Evaluation of the Etest for determining the in-vitro susceptibilities of Prevotella intermedia isolates to metronidazole

J Antimicrob Chemother 1999; 43: 610–611

Pierre P. Poulet*, Danièle Duffaut and Jean P. Lodter

Department of Oral Biology, School of Dental Medicine, Paul Sabatier University, Toulouse, France

Sir,

The bacterial composition of the subgingival flora varies widely from individual to individual and from pocket to pocket in the same individual. Certain pathogens, particularly Prevotella intermedia, are associated with severe forms of periodontal disease. 1 Not all patients respond equally to periodontal treatment and antibiotics can enhance the effects of surgical intervention in those with rapidly progressive or refractory periodontitis. 2 One of the most frequently prescribed antibiotics is metronidazole, resistance to which is rarelyencountered, although treatment with high dosages may be necessary to eradicate somepathogens. In common with the causes of infection at other sites, periodic surveys of thesusceptibilities of the aetiological agents of periodontitis are necessary in order to detectchanges in the patterns of resistance and thereby to facilitate optimal antibiotic therapy. Thepresent study was undertaken to determine the in-vitro susceptibilities of P. intermedia isolates to metronidazole and to evaluate the efficacy of the Etest for this purpose.

Subgingival plaque samples were obtained from patients with periodontitis and inoculatedon to solid medium within 3 h; the plates were incubated under anaerobic conditions at 37°C for 3–7 days. Isolates of P. intermedia (n = 13) were identified according to standard laboratory procedures. Susceptibility to metronidazole wasdetermined by an agar dilution method recommended by the NCCLS 3 and by the Etest method (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions. The medium in both cases was Columbia agar (bioMérieux, Marcy l'Etoile, France) supplemented with 10% sheep blood, haemin (5 mg/L) and menadione (0.5mg/L), and the inoculum for the agar dilution method was 10 5 cfu. P. intermedia isolates CIP 103607 and CIP 6322 (Collection of the Pasteur Institute, Paris) were used ascontrols and all plates were incubated for 48 h at 37°C in an anaerobic chamber.

The mean MIC for the 13 isolates, as determined by the reference agar dilution method, was0.98 mg/L (range 0.25–2 mg/L), whereas the mean MIC determined by the Etest was 0.82mg/L (range 0.047–2 mg/L). In 33% of cases, the MICs determined by the two methods were the same. Of the remaining isolates, the MICs for 87% and 93% were within one and two two-fold dilutions, respectively. For only one strain was the difference between the MICs determined by the two methods more than four two-fold dilutions (MIC of 0.047 mg/L with the Etest and 0.25 mg/L with the agar dilution method).

We have shown in the present study that the MICs of metronidazole for clinical isolates of P. intermedia are broadly similar, regardless of whether they are determined by the Etest or the agar dilution method; 93% of results differed by no more than two two-fold dilutions. Others have reported similar results, i.e. concordance or a difference of only one or two two-fold dilutions in34.3%, 74% and 90% of strains, respectively. 4 We conclude that the Etest is a simple, rapid and reliable method of determining the MICs ofmetronidazole for P. intermedia isolates. However, its relatively high cost will preclude it from being used as a routinesusceptibility testing method.

The MIC 100 of metronidazole for the 13 isolates (2 mg/L), based on MICs determined by the agar dilution method, is in agreement with the susceptibility data reported by other investigators 5,6 and is less than the MIC breakpoint (8 mg/L) recommended by the NCCLS. 3 The highest MIC recorded by us, 2 mg/L, was one two-fold dilution higher than that reported by Sutter et al. 6 However, those investigators included isolates other than P. intermedia and did not distinguish one black pigmented species from another.

Although only a small number of isolates were evaluated, the present study demonstrates theexcellent in-vitro activity of metronidazole against P. intermedia, with 100% of the isolates tested being susceptible to this agent on the basis of the recommended MIC breakpoint. This observation is in accord with multicentre studies in the USA which determined the susceptibilities of much larger numbers of isolates, and confirms that the isolationof a strain belonging to this species that exhibits resistance to metronidazole is a very rare event.

Acknowledgments

This study was supported by Rhône–Poulenc Rorer, France. We thank Professor Giorgio Cimasoni, Dental School, University of Geneva, Switzerland, for his valuable criticism of the manuscript, Professor Henry Dabernat, Microbiology Laboratory, Purpan Hospital, Toulouse, France, and N. Mallet for technical assistance.

Notes

* Correspondence address. Buccal Biology Laboratory, Faculty of Dentistry, 3 chemin desMaraîchers, 31063 Toulouse cedex, France. Tel: +33-5-62-17-36-08; Fax: +33-5-61-06-51-13. Back

References

1 . van der Weijden, G. A., Timmerman, M. F., Reijerse, E., Wolffe, G. N., van Winkelhoff, A.J. & van der Velden, U. (1994). The prevalence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in selected subjects with periodontitis. Journal of Clinical Periodontology 21, 583–8.[ISI][Medline]

2 . Loesch, W. J., Schmidt, E., Smith, B. A., Morrison, E. C., Caffesse, R. & Hujoel, P. (1991).Metronidazole in periodontitis II: effects upon treatment needs. Journal of Periodontology 62, 247–57.[ISI][Medline]

3 . National Committee for Clinical Laboratory Standards. (1993). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Third Edition: Approved Standard M11-A3. NCCLS, Villanova, PA.

4 . Citron, D. M., Ostovari, M. I., Karlsson, A. & Goldstein, E. J. C. (1991). Evaluation of theEtest for susceptibility testing of anaerobic bacteria. Journal of Clinical Microbiology 29, 2197–203.[ISI][Medline]

5 . Abu Fanas, S. H., Drucker, D. B., Hull, P. S., Reeder, J. C. & Ganguli, L. A. (1991).Identification, and susceptibility to seven antimicrobial agents, of 61 Gram-negative anaerobicrods from periodontal pockets. Journal of Dentistry 19, 46–50.[ISI][Medline]

6 . Sutter, V. L., Jones, M. J. & Ghoneim, A. T. M. (1983). Antimicrobial susceptibilities of bacteria associated with periodontal disease. Antimicrobial Agents and Chemotherapy 23, 483–6.[ISI][Medline]





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