Susceptibility testing of fastidious organisms.

Sir, The NCCLS has published guidelines for the susceptibility testing of fastidious organisms. Following these guidelines for the assessment of a new trinem, sanfetrinem (GV 104326), against Neisseria spp. we obtained unexpectedly high MICs and therefore undertook a study to compare MICs obtained on a variety of media. The media compared were as follows: medium A, IsoSensitest agar (Unipath, Basingstoke, UK) supplemented with 20 mg/L NAD (Sigma, Poole, UK) and 5% whole horse blood (E and O, Bonnybridge, UK); medium B, Mueller–Hinton agar (Unipath) supplemented with 20 mg/L NAD and 5% whole horse blood; medium C, Mueller–Hinton agar supplemented with 20 mg/L NAD and 2% (w/v) haemoglobin (Unipath); medium D, GC agar base (Unipath) supplemented according to NCCLS recommendations excluding L-cysteine; medium E, GC agar base supplemented with 2% (w/v) haemoglobin and Vitox (Unipath); medium F, GC agar base supplemented according to NCCLS recommendations including Lcysteine; medium G, Mueller–Hinton agar supplemented with 5% whole horse blood and Vitox. The antibiotics studied were sanfetrinem (GlaxoWellcome, Greenford, UK), spectinomycin (Upjohn, Crawley, UK), meropenem (Zeneca, Macclesfield, UK), ciprofloxacin (Bayer AG, Wuppertal, Germany), cefixime (Cyanamid, Gosport, UK) and benzylpenicillin (SmithKline Beecham, Welwyn Garden City, UK). The organisms investigated were ten clinical strains of Neisseria gonorrhoeae and control strain NCTC 12700 (ATCC 49226) and five clinical strains of Neisseria meningitidis. The methodology was that described in the NCCLS guidelines and in the Working Party Report of the BSAC. Briefly, the final inoculum was 10 cfu/spot, incu bation was at 35–37°C in 4–6% CO 2 in air for 20–24 h and the MIC was defined as the lowest concentration of antibiotic inhibiting growth, up to three colonies being ignored. In the Figure are plotted the mode MICs for the 11 N. gonorrhoeae strains tested for each of the media investigated. The graph clearly illustrates a variation in results obtained depending on the medium used. The NCCLS recommends that a medium free of L-cysteine should be used when testing clavulanate and carbapenem antibiotics and the results were in agreement with this recommendation. MICs obtained were significantly raised with meropenem and sanfetrinem and to a lesser extent with benzylpenicillin when using media E , F and G (all containing cysteine). When sanfetrinem MIC data for media with and without the addition of L-cysteine were compared statistically (paired t-test) a P value of 0.0001 was obtained, indicat ing that there was a significant statistical difference. When medium D (NCCLS reference medium) and medium A (BSAC Working Party recommendation) were compared there was no statistical difference in results (P 0.4294). A similar pattern of results was obtained for N. meningitidis. It is also worthy of note that all strains grew on medium A, whereas one clinical isolate of N. gonorrhoeae failed to grow on the NCCLS recommended media (F and G). These data suggest that Iso-Sensitest agar supplemented with 20 mg/L NAD and 5% whole horse blood would be appropriate for sensitivity testing compounds affected by the presence of L-cysteine. Previous data published from this department have also shown that this medium is suitable for testing other fastidious organisms such as Streptococcus pneumoniae and Haemophilus influenzae. The results from a recent BSAC questionnaire have shown that there is a general consensus that there is a need to standardize the testing of fastidious organisms. These data suggest that supplemented Iso-Sensitest agar, the medium chosen by the BSAC Working Party on antibiotic sensitivity testing, would be appropriate and that MIC results obtained with this medium would be comparable to those obtained on the NCCLS recommended media.