In Vitro Activity of Metronidazole against Entamoeba Gingivalis

Farid Eloufir; Saber Khelaifia; Gérard Aboudharam; Miche Drancourt

doi:10.4172/2332-0877.1000170

ISSN: 2332-0877

Journal of Infectious Diseases & Therapy

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In Vitro Activity of Metronidazole against Entamoeba Gingivalis

Farid Eloufir^#, Saber Khelaifia^#, Gérard Aboudharam and Michel Drancourt^*
Aix Marseille University, URMITE, Marseille, France
^#These authors equally contributed.
Corresponding Author :	Michel Drancourt Faculty of Medicine Research Unit of Infectious and Tropical Diseases Emerging 27, Boulevard Jean Moulin, Marseille cedex 5- France Tel: 33 4 91 38 55 17 Fax: 33 4 91 38 77 72 E-mail: michel.drancourt@univ-amu.fr
Received August 11, 2014; Accepted September 25, 2014; Published September 30, 2014
Citation: Eloufir F, Khelaifia S, Aboudharam G, Drancourt M (2014) In Vitro Activity of Metronidazole against Entamoeba Gingivalis. J Infect Dis Ther 2:170. doi:10.4172/2332-0877.1000170
Copyright: © 2014 Eloufir F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Entamoeba gingivalis has been the first ever discovered amoeba in humans in the dental plaque [1]. Its role in the periodontal disease remains controversial, but studies found a significant association between E. gingivalis and the severity of periodontal disease [1,2]. One clinical study showed a decreased frequency of E. gingivalis in patients with periodontal disease receiving metronidazole, compared to untreated patients [1]. However, the in vitro activity of metronidazole against E. gingivalis is unknown.

Therefore, we investigated the in vitro activity of metronidazole against E. gingivalis ATCC 30927 type strain (American Type Culture Collection, Rockville, USA). Amoeba were cultured in modified liquid medium Peptone Yeast Glucose (PYG) (Gibco®, Saint Aubin, France) incubated at 37°C in Hungate tubes (Dutscher, Issy-les-Moulineaux, France) under 2-bar of N₂/CO₂ (80%-20%). The activity of metronidazole (B. Braun Medical SAS, Boulogne, France) was ensured by testing the reference Methanobrevibacter smithii strain ATCC 35061T DSMZ 861 as previously described [3]. In vitro susceptibility test was carried out by inoculating 105 cells of E. gingivalis into 8 mL of fresh medium containing 0.5, 1, 2, 4, 8 or 40 mg/L of metronidazole. Non-inoculated negative controls tubes contained identical concentration of metronidazole, and three inoculated tubes without metronidazole were used as positive controls. All tubes were then incubated at 37°C with agitation. E. gingivalis cells were counted after 3-day incubation, every day for 10 days, by using 32x82.5 mm slides (Hycor Biomedical, Penicuik, United Kingdom) and observed by using an optical microscope at X10 and X40 magnifications. The minimal inhibitory concentration (MIC) was defined as the lowest metronidazole concentration inhibiting growth of E. gingivalis. Cell viability was assessed after metronidazole treatment by centrifuging cells at 5000Xg for 20 minutes, washing the cell pellet three times with medium, sub-culturing in metronidazole-free medium, and observing cells under the microscope as above stated. Experiments were done in triplicate.

The activity of the metronidazole suspension used here, was confirmed by observing growth inhibition of M. smithii in agreement with previously reported observations [3]. While negative control tubes remained sterile, positive control tubes containing an initial 104 E. gingivalis cells/mL suspension yielded an exponential growth kinetics over 8 days to reach a stationary phase after 8-day incubation (Figure 1). In this system, the doubling time was of two days (Figure 1). The MIC of metronidazole was determined as 4 mg/L as metronidazole concentrations of 0.5, 1 and 2 mg/L did not inhibit the growth of E. gingivalis (Figure 1) contrary to concentrations of 4 mg/L, 8 mg/L and 40 mg/L. Subculturing E. gingivalis exposed for 10 days in to 4 mg/L metronidazole, failed.

Here, validating data by appropriate controls, we tested the in vitro susceptibility of E. gingivalis to metronidazole, a molecule previously demonstrated to be active against anaerobic bacteria and parasites [4]. We observed that growth of E. gingivalis was inhibited by metronidazole at concentration ≥ 4 mg/L. More probably, metronidazole was killing E. gingivalis cells at concentration as low as 4 mg/L, as revealed by the absence of growth after subculturing. These data therefore support the in vitro effectiveness of metronidazole against E. gingivalis. It is not possible to directly extrapolate these in vitro data, to treatment of E. gingivalis infections. However, several studies showed that oral metronidazole 750-1.500 mg, yielded plasma, saliva and gingival fluid concentration of 8.7-15 ± 7.4 mg/L [5]. Moreover, a unique clinical study found a decreased frequency from 64% to 26% of E. gingivalis in periodontal disease after oral metronidazole, 750 mg a day for 7 days [1]. In this study, the number of E. gingivalis trophozoites was also significantly reduced from 15.9 ± 4.48 to 6 ± 1.5 per microscopic field, after metronidazole treatment [1].

Combining these clinical data with our in vitro susceptibility data indicates that a standard regimen of oral metronidazole would yield an effective local concentration of metronidazole against E. gingivalis cells in periodontal lesions. Therefore, these cumulative data support the use of metronidazole and derivatives in the treatment of periodontal disease.

Funding

This study was funded by URMITE, Marseille, France.