteroides fragilis group isolates 320 in Europe: 20 years of experience
  • Anaerobic Bacteria
Anaerobic Bacteria. Antimicrobial susceptibility of Bacteroides fragilis group isolates 320 in Europe: 20 years of experience. Clin Microbiol Infect. 2011;17(3):371-379.