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Antimicrobial Agents and Chemotherapy, May 2002, p. 1218-1225, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1218-1225.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to Membrane-Active Peptides and Enrofloxacin in Human Tissue Cell Cultures

Ran Nir-Paz,^1, Marie-Christine Prévost,¹ Pierre Nicolas,² Alain Blanchard,³ and Henri Wróblewski^4*

Unité d'Oncologie Virale, Institut Pasteur,¹ Laboratoire de Bioactivation des Peptides, UMR CNRS 7592, Institut Jacques Monod, Université de Paris 7, Paris,² and Institut de Biologie Moléculaire Végétale, INRA, Villenave d'Ornon,³ UMR CNRS 6026, Université de Rennes I, Campus de Beaulieu, Rennes, France⁴

Received 2 October 2001/ Returned for modification 6 November 2001/ Accepted 21 December 2001

Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 µM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 µM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 µM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 µM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.

Present address: Division of Internal Medicine, Hadassah University Hospital, Ein Karem, Jerusalem, Israel.

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