Adaptation of Mycoplasmas to Antimicrobial Peptides: Development of Melittin Resistance in Acholeplasma laidlawii Is Associated with Changes in Genomic and Proteomic Profiles and in Virulence.

For the first time it is shown that the development of resistance to melittin in Acholeplasma laidlawii, a mycoplasma that is widely spread in nature and that is the main contaminant of cell cultures and vaccines, is associated with significant changes in the genomic profile, in cellular and vesicular proteomes, as well as in virulence.

Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target.

This study demonstrated that extracellular membrane vesicles are involved with the development of resistance to fluoroquinolones by mycoplasmas (class Mollicutes). This study assessed the differences in susceptibility to ciprofloxacin among strains of Acholeplasma laidlawii PG8. The mechanisms of mycoplasma resistance to antibiotics may be associated with a mutation in a gene related to the target of quinolones, which could modulate the vesiculation level. A. laidlawii extracellular vesicles mediated the export of the nucleotide sequences of the antibiotic target gene as well as the traffic of ciprofloxacin. These results may facilitate the development of effective approaches to control mycoplasma infections, as well as the contamination of cell cultures and vaccine preparations.

Nonthermal plasma affects viability and morphology of Mycoplasma hominis and Acholeplasma laidlawii.

AIM: To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall-lacking Mollicutes bacteria. METHODS AND RESULTS: 10(8) CFU ml(-1) agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30-300 s. The maximal 10- and 100-fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10(5) and 10(3) drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l(-1) antioxidant butylated hydroxytoluene decreased mortality by a factor of 25-200. UV radiation alone caused 25-85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H2O2 did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. CONCLUSIONS: Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.

Structure-activity relationships of the antimicrobial peptide gramicidin S and its analogs: aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes.

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely ß-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.

[Effect of low temperature plasma on various mycoplasma species].

AIM: Study the influence of low temperature (cold) electrolyte plasma (CEP) on survivability of some mycoplasma strains growing in agar as well as mycoplasma that most frequently contaminate transplantable human cell lines of normal and malignant origin with the aim of decontamination. MATERIALS AND METHODS: Mycoplasma hominis, Mycoplasma arginini and Aholeplasma laidlawii grown in agar and mycoplasma that contaminated transplantable human cell lines of normal (MT4) and malignant (HeLa) origin. Plasma source--Plasmatom device that generates CEP at normal atmosphere pressure and environment temperature. Exposure to plasma was carried out with adherence to the same modes for all the variants of biological substrate. The duration of exposure was selected randomly from 15 to 300 seconds. RESULTS: A pronounced bactericidal effect of high doses of CEP on all the tested mycoplasma variants exposed immediately after seeding into agar was shown. However after a passage a residual number of survived colonies was registered. Passage of colonies exposed in grown state even to high doses of CEP also showed survival of a residual number of bacteria in all the tested mycoplasma species. Exposure of M. hominis immediately after seeding to low doses of CEP resulted in formation of unusual mini-colonies identical to those isolated from humans infected by the same mycoplasma. During microbiological seeding into agar of cultural fluid from 2 spontaneously contaminated strains of transplantable human cells and exposed to CEP growth ofmycoplasma was not detected. CONCLUSION: CEP has pronounced bactericidal properties on various mycoplasma strains growing in both agar and contaminating eukaryotic cells. However even at high doses of exposure to CEP an insignificant part of bacterial cells growing in agar still survives. This may indicate a high degree of heterogeneity and adaptation of mycoplasma subjected to even such hard exposure as cold plasma with plasma-chemical mechanism of destruction of biological substrate.

Nutritional effects of culture media on mycoplasma cell size and removal by filtration.

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 microm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 microm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 microm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 microm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 microm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 microm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.

[Effect of wheat germ agglutinin on acholeplasma--the pathogen of phytomycoplasmosis].

Introduction of wheat germ agglutinin (WGA) to cultural medium of pale green dwarf agent of wheat Acholeplasma laidlawii var. granulum str. 118 gives rise to pleiotropic responses of acholeplasma: activation of growth process, an increase of common protein in comparison with control, a decrease of hemagglutinating activity which results in a decrease of the adhesion properties of pathogen.

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: differential scanning calorimetric and Fourier transform infrared spectroscopic studies.

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into alpha-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D(2)O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2 congruent with citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.

[Effect of silver nitrate, adenosine-5'-monophosphate and phosphoenolpyruvate on activity of extracellular fructose bisphosphatase of Acholeplasma laidlawii var. granulum strain 118].

The reactions of glycolysis or gluconeogenesis proceed in good coordination in the cells of microorganisms, and each stage of these processes is distinctly regulated. Under such conditions fructose-bisphosphatase (FBPase) activity (the enzyme level being constant in the cells of microorganisms) is inhibited by adenosine-5'-monophosphate (AMP) and is activated by phosphoenolpyruvate (PEP) depending on the kind of the source of carbon (glycolytic or glyconeogenic) used for microorganism growth. It is evident that the corresponding regulation of FBPase should be absent in the extracellular environment where one cannot observe a distinct coordination of functioning of the enzyme systems. The investigation results prove that both AMP and PEP, under their individual testing in concentrations up to 20 microM did not practically affect activity of extracellular FBPase, and at higher concentrations they sharply decreased its activity (200 microM AMP by 70%, and PEP - by 75%). Under joint use of PEP and AMP (in concentration 200 microM and 500 microM) one could observe mutual neutralization of the effect of these substances on FBPase; as a result, its activity decreased only by 15% under AMP concentration of 500 microM, and by 25% at AMP concentration of 200 microM, that is in complete agreement with the data of individual testing of the above substances. PEP in high concentrations has displayed itself as a more active repressor of FBPase activity than AMP. AgNO3 in concentrations to 20 microM has manifested itself as a moderate stimulator of FBPase activity and even in the concentration of 200 microM it decreased the enzyme activity by 50% only. The data obtained are rather different than those described in literature for cellular FBPases of microorganisms. It is known that AMP is a powerful inhibitor of its FBPases activity (Ki = 5 microM) while PEP activates it (Ka = 20 microM).

Cholesterol protects Acholeplasma laidlawii against oxidative damage caused by hydrogen peroxide.

The aim of this study was to determine whether cholesterol, added to the cell growth medium or to cell suspension buffer, could protect Acholeplasma laidlawii cells against the toxic effects of hydrogen peroxide (H(2)O(2)). Variable concentrations of cholesterol (0.05-1.0 mg/ml) were added to the A. laidlawii suspension buffer and to the growth medium. Cells were then washed carefully and incubated with 0.001% (v/v) H(2)O(2) at 37 degrees C for 30 min and the viability was determined. The results indicated that cells were more viable in the presence of cholesterol than were cells grown in the absence of cholesterol. In addition, the oxygen uptake rate resulting from the oxidation of 5.5 mmol/L glucose was 2-fold and 4-fold higher for cells grown in medium supplemented with 0.05 and 0.50 mg/ml cholesterol, respectively, compared to cells grown in a medium with no added cholesterol. These findings indicate that cholesterol might play a role in protecting Mollicutes against the oxidative damage caused by H(2)O(2).

Structure-activity relationships of diastereomeric lysine ring size analogs of the antimicrobial peptide gramicidin S: mechanism of action and discrimination between bacterial and animal cell membranes.

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.

Colistin sulfate as a suitable substitute of thallium acetate in culture media intended for mycoplasma detection and culture.

Thallium acetate in concentrations of 500 to 1000 mg/l is tolerated in the culture by the most mollicutes of the orders Mycoplasmatales and Acholeplasmatales and by this reason it is added in the culture media as a selective element for the detection and propagation of mycoplasmas and acholeplasmas. Because of the high toxicity of thallium acetate and its accumulation in the environment, thallium acetate is not biodegradable, an alternative was searched. The results and analysis of tests with nine mollicute species are presented here. It is recommended to replace thallium acetate in the formulations where it is used and colistin sulfate is proposed as its substitute.

Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes.

Cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)), a synthetic tetradecameric ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S (GS), retains the strong antimicrobial activity of GS but is 15-20 times less hemolytic. To characterize its interaction with lipid membranes and to understand the molecular basis of its capacity to lyse bacterial cells, in preference to erythrocytes, we have investigated the interactions of GS14dK(4) with detergent micelles and with lipid bilayer model membranes by Fourier transform infrared spectroscopy and compared our results with those of a similar study of GS [Lewis, R. N. A. H., et al. (1999) Biochemistry 38, 15193-15203]. In both aqueous and organic solvent solutions, GS14dK(4) adopts a beta-sheet conformation that is somewhat distorted and more sensitive to the polarity of its environment than GS. Like GS, GS14dK(4) is completely or partially excluded from gel-state lipid bilayers but interacts strongly with liquid-crystalline lipid bilayers and detergent micelle, and interacts more strongly with more fluid liquid-crystalline lipid systems. However, its interactions are more strongly influenced by membrane lipid order and fluidity, and unlike GS, it is essentially excluded from cholesterol-containing phospholipid bilayers. Also, GS14dK(4) is excluded from cationic lipid bilayers, but partitions more strongly and/or penetrates more deeply into anionic lipid bilayers than into those composed of either zwitterionic or nonionic lipids. Anionic lipids also facilitate GS14dK(4) interactions with multicomponent lipid bilayers which are predominantly zwitterionic or nonionic. Although GS14dK(4) generally penetrates and/or partitions into zwitterionic or uncharged lipid bilayers less strongly than does GS, its greater size and altered distribution of positive charges make it intrinsically more perturbing with regard to membrane organization once associated with lipid bilayers. This fact, combined with its relatively strong interactions with anionic phospholipids, may explain why GS14dK(4) retains relatively high antimicrobial activity. However, its low hemolytic activity is probably largely attributable to its low propensity to penetrate and/or partition into cholesterol-containing zwitterionic lipid membranes.

The effects of ring-size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B.

We have examined the effects of three ring-size analogs of the cyclic beta-sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall-less Gram-positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high-sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid-crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring-size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring-size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring-size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall-less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring-size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.

Effects of synthetic cecropin analogs on in vitro growth of Acholeplasma laidlawii.

Four synthetic peptides (Peptidyl MIMs; Demeter Biotechnologies, Inc.) were evaluated for their in vitro activity against Acholeplasma laidlawii. Fifty percent effective concentration values ranged from 1 to 15 microM. Three of these compounds are more lethal than cecropin B against A. laidlawii.

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii]. / Uchastie transportno-bar'ernykh funktsii plazmaticheskoi membrany v razvitii rezistentnosti k ftorkhinolonam (tsiprofloksatsinu) u Acholeplasma laidlawii.

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

Bactericidal/permeability-increasing protein inhibits growth of a strain of Acholeplasma laidlawii and L forms of the gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes.

Bactericidal/permeability-increasing protein (BPI) inhibited growth of cell wall-deficient Acholeplasma laidlawii and L forms of certain strains of Staphylococcus aureus and Streptococcus pyogenes. However, the same strains of S. aureus and S. pyogenes with intact cell walls were not susceptible to the growth-inhibitory effects of BPI.

[Changes in the properties of the plasma membrane of Acholeplasma laidlawii under the action of tetracycline]. / Izmenenie svoistv plazmaticheskoi membrany Acholeplasma laidlawii pri deistvii tetratsiklina.

The role of physico-chemical rearrangements in the cell plasmalemma of Acholeplasma laidlawii in the development of resistance to tetracycline was investigated. The cells of A.laidlawii were shown to be tolerant to tetracycline and to preserve a rather high titre of the cells even at a concentration of the antibiotic in the inoculation medium much higher than the MIC. The results of the investigation of the structural rearrangements in the plasmatic membrane of the cells grown in the presence of tetracycline revealed changes in the lipid flow in the surface layer and an increase in the cholesterol and phospholipid contents. The size of the changes depended on the time of tetracycline addition and the phase of the culture growth.

[Formation of M. hominis and A. laidlawii resistance to fluoroquinolones]. / O formirovanii rezistentnosti k ftorkhinolonam u M. hominis i A. laidlawii.

Mycoplasma hominis and Acholeplasma laidlawii cultures resistant to antibacterial fluoroquinolone drugs ciprofloxacin (Cpf), ofloxacin (Ofl), and lomefloxacin (Lmf) were prepared by selection in liquid nutrient medium with ascending concentrations of Cpf. Resistant mycoplasma clones contained point mutations in the gyrase. A gene region determining quinolone resistance (QRDR gyrA): M. hominis contained C-->T transition resulting in substitution of Ser(83) for Leu and A. laidlawii G-->A resulting in substitution of Asp (91) for Asn. The phenomena of mutation formation during mycoplasma culturing in the presence of fluoroquinolones is studied. In the presence of Cpf in culture medium in concentrations of up to 10 micrograms/ml (for M. hominis) and 1 microgram/ml (for A. laidlawii) the mycoplasma populations contained cells with both altered and wild genotype. Culturing in the presence of higher Cpf concentrations resulted in elimination of cells nonmutant for QRDR gyrA. Besides in vitro studies, we analyzed clinical strains of M. hominis in the presence of different Cpf concentrations. M. hominis clones resistant to Cpf varying in genotypes were detected. These data permit a conclusion that the mechanism of fluoroquinolone resistance formation in mycoplasma includes several stages.

Antisignature oligonucleotides and their analogs as inhibitors of mollicutes-cofactors of HIV.

Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.