Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nir-Paz, R. Articles by Wróblewski, H. Search for Related Content PubMed PubMed Citation Articles by Nir-Paz, R. Articles by Wróblewski, H.

 Previous Article  |  Next Article 

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the past decade, bacterial antibiotic resistance has become a major medical issue (16, 26, 41). Infections with bacteria resistant to multiple antibiotics are associated with higher rates of morbidity and mortality (7, 9, 26), specifically in patients suffering from chronic diseases such as cystic fibrosis. Therefore, there is an urgent need for new antibiotics and/or treatment strategies to overcome the greater difficulty in controlling bacterial infections and in reducing hospitalization times and hospitalization-related expenses (7, 9, 26).

Since the most widely used defense system against microorganisms in nature involves membrane-active peptides (17, 25), these molecules might offer exciting opportunities in the face of the declining efficacies of conventional antibiotics in antimicrobial chemotherapy (17, 30). Antimicrobial membrane-active peptides are indeed produced by animals (including humans), plants, fungi, and bacteria; and despite their structural diversities, they display wide spectra of activity. They seem to act primarily on the plasma membranes of target cells (11), but it has recently been shown that some defense peptides may also interact with intracellular macromolecules (see, e.g., references 29 and 43). Although several peptides have been tested in clinical trials, we still need to improve our knowledge of the mechanisms governing their actions and the factors responsible for the spectra of activity of these antibiotics. Furthermore, only a few in vitro and in vivo models are available for evaluation of their activities and efficacies (3, 33). In vitro assays such as those used to determine MICs and minimal bactericidal concentrations (MBCs) do not necessarily reflect the true activities and efficacies of antibiotics against bacteria in the host (18-20). This discrepancy results from various factors such as the ability of certain bacteria to penetrate host cells, the binding of antibiotics to host (lipo)proteins and cells, and the different bacterial concentrations in hosts and cultures.

In order to take these factors into account, we chose an in vitro model that simulates the complexity of the interactions among membrane-active peptides, human cells, culture medium, and infecting bacteria. This experimental system was based upon human HeLa cells cultured in a serum-containing medium and infected with mycoplasmas. Mycoplasmas were chosen because of their ability to cause persistent infections in cell lines without the induction of cell death, thus enabling study of the effects of peptides without encountering bacterial overgrowth. All known mycoplasmas (class Mollicutes) are parasites of various animal hosts; and several species cause respiratory, arthritic, and urogenital diseases associated with severe morbidity and mortality in vertebrate animals (31), including humans (36). Mycoplasmoses are most often progressive, chronic diseases difficult to control due to (i) the innate resistance of mycoplasmas to murein synthesis inhibitors since they are devoid of a cell wall (31) and (ii) the development of resistance to other antibiotics through mutations or the acquisition of mobile genetic elements (35). Hence, there is a growing interest in new antibiotics and/or treatment strategies able to protect humans and animals, as well as tissue cell cultures, against mycoplasmas. Mycoplasma hyorhinis and Mycoplasma fermentans were chosen for this study because they are both common contaminants of cell cultures and are potentially pathogenic for swine and humans, respectively (31, 36).

Using the HeLa cell-mycoplasma model system, we have evaluated the antimycoplasma activities of four peptides: (i) dermaseptin B2, a 33-residue linear peptide isolated from the frog Phyllomedusa bicolor (8); (ii) alamethicin, a 20-residue -methylalanine-containing peptide produced by the fungus Trichoderma viride (10, 27); (iii) surfactin, an 8-residue cyclic lipopeptide from Bacillus subtilis (2); and (iv) gramicidin S (or tyrocidin S), a 10-residue peptide which is produced by Bacillus brevis and which, similar to surfactin, is also cyclic but not acylated (22). We also compared the activities of these peptides with that of enrofloxacin, a fluoroquinolone that is used in veterinary practice and that is known to have potent activity against mycoplasmas (23). In addition, we have attempted to develop a method, based upon the use of these peptides, for the eradication of mycoplasmas from human tissue cell cultures.

(This work was done as part of the requirements of the Scientific Council of the Israel Medical Association for completion of the specialty in internal medicine by Ran Nir-Paz.)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptides and enrofloxacin. Dermaseptin B2 (3,182 Da) was synthesized as described previously (8), whereas surfactin (1,036 Da), alamethicin (1,964 Da), and gramicidin S (1,141 Da) were purchased from Sigma (St. Louis, Mo.). Enrofloxacin (359.4 Da; Baytril) was a gift from Bayer AG (Leverkusen, Germany).

Cell line and mycoplasmas. The HeLa cervical carcinoma cell line was cultured in RPMI medium (Sigma) supplemented with 10% fetal calf serum (Gibco BRL, Life Technologies, Paisley, United Kingdom) and 5 mM glutamine (Gibco BRL). Cells were cultured in 25-cm² flasks (Costar, Cambridge, Mass.); antibiotic treatments, however, were performed in six-well plates (Costar). M. hyorhinis (strain PG29^T) and M. fermentans (strain PG18^T) were grown on glucose-enriched PPLO broth, as described previously (14). Chronic infection of HeLa cells was achieved by inoculating 10⁵ HeLa cells with 10⁵ CFU of mycoplasmas. The chronicity of infection was confirmed by PCR and staining with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI), as described below. Both infected and noninfected HeLa cells were harvested with a cell scraper (Costar) and gentle resuspension in growth medium.

Determination of MICs and MBCs. The antimycoplasma activities of the peptides were assayed as described previously (6). For the determination of MICs, mycoplasmas (initial concentration, 10⁶ CFU/ml) were cultured in 96-well plates in the presence of twofold serial dilutions of the different antibiotics (0 to 100 µM). A change in the color of the phenol red indicator due to medium acidification was used as an indicator of bacterial growth. MBCs were determined as described previously (6) by plating 20 µl of mycoplasma cells (2 x 10⁴ CFU) that had been treated for 2 h with enrofloxacin or peptides or cells that were not treated on agar medium for detection of colony formation. The MICs and the MBCs were defined as the lowest concentrations that totally inhibited growth or that killed 99.9% of the mycoplasma cells, respectively.

Mycoplasma detection. Mycoplasmas were detected by staining of DNA with fluorescent dyes and PCR. In the first case, HeLa cells were grown in chamber slides (Nunc, Naperville, Ill.) until they were 70 to 90% confluent. The medium was removed and the cells were fixed with a solution containing 60% ethanol, 30% chloroform, and 10% acetic acid. Thereafter, the cells were incubated with 0.1 µg of DAPI (Boehringer, Mannheim, Germany) per ml (32) for 15 min at room temperature and were observed with a Leitz Laborlux fluorescence microscope (Ernst Leitz, Wetzlar, Germany) with appropriate filters. For the detection of mycoplasmas by PCR amplification of ribosomal DNA, cultured cells were scraped from the culture plate, suspended in culture medium, rinsed with phosphate-buffered saline, and lysed by incubation at 56°C for 2 h in a solution containing 120 µg of proteinase K per ml, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 0.5% Tween 20, and 0.5% Triton X-100. The samples were subsequently incubated for 10 min at 96°C and then stored at -20°C. Primers GPO1 and MGSO were used for the PCR amplification of a region of DNA encoding 16S rRNA, as described previously (38). Briefly, 5 µl of cell lysate was added to 45 µl of the following PCR mixture (final concentrations): 10 µM each primer (primers GPO1 and MGSO), 200 µM (each) deoxynucleoside triphosphate, 1 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Cleveland, Ohio), and 3 mM MgCl₂ in PCR buffer (Amersham Pharmacia Biotech). The thermal profile included an initial denaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C, annealing at 55°C, and elongation at 72°C (1 min each). A final elongation step was performed for 10 min at 72°C. The amplified PCR products were separated by standard 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The cells were considered contaminated with mycoplasmas if a 715-bp specific DNA product was visualized.

Antibiotic treatment of cell cultures. Surfactin and dermaseptin B2 were dissolved in sterile phosphate-buffered saline immediately before use. Surfactin was used at a final concentration of 40.4 µg/ml in medium for two passages of HeLa cells, as described previously (39). Dermaseptin B2 was used at a final concentration of 79.6 µg/ml for one passage of HeLa cells. Alamethicin was dissolved in methanol and was then added to cultured cells at different final concentrations (15 to 100 µM, i.e., 29.5 to 196.4 µg/ml) and different exposure times (0.5 or 24 h). Gramicidin S was dissolved in ethanol and was used at concentrations of 1 to 30 µM (1.1 to 34.2 µg/ml) for 0.5 or 24 h, as indicated below. Methanol and ethanol concentrations never exceeded 1% (by volume). Enrofloxacin was used at a final concentration of 70 µM (25 µg/ml). When enrofloxacin was used alone, treatment was for 8 to 10 days (12), whereas when it was used in combination with peptide antibiotics, the treatment was reduced to 24 h. All antibiotics were added at the desired concentrations to mycoplasma-infected and noninfected tissue cell cultures at about 70% cell confluence. The noninfected treated cells served as a negative control and a treatment toxicity control. All cell lines were tested for mycoplasma infection by the methods described above at least four passages after the end of the treatment to prevent false-negative detection of mycoplasmas (see Fig. 1).

View larger version (41K): [in this window] [in a new window]

FIG. 1. Antibiotic treatment and detection of mycoplasmas. (a) Illustration of the protocol used in this study. Mycoplasmas were sought after at least four passages following experimental infection of the HeLa cells to verify the chronicity of infection. Thereafter, antibiotic treatment was applied and was terminated at the appropriate time by replacement of medium. The cells were then cultivated for at least four passages before they were checked again for the presence of mycoplasmas. (b) DAPI staining of mycoplasma-free cultures; only fluorescent nuclei can be seen. (c) Tissue cell cultures infected with M. hyorhinis: the typical "starry sky" appearance between the cell nuclei is due to the proliferation of mycoplasma cells. (d) PCR analysis of DNA extracted from tissue cell cultures. Lane 1, HeLa cells without experimental mycoplasma infection; lane 2, HeLa cells infected with M. hyorhinis and showing amplification of a 715-bp segment; lane 3, cured HeLa cells after treatment with alamethicin at 25 µM (49.1 µg/ml) for 24 h.

Electron microscopy. Confluent HeLa cells were fixed with 2.5% glutaraldehyde after incubation with the specific antibiotic for 1 h at 37°C. The fixed cells were dehydrated in ethanol and embedded in Epon by standard techniques (28). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a JEOL EX 1200 electron microscope operating at an accelerating voltage of 80 kV.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antimycoplasma activities of the peptides and enrofloxacin. The MICs and MBCs of the peptides are listed in Table 1. Alamethicin, dermaseptin B2, and gramicidin S inhibited the growth of both M. fermentans PG18 and M. hyorhinis PG29, with MICs ranging from 12.5 µM (the MIC of alamethicin for M. hyorhinis) to 50 µM, while surfactin was ineffective even at 100 µM. MBCs were determined in the presence and in the absence of serum. The data reveal that in the absence of serum, alamethicin, dermaseptin B2, and surfactin killed both mycoplasmas (MBC range, 12.5 to 50 µM), whereas dermaseptin B2 killed M. hyorhinis (MBC range, 50 to 100 µM) and was bacteriostatic toward M. fermentans at concentrations 100 µM. The presence of serum (either in the culture medium or in buffer) decreased the activities of the peptides by two- to fourfold, while bovine serum albumin (BSA) did not interfere with the activities of the peptides. In comparison, enrofloxacin inhibited the growth of both mycoplasmas, with the MICs of enrofloxacin being lower than those of the peptides (enrofloxacin MICs, 0.49 µM for M. fermentans and 1.95 µM for M. hyorhinis). The MBCs of this fluoroquinolone were 7.8 and 125 µM, respectively, and were unaffected by the presence of serum or BSA.

View this table: [in this window] [in a new window]

TABLE 1. Antimycoplasma activities of enrofloxacin and membrane-active peptides

Establishment of chronic mycoplasma infection in HeLa cell cultures. Chronic infection of HeLa cells was achieved by infecting the cells with mycoplasmas as described in Materials and Methods. The chronicity of infection was evaluated at least four passages after inoculation, as shown in Fig. 1a. A spontaneous cure of mycoplasma infection did not occur after 30 consecutive passages of experimentally infected cells, and no accidental mycoplasma contamination of noninfected HeLa cells was detected. Examples of representative results obtained by both methods of mycoplasma detection (DAPI staining and PCR) are shown in Fig. 1b to d. In the 150 cell cultures used in this study, there were no discrepancies between the results obtained by DAPI staining and those obtained by PCR methods.

Activities of different peptides on HeLa cell cultures infected with mycoplasmas. The four peptides and enrofloxacin were evaluated for their effects on the infected HeLa cells. The concentrations used for these experiments were, whenever possible, below the cytolytic concentrations for the HeLa cells and above the MICs for the mycoplasmas. In addition to mycoplasma detection by PCR and DAPI staining, the effects of the different peptides on cell morphology and ultrastructure were observed by electron microscopy. Representative photographs are shown in Fig. 2 and 3.

View larger version (149K): [in this window] [in a new window]

FIG. 2. Effects of membrane-active peptides and enrofloxacin on M. fermentans infecting HeLa cells, as shown by electron microscopy. (a) Control consisting of HeLa cells infected with M. fermentans and with no additional treatment. Cells fixed after 1 h of treatment with surfactin at 40 µM (41.4 µg/ml) (b), dermaseptin B2 at 25 µM (79.6 µg/ml) (c), alamethicin at 25 µM (49.1 µg/ml) (d), gramicidin S at 5 µM (5.7 µg/ml) (e), or enrofloxacin at 70 µM (25.1 µg/ml) (f) are shown. Mycoplasma cells displaying a normal morphology are indicated by arrows with solid arrowheads, while swelled or lysed mycoplasma cells are indicated by arrows with open arrowheads.

View larger version (139K): [in this window] [in a new window]

FIG. 3. Effects of membrane-active peptides and enrofloxacin on M. hyorhinis infecting HeLa cells, as shown by electron microscopy. (a) Control consisting of HeLa cells infected with M. hyorhinis with no additional treatment. Cells fixed after 1 h of treatment with surfactin at 40 µM (41.4 µg/ml) (b), dermaseptin B2 at 25 µM (79.6 µg/ml) (c), alamethicin at 25 µM (49.1 µg/ml) (d), gramicidin S at 5 µM (5.7 µg/ml) (e), or enrofloxacin at 70 µM (25.1 µg/ml) (f) are shown. Mycoplasma cells displaying a normal morphology are indicated by arrows with solid arrowheads, while swelled or lysed mycoplasma cells are indicated by arrows with open arrowheads.

In the absence of peptide or enrofloxacin, HeLa cells exhibited irregular contours (Fig. 2a and 3a), and the cells of both M. fermentans (Fig. 2a) and M. hyorhinis (Fig. 3a) were elongated and pleiomorphic, with dense cytoplasms. Treatment of the infected cultures with 40 µM surfactin was apparently harmless to HeLa cells, but it was also harmless to the mycoplasmas (Fig. 2b and 3b). Deformation and lysis of most mycoplasma cells occurred upon treatment with 25 µM dermaseptin B2, but in these conditions, HeLa cells were also altered (Fig. 2c and 3c). More promising results were obtained with alamethicin and gramicidin S. Indeed, 25 µM alamethicin split or lysed M. fermentans cells (Fig. 2d) and swelled M. horhinis cells (Fig. 3d), and 5 µM gramicidin S promoted mycoplasma cell swelling in both species (Fig. 2e and 3e), whereas HeLa cells resisted these two treatments. Finally (Fig. 2f and 3f), no effect of treatment with 70 µM enrofloxacin on either HeLa cells or mycoplasmas could be detected by electron microscopy. Since all pictures were taken after only 1 h of treatment, the results demonstrate the high affinities of the peptides for mycoplasmas and their fast actions even in the presence of high serum concentrations and in the presence of human cells.

Efficacies of alamethicin and gramicidin S in eliminating mycoplasma infection. Since interesting results were obtained with alamethicin and gramicidin S, further evaluations were performed with these two molecules. For alamethicin, two types of treatments were performed: (i) treatment for 24 h with 15 to 25 µM peptide or (ii) treatment for 0.5 h with 30 to 100 µM peptide (Table 2). It should be noted that although the high concentrations in both groups resulted in high rates of mortality for the HeLa cells and increased the time required for cell division from 3 to 11 days, the surviving cells were still able to multiply thereafter. The 0.5-h treatment with alamethicin alone was not effective in eradicating either M. fermentans or M. hyorhinis even when the peptide was used at a concentration of 100 µM (i.e., more than four times the MIC), and a longer exposure with a lower concentration of alamethicin (25 µM) caused only a partial cure for HeLa cells infected with M. hyorhinis. The same concentration had no effect on M. fermentans-infected cells, although the MIC for this bacterium was lower (12.5 µM). Treatment with gramicidin S alone did not result in the eradication of mycoplasmas from the infected cells (Table 3). A concentration above the MIC was tested only for M. fermentans because for M. hyorhinis-infected HeLa cells, a concentration above the MIC (25 µM) resulted in lethality for the HeLa cells.

View this table: [in this window] [in a new window]

TABLE 2. Efficacy of alamethicin in the treatment of mycoplasma-infected HeLa cell cultures

View this table: [in this window] [in a new window]

TABLE 3. Efficacy of gramicidin S in the treatment of mycoplasma-infected HeLa cell cultures

Influence of addition of enrofloxacin on efficacies of alamethicin and gramicidin S treatments. The combination of enrofloxacin with alamethicin or gramicidin S led to the more effective eradication of M. fermentans and M. hyorhinis from infected HeLa cells (Tables 2 and 3). Treatment with 70 µM enrofloxacin alone for 24 h was not sufficient to cure the cultured cells of the infecting mycoplasmas. This treatment resulted in cure only after 8 days. Interestingly, when enrofloxacin was added to HeLa cells infected with M. fermentans and the cells were treated with alamethicin at high concentrations (50 and 100 µM) for only 0.5 h, three of four cell cultures were cured of the mycoplasmas. The same effect was not observed for HeLa cells infected with M. hyorhinis. Indeed, cure of M. hyorhinis-infected cells was obtained only by treatment with alamethicin and enrofloxacin in combination for an extended period of time (24 h). The addition of enrofloxacin to the treatment with gramicidin S (15 µM) resulted in cure for only one culture of HeLa cells infected with M. fermentans after 24 h. Such an effect was not observed with M. hyorhinis due to the toxicity of gramicidin S for HeLa cells at higher concentrations (i.e., concentrations above 15 µM).

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown in previous studies that although mollicutes (acholeplasmas, mycoplasmas, and spiroplasmas) are generally sensitive to membrane-active peptides, mycoplasmas are particularly resistant to animal antimicrobial peptides (1, 5, 6, 13, 24). The MIC and MBC data obtained in this work (Table 1) indicate that M. fermentans and M. hyorhinis are more susceptible to alamethicin, gramicidin S, and surfactin than to dermaseptin B2 and hence support the hypothesis that mycoplasma resistance to animal peptides is dictated by their obligatory parasitism (5, 6). We further observed that the antimycoplasma activities of the four peptides used in this work were hampered by the presence of serum in the culture medium. This effect was likely caused by serum lipoproteins since these complexes are capable of binding membrane-active peptides and hence decrease their apparent antimicrobial activities (34, 40). Thus, as clearly seen from our results and those of Hindler et al. (18), the antibiotic MIC does not necessarily predict the concentration at which cure will occur in tissue cell cultures not only in the case of membrane-active peptides but also in the case of conventional antibiotics. One practical consequence of this phenomenon was the difficulty in finding conditions that allowed the peptides to kill the mycoplasmas without damaging the HeLa cells.

Enrofloxacin, which has an MIC of 2 µM (Table 1), could cause cure in our model when it was used at a concentration of 70 µM, but only for prolonged periods (8 days) (see also reference 12). Treatment with enrofloxacin for shorter periods such as 24 h did not result in cure. The same was true for the peptides: concentrations that were greater than or equal to the MICs or the MBCs were hardly capable of curing chronically infected HeLa cells. Furthermore, at these concentrations the peptides were too toxic to the cells for testing of the treatment to be conducted for longer periods. This was notably the case with dermaseptin B2, which proved toxic to HeLa cells at concentrations at which it was harmless to the mycoplasmas. Although Vollenbroich et al. (39) reported that it is feasible to eliminate M. hyorhinis and Mycoplasma orale from mammalian cell cultures with surfactin, we found that this peptide was ineffective against M. fermentans and M. hyorhinis. This fact is fully consistent with our findings in that in the presence of serum, surfactin concentrations 100 µM were unable to inhibit the growth of either mycoplasma species.

We were, however, able to obtain better results with alamethicin and gramicidin S, specifically, when they were used in association with a fluoroquinolone. Alamethicin alone was able to eradicate the infecting mycoplasmas in some cases, but this effect was more consistently obtained when the two peptides were used in combination with enrofloxacin. Under these conditions, the elimination process was rapid and could occur after only half an hour of exposure of M. fermentans to alamethicin. Consistent with previous studies performed with spiroplasma cells (4-6), alamethicin and gramicidin S promoted mycoplasma cell splitting or swelling, followed by cell lysis after 1 h of treatment, as illustrated by electron microscopy (Fig. 2 and 3). The ability of membrane-active peptides to induce such a rapid effect under these conditions supports the contention that they may play a role in the elimination of bacterial infections in chronically infected immunocompromised patients (e.g., patients with cystic fibrosis) (3) or bacterial infections on indwelling devices.

A synergistic effect between polycationic peptides and conventional antibiotics against gram-positive and gram-negative bacteria has recently been shown (15, 42). In our study, however, it was not possible to specify whether the association of enrofloxacin and alamethicin or gramicidin S is synergistic or additive since we rechecked the surviving bacteria after four passages (Fig. 1a) to allow even a minor residing population of mycoplasmas to be detected.

HeLa cells are a cervical carcinoma cell line. Further studies with such systems should test the effects of peptides against organisms in more specialized cell lines derived from organs which are the sites of bacterial colonization, such as the airways or skin epithelial cells. In combination with more specialized cell lines, other pathogenic bacteria should be tested as well. Such models, which have recently been developed to determine the MICs for obligatory intracellular bacteria (21) or to study pathogenicity mechanisms (21, 37), might also prove useful for testing of the efficacies of membrane-active peptides.

In conclusion, we have tested the effectiveness of different membrane-active peptides in eliminating mycoplasma infections from a tissue cell culture model. Alamethicin and gramicidin S were able to effectively cure such infections if enrofloxacin was added to the culture medium. This suggests that the association of membrane-active peptides with intracellularly acting antibiotics should promote the more efficient cure of infections. This work also shows the usefulness of the proposed model system, based on tissue cell cultures, which, at least to some extent, fills the gap between standard in vitro assays and in vivo experiments which are performed with animals and which are prerequisites for clinical trials.

 
Ran Nir-Paz was recipient of a Chateaubriand Scholarship from the French government. This work was supported by the "Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires" (Ministère de l'Education Nationale, de la Recherche et de la Technologie, Paris, France), the Groupement de Recherches CNRS 790 "Protéines et Peptides Membranotropes", and the Langlois Foundation.

The expert assistance of Jean-Jacques Montagne with peptide synthesis is gratefully acknowledged. We thank Albert Simhon for critically reading the manuscript.

 
* Corresponding author. Mailing address: Université de Rennes I, UMR CNRS 6026, Campus de Beaulieu, F-35042 Rennes Cedex, France. Phone: 33 2 23 23 61 38. Fax: 33 2 23 23 61 39. E-mail: wroblews{at}univ-rennes1.fr.

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

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Amiche, M., A. A. Seon, H. Wróblewski, and P. Nicolas. 2001. Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family. Eur. J. Biochem. 267:4583-4592.[Medline]
2
2. Arima, K., A. Kakinuma, and G. Tamura. 1968. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31:488-494.[CrossRef][Medline]
3
3. Bals, R., D. J. Weiner, R. L. Meegalla, and J. M. Wilson. 1999. Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model. J. Clin. Investig. 103:1113-1117.[Medline]
4
4. Béven, L., O. Helluin, G. Molle, H. Duclohier, and H. Wróblewski. 1999. Correlation between anti-bacterial activity and pore sizes of two classes of voltage-dependent channel-forming peptides. Biochim. Biophys. Acta 1421:53-63.[Medline]
5
5. Béven, L., D. Duval, S. Rebuffat, F. G. Riddell, B. Bodo, and H. Wróblewski. 1998. Membrane permeabilisation and antimycoplasmic activity of the 18-residue peptaibols, trichorzins PA. Biochim. Biophys. Acta 1372:78-90.[Medline]
6
6. Béven, L., and H. Wróblewski. 1997. Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes. Res. Microbiol. 148:163-175.[Medline]
7
7. Carmeli, Y., N. Troillet, A. W. Karchmer, and M. H. Samore. 1999. Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa. Arch. Intern. Med. 159:1129-1132.
8
8. Charpentier, S., M. Amiche, J. Mester, V. Vouille, J. P. Le Caer, P. Nicolas, and A. Delfour. 1998. Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics. J. Biol. Chem. 293:14690-14697.
9
9. Cohen, M. L. 1992. Epidemiology of drug resistance: implications for a post-antimicrobial era. Science 257:1050-1055.
10
10. Duclohier, H., and H. Wróblewski. 2001. Voltage-dependent pore formation and antimicrobial activity by alamethicin and analogues. J. Membr. Biol. 184:1-12.[CrossRef][Medline]
11
11. Epand, R. M., and H. J. Vogel. 1999. Diversity of antimicrobial peptides and their mechanisms of action. Biochim. Biophys. Acta 1462:11-28.[Medline]
12
12. Fleckenstein, E., C. C. Uphoff, and H. G. Drexler. 1994. Effective treatment of mycoplasma contamination in cell lines with enrofloxacin (Baytril). Leukemia 8:1424-1434.[Medline]
13
13. Fleury, Y., V. Vouille, L. Béven, M. Amiche, H. Wróblewski, A. Delfour, and P. Nicolas. 1998. Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family. Biochim. Biophys. Acta 1396:228-236.[Medline]
14
14. Freundt, E. A. 1983. Culture media for classical mycoplasmas, p. 129-136. In S. Razin and J. G. Tully (ed.), Methods in mycoplasmology, vol. 1. Mycoplasma characterization. Academic Press, Inc., New York, N.Y.
15
15. Giacometti, A., O. Cirioni, M. A. Del Prete, A. Mataloni Paggi, M. M. D'Errico, and G. Scalise. 2000. Combination studies between polycationic peptides and clinically used antibiotics against gram-positive and gram-negative bacteria. Peptides 21:1155-1160.[CrossRef][Medline]
16
16. Guillemot, D. 1999. Antibiotic use in humans and bacterial resistance. Curr. Opin. Microbiol. 2:494-498.[CrossRef][Medline]
17
17. Hancock, R. E. W., and M. G. Scott. 2000. The role of antimicrobial peptides in animal defenses. Proc. Natl. Acad. Sci. USA 97:8856-8861.[Abstract/Free Full Text]
18
18. Hindler, J. A., B. J. Howard, and J. F. Keiser. 1994. Antimicrobial agents and antimicrobial susceptibility testing, p. 145-195. In B. J. Howard, J. F. Keiser, T. F. Smith, A. S. Weissfeld and R. C. Tilton (ed.), Clinical and pathogenic microbiology, 2nd ed. Mosby-Year Book, St. Louis, Mo.
19
19. Hoeprich, P. D. 1994. General principles of systemic anti-infective chemotherapy, p. 222-240. In P. D. Hoeprich, M. C. Jordan, and A. R. Ronald (ed.), Infectious diseases—a treatise of infectious processes, 5th ed. J. B. Lippincott, Philadelphia, Pa.
20
20. Isenberg, D. H. 1998. Clinical microbiology, p. 123-145. In S. L. Gorbach, J. G. Bartlet, and N. R. Blacklow (ed.), Infectious diseases, 2nd ed. The W. B. Saunders Co., Philadelphia, Pa.
21
21. Ives, T. J., P. Manzewitsch, R. L. Regnery, J. D. Butts, and M. Kebede. 1997. In vitro susceptibilities of Bartonella henselae, B. quintana, B. elizabethae, Rickettsia rickettsii, R. conorii, R. akari, and R. prowazekii to macrolide antibiotics as determined by immunofluorescent-antibody analysis of infected Vero cell monolayers. Antimicrob. Agents Chemother. 41:578-582.[Abstract]
22
22. Izumiya, N., T. Kato, H. Aoyagi, M. Waki, and M. Kondo. 1979. Gramicidin S and tyrocidines, p. 49-107. In Synthetic aspects of biologically active cyclic peptides. Kodansha, Tokyo, Japan, and Halsted Press, New York, N.Y.
23
23. Kobayashi, H., N. Sonmez, T. Morozumi, K. Mitani, N. Ito, H. Shiono, and K. Yamamoto. 1996. In vitro susceptibility of Mycoplasma hyosynoviae and M. hyorhinis to antimicrobial agents. J. Vet. Med. Sci. 58:1107-1111.[Medline]
24
24. Leclerc, G., C. Goulard, Y. Prigent, B. Bodo, H. Wróblewski, and S. Rebuffat. 2001. Sequences and antimycoplasmic properties of longibrachins LGB II and LGB III, two novel 20-residue peptaibols from Trichoderma longibrachiatum. J. Nat. Prod. 64:164-170.[CrossRef][Medline]
25
25. Matsuzaki, K. 1999. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta 1462:1-10.[Medline]
26
26. McGowan, J. E., Jr., and J. Carlet. 1998. Antimicrobial resistance: a worldwide problem for health care institutions. Am. J. Infect. Control 26:541-543.[CrossRef][Medline]
27
27. Meyer, C. E., and F. Reusser. 1967. A polypeptide antibacterial agent isolated from Trichoderma viride. Experientia 23:85-86.[CrossRef][Medline]
28
28. Neyrolles, O., J.-P. Eliane, S. Ferris, R. Ayr Florio da Cunha, R., M.-C. Prevost, E. Bahraoui, and A. Blanchard. 1999. Antigenic characterization and cytolocalization of P35, the major Mycoplasma penetrans antigen. Microbiology 145:343-355.[Abstract/Free Full Text]
29
29. Otvos, L., I. O, M. E. Rogers, P. J. Consolvo, B. A. Condie, S. Lovas, P. Bulet, and M. Blaszczyk-Thurin. 2000. Interactions between heat shock proteins and antimicrobial peptides. Biochemistry 39:14150-14159.[CrossRef][Medline]
30
30. Rautenbach, M., and J. W. Hastings. 1999. Cationic peptides with antimicrobial activity—the new generation of antibiotics? Chim. Oggi-Chem. Today Nov./Dec.:81-89.
31
31. Razin, S., D. Yogev, and Y. Naot. 1998. Molecular biology and pathogenicity of mycoplasmas. Microbiol. Mol. Biol. Rev. 62:1094-1156.[Abstract/Free Full Text]
32
32. Russell, W. C., C. Newman, and D. H. Williamson. 1975. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 253:461-462.[CrossRef][Medline]
33
33. Sawa, T., K. Kurahashi, M. Ohara, M. A. Gropper, V. Doshi, J. W. Larrick, and J. P. Wiener-Kronish. 1998. Evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic CAP18 fragment against Pseudomonas aeruginosa in a mouse model. Antimicrob. Agents Chemother. 42:3269-3275.[Abstract/Free Full Text]
34
34. Sorensen, O., T. Bratt, A. H. Johnsen, M. T. Madsen, and N. Borregaard. 1999. The human antibacterial cathelicidin, hCAP-18 is bound to lipoproteins in plasma. J. Biol. Chem. 294:22445-22451.
35
35. Taylor-Robinson, D., and C. Bébéar. 1997. Antibiotic susceptibilities of mycoplasmas and treatment of mycoplasmal infections. J. Antimicrob. Chemother. 40:622-630.[Abstract/Free Full Text]
36
36. Taylor-Robinson, D., and J. Bradbury. 1998. Mycoplasma diseases, p. 1013-1037. In L. Collier, A. Balows, and M. Sussman (ed.), Topley and Wilson's microbiology and microbial infections, 9th ed., vol. 3. Bacterial infections (W. J. Hausler and M. Sussman, ed.). Arnold, London, Great Britain.
37
37. Ulrich, M., S. Herbert, J. Berger, G. Bellon, D. Louis, G. Munker, and G. Doring. 1998. Localization of Staphylococcus aureus in infected airways of patients with cystic fibrosis and in a cell culture model of S. aureus adherence. Am. J. Respir. Cell. Mol. Biol. 19:83-91.[Abstract/Free Full Text]
38
38. Van Kuppeveld, F. J., J. T. van der Logt, A. F. Angulo, M. J. van Zoest, W. G. Quint, H. G. Niesters, J. M. Galama, and W. J. Melchers. 1992. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl. Environ. Microbiol. 58:2606-2615.[Abstract/Free Full Text]
39
39. Vollenbroich, D., G. Pauli, M. Ozel, and J. Vater. 1997. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl. Environ. Microbiol. 63:44-49.[Abstract]
40
40. Wang, Y., B. Agerberth, A. Lothgren, A. Almstedt, and J. Johansson. 1998. Apolipoprotein A-I binds and inhibits the human antibacterial/cytotoxic peptide LL-37. J. Biol. Chem. 293:33115-33118.
41
41. Wenzel, R. P. 1999. Surveillance and control of pathogens of epidemiologic importance—introduction. Clin. Infect. Dis. 29:238.
42
42. Zhang., L., R. Benz, and R. E. W. Hancock. 1999. Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides. Biochemistry 38:8102-8111.[CrossRef][Medline]
43
43. Zhang., L., P. Dhillon, H. Yan, S. Farmer, and R. E. W. Hancock. 2000. Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:3317-3321.[Abstract/Free Full Text]

---

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.

This article has been cited by other articles:

• Fassi Fehri, L., Wroblewski, H., Blanchard, A. (2007). Activities of Antimicrobial Peptides and Synergy with Enrofloxacin against Mycoplasma pulmonis. Antimicrob. Agents Chemother. 51: 468-474 [Abstract] [Full Text]  
• Fehri, L. F., Sirand-Pugnet, P., Gourgues, G., Jan, G., Wroblewski, H., Blanchard, A. (2005). Resistance to Antimicrobial Peptides and Stress Response in Mycoplasma pulmonis. Antimicrob. Agents Chemother. 49: 4154-4165 [Abstract] [Full Text]  
• Simmons, W. L., Denison, A. M., Dybvig, K. (2004). Resistance of Mycoplasma pulmonis to Complement Lysis Is Dependent on the Number of Vsa Tandem Repeats: Shield Hypothesis. Infect. Immun. 72: 6846-6851 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nir-Paz, R. Articles by Wróblewski, H. Search for Related Content PubMed PubMed Citation Articles by Nir-Paz, R. Articles by Wróblewski, H.