Gentamicin diffusion in Mueller–Hinton agar plates from different manufacturers

B. Comuzzia, C. Arcellonia,*, R. Vaianib and R. Paronia

a Laboratory of Separative Techniques and b Microbiology Service, Department of Laboratory Medicine, Scientific Institute H. S. Raffaele and School of Medicine, Via Olgettina 60, 20132 Milan, Italy

Sir,

The diameter of inhibition zones in agar diffusion susceptibility test is the result of several variables related to the antibiotic–bacterium pair, standard method and composition of the medium.1 The evaluation of different lots of Mueller–Hinton agar (MHA) and the development and application of reference media in the agar diffusion test has been taken into consideration by the NCCLS2 and European microbiological societies.3

The quality controls made by the media manufacturers are based on physical tests of agar matrix composition and on bioassays using organisms with known characteristics: the produced media must be in the range of the NCCLS quality controls for the ATCC strains and antibiotics. The disc diffusion test is reliable for judging the performance of agar media, as it is the resultant of many variables that interact with each other, and is simple and well tried. However, we think that to evaluate the performance of different media, the interactions between antibiotics and agar composition should be studied also in the absence of bacterial growth.

After developing a chromatographic method,4 we present a comparison of the diffusion kinetics of gentamicin in MHA from different suppliers by high performance liquid chromatography (HPLC) to highlight the need for a reference medium that would allow manufacturers to compare each new lot of MHA and test the reproducibility of the performances.

Four agar samples (3 µL) were harvested with calibrated glass capillaries (1 mm internal diameter, Coulter Electronics, UK) and placed in an Eppendorf tube with acetonitrile (100 µL), tris(hydroxymethyl) aminomethane (Tris) (165 mM, 50 µL) and 1-fluoro-2,4-dinitrobenzene (FDNB) (269 mM in acetonitrile, 20 µL). After heating (80°C for 15 min), 150 µL were injected into the HPLC column (C18, Beckman, CA, USA) and eluted with 8.25 mM Tris (pH 7.0):acetonitrile (25/75, v/v). The performance standards of the method were: (i) linearity between 1 and 100 mg/L; (ii) sensitivity in agar matrix of 1 mg/L; and (iii) recovery from agar of 80–85% compared with aqueous standards.4

Standard gentamicin (Schering Plough; Milan, Italy) had a potency of 684 µg/mg. For the antibiotic diffusion, gentamicin sensi-discs (10 µg; Becton Dickinson, Cockeysville, MD, USA) of the same lot (no. B1006-708545) were used. Plates of MHA (90 mm diameter, 4 mm agar depth) from Becton Dickinson (lot no. E83AA), Oxoid (Basingstoke, Hampshire, UK; lot no. B3374), bioMérieux (Marcy l’Étoile, France; lot no. 719867401) and from a local company (DID, Milan, Italy; lot no. 0602706A) were tested. Sampling was performed after 2, 4, 6, 15 and 24 h of incubation at 35 ± 0.5°C, and at 3 (edge), 6 and 9 mm from the centre of the disc. The kinetics were performed in triplicate. Differences between samples were evaluated using Student's t-test: a P value <=0.05 was considered significant.

The chromatographic method was able to separate all five peaks belonging to gentamicin sulphate derivatives (C1, C1a, C2, C2a and C2b).4 For each component, a good correlation was obtained evaluating the linearity between the peak area and the gentamicin concentration in the 1–100 mg/L range. The ratio between the peak areas of the five components was maintained in the 2–24 h time interval.

The composition of the gentamicin used for the preparation of discs and that of the standard powder were compared by putting a disc (10 µg gentamicin) in water (0.5 mL) and by injecting a 100 µL aliquot into the HPLC column after 30 min. The chromatographic profile and the distribution (%) of the gentamicin components were similar.

The profiles of the kinetics obtained with plates from different manufacturers are shown in the Figure.Go At 3 mm (a), the kinetics showed very different gentamicin concentrations at each time point, although the trends were similar. At 6 mm (b), after 4 h the antibiotic concentrations were all significantly different (from 1.36 ± 0.36 mg/L in the Becton Dickinson plates to 10.79 ± 1.52 mg/L in the bioMérieux plates). At 9 mm (c), after 15 h the differences between the four agar matrices were more evident and remained so after 24 h (P <= 0.05).



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Figure. Gentamicin kinetics of diffusion in uninoculated plates, sampling at 3 mm (a), 6 mm (b) and 9 mm (c) from the disc centre. At each distance tested, the samples marked with an asterisk differ significantly from the others at the same time point (P <= 0.05). (•) Becton Dickinson, ({circ}) DID, ({blacksquare}) Oxoid, ({square}) bioMérieux.

 
The most important finding of this study is that after 24 h and at 9 mm from the disc centre, which is the distance close to the diameter of the inhibition zone for Pseudomonas aeruginosa ATCC 27853 and gentamicin (16–21 mm, from the NCCLS), the concentrations found in agar from the four manufacturers were significantly different.

We can expect, as already observed,4 that although the four matrices produce different inhibition zones, the concentration of gentamicin remains constant at the edge of the zone. The concentration by HPLC, however, does not correspond to the MIC (the amount required to inhibit bacterial growth) but to the ‘residual’ amount of antibiotic in agar.

Recently, it was noticed that the performance of Isosensitest agar is thought to have changed over time with consequences for the susceptibility tests for aminoglycosides against P. aeruginosa.5 The findings of this paper reinforce the need for improved standards of media performance and illustrate the usefulness of analytical tests coupled to the microbiological ones in attaining a complete overview of the characteristics of the media.

The HPLC technique may be useful for manufacturers and control agencies as an additional parameter to define better the control limits and improve the quality of media for sensitivity tests. By introducing the appropriate modifications, the protocol presented here could be used to investigate many other antibiotic families such as the quinolones and/or daptomycin.6

Notes

J Antimicrob Chemother 2001; 47: 496–498

* Correspondence address. Laboratory of Separative Techniques, IRCCS H. S. Raffaele Milan, Italy. Tel: +39-02-2643-2714; Fax: +39-02-2643-2640; E-mail: arcelloni.cinzia{at}hsr.it Back

References

1 . Barry, A. L. & Effinger, L. J. (1974). Performances of Mueller– Hinton agars prepared by three manufacturers. American Journal of Clinical Pathology 62, 113–7.[ISI][Medline]

2 . National Committee for Clinical Laboratory Standards. (1996). Protocols for Evaluating Dehydrated Mueller–Hinton Agar: Approved standard M6-A. NCCLS, Villanova, PA.

3 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

4 . Comuzzi, B., Arcelloni, C., Vaiani, R. & Paroni, R. (2001). Quantification of gentamicin in Mueller–Hinton agar by high-performance liquid chromatography. Journal of Chromatography B: Biomedical Science and Applications 753, 151–6.

5 . Andrews, J. M. (2000). Effect of medium composition on the MIC breakpoint for gentamicin. Journal of Antimicrobial Chemotherapy 46, 851–2.[Free Full Text]

6 . Steward, C. D., Stocker, S. A., Swenson, J. M., O'Hara, C. M., Edwards, J. R., Gaynes, R. P. et al. (1999). Comparison of agar dilution, disc diffusion, MicroScan and Vitek antimicrobial susceptibility testing methods to broth microdilution for detection of fluoroquinolone-resistant isolates of the family Enterobacteriaceae. Journal of Clinical Microbiology 37, 544–7.[Abstract/Free Full Text]





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