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The impact of glycine, added to the cultivation medium, on resistance of Staphylococcus aureus strains to actinomycin D and gramicidin S was studied. The antibiotic resistant strains were isolated after cultivation of the susceptible S. aureus strain 209P on media with increasing concentrations of actinomycin or gramicidin. When the strains were grown on the glycine-containing medium. i. e. under the conditions providing replacement of D-alanine by glycine in the C-end dipeptides of peptidoglycanes, the resistance of the staphylococci to actinomycin markedly decreased. However, in the resistant cells, characterized by significant thickening of the cell walls, the peptidoglycane quantity per a biomass unit did not lower, that was evident of preservation of the wall thickness. At the same time, with addition of glycine to the medium there was observed increased ability of the cells to bind actinomycin. When the gramicidin-adapted strains were grown on the glycine-containing medium, their resistance to the antibiotic did not change. The modification of the peptidoglycane C-end dipeptides probably lowered the protective role of the thicker walls of the cells on their contact with actinomycin but not gramicidin.

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Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.

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Staphylococcus aureus strains, resistant to actinomycin D (AMD) and to gramicidin S (GS) were selected by S. aureus 209P passing on the media containing the above mentioned drugs. Strain R80 resistant to AMD and strain R9 resistant to GS and AMD and described before didn't perform enzyme inactivation of AMD. Cells of both strains had diminished ability to bind exogenous AMD. Electron microscopy investigation revealed that cells of R80 strain had thickened cell walls and they are characterized by more electron density then cells of R9 strain and of parent strain. Adaption to AMD and GS influenced also on functions of some staphylococcal surface proteins--the activity of endogenous coagulase (clumping factor) was found only in R9 strain. Exogenous coagulase was present in all the strains, but development of resistant to AMD and GS diminished this enzyme activity. It is concluded that development of resistance to AMD and GS causes substantial changes in staphylococcal cell wall, but the type of these changes differ.

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Streptomyces chrysomallus is known as an organism producing macrotetrolides (MTL) and actinomycin C. The dynamics of the MTL biosynthesis by some variants of S. chrysomallus in the process of their growth in liquid media was studied. In parallel the ability of the culture mycelium (washed or suspended in physiological solution) to bind exogenous actinomycin D (AMD) was estimated. An inverse correlation between the dynamics of MTL biosynthesis and the rate of the AMD binding by the washed mycelium during the whole period of the culture development was observed: a decrease in the culture ability to bind AMD corresponded to active biosynthesis of MTL and an increase in the culture ability to bind AMD corresponded to lower biosynthesis of MTL. It was suggested that the active biosynthesis of MTL correlated not only with a decrease in the ability of the suspended mycelium to bind AMD but also with a decrease in binding of actinomycin synthesized and excreted to the medium by the culture. A decrease in the reflux of the synthesized antibiotic to the cells was likely one of the components of the system of the S. chrysomallus insensitivity to its own antibiotic.

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Binding of exogenous actinomycin D (AMD) by washed mycelium of streptomycetes i.e. variants of Streptomyces chrysomallus producing and not producing actinomycins and Streptomyces lividans not synthesizing the antibiotics was studied. Dependence of the bound quantity of AMD on its concentration, incubation time and temperature, energy source availability, influence of respiration inhibitors and the membranotropic antibiotic gramicidin S was shown. The intracellularly localized portion of the bound AMD likely penetrated to the cells by diffusion and was strongly bound presumably to DNA in the AMD sensitive S.lividans and to the specific intracellular actinomycin-binding proteins in the AMD resistant variants of S.chrysomallus. The ratio of AMD strongly bound by the mycelium and AMD easily washed with physiological solution and probably localized on the surface was determined. The ratio depended on sensitivity of the culture to AMD and for the variants of S.chrysomallus on the age of the culture and its ability to synthesize actinomycins.

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The affect of preliminary irradiation of staphylococcus culture by electromagnetic radiation of extremely high frequency (42, 54, 66 + 78 GHz) of nonthermal intensity on the bacteria growth on the media containing various antibiotics is studied. The reliable change in bacteria sensitivity toward 5 antibiotics, mainly having membranotropic properties is observed in the experiments using 14 antibiotics with various mechanisms of action. It has been established that in the presence of subbactericide concentrations of active antibiotics the irradiation could result in both further suppression of bacteria growth and its stimulation. As shown, the development of these effects takes place even in a matter of minutes of preliminary irradiation, and weak changes are observed at further increase of this period up to 60 min.

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The absorption of actinomycin D by the cell suspension of Staphylococcus aureus via diffusion linearly depended on the antibiotic concentration in the suspension within the ranges of 2 to 15 micrograms/ml. The absorption of active actinomycins C2, C3 and Au6 was the same as that of actinomycin D. The Staphylococcus intact membranes limited the inlet of the actinomycins to the cells since the membranotropic substances such as gramicidin S and its derivatives and thyrocidin increased their absorption by 30-70 per cent. The absorption of a low active actinomycin D0 and inactive actinomycinic acid even after the exposure to the membranotropic substances was not detectable. These compounds did not form any complexes with DNA. The level of the absorption of the actinomycins by the cells was likely defined by their ability to complex with DNA.

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Factors defining actinomycin D resistance in Staphylococcus aureus resistant to gramicidin S were investigated. The results of the thin layer chromatography, high-voltage electrophoresis and bioautography showed that the resistant cells did not inactivate actinomycin D by the hydrolysis of the lactone bond in the antibiotic molecule. The estimation of the cell ability to bind actinomycin D revealed that the antibiotic binding to the resistant cells was lower by 70-75 per cent as compared to the cells of the susceptible strains. Gramicidin S impaired the intactness of the cytoplasmic membranes and increased the absorption of actinomycin D by the susceptible cells and to a much lesser extent by the cells of the resistant strains. Actinomycin D bound by the susceptible cells could not be washed out with a buffer solution. It could be separated from the cells only by extraction with an organic solvent. Comparative electron microscopy of the susceptible and resistant cells demonstrated that the cell walls in the resistant strains were 1.5-2-fold thicker than the cell walls in the susceptible strains. The actinomycin D resistance of the Staphylococcus strains resistant gramicidin S was likely conditioned by the barrier properties of the morphologically changed cell walls.

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Strains of Staphylococcus aureus 209P growing in the presence of 20 micrograms/ml of gramicidin S were isolated after the successive subculture on a liquid medium with increasing concentrations of the antibiotic. The resistance was stable and preserved after the subculture on media not containing the antibiotic. The development of the resistance to gramicidin S did not lower the cell sensitivity to a large number of antibiotics known as inhibitors of the cell wall synthesis, protein synthesis and RNA-polymerase reaction. There was observed the development of moderate resistance to actinomycin D but not to other antibiotics interacting with DNA. The gramicidin resistant strains were also resistant to tyrocidine, a membrane active polypeptide. By the amount of the bound gramicidin S the cells of the sensitive and resistant strains did not practically differ.

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Polarographic determination of the rate of endogenic respiration of the cells of Staphylococcus aureus 209P showed that the respiration activity of the cells of the strain resistant to 20 micrograms/ml of gramicidin S was 20 to 30 per cent lower than that of the sensitive strain. The rate of oxygen consumption in oxidation of NADH by the membrane preparations of the resistant cells was also 25 to 30 per cent lower. By comparison with the initial sensitive strain the activity of endogenic DPI-reductases of the intact cells and NADH-dehydrogenases of the membranes of the resistant strain was also lower. The velocity of the valine transport to the resistant cells was much lower than that of the amino acid transport to the cells of the sensitive strain. Development of gramicidin S resistance in the staphylococcal strain was likely accompanied by a decrease in the activity of the energy metabolism in the membranes.

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The absence of pyruvate and insignificant levels of alpha-keto-glutarate in the mycelium during the fermentation cycle were characteristic of a highly active erythromycin-producing strain of Saccharopolyspora erythraea. Alpha-keto-glutarate partially excreted to the fermentoffon broth. The activity of pyruvate decarboxylase and alpha-keto-glutarate decarboxylase was detected in the cells during the entire period of the cultivation. The same regularities were observed in the chloramphenicol resistant mutant of the strain. The mycelium of a low productive strain of S.erythraea contained not only alpha-keto-glutarate but also pyruvate and excreted large amounts of keto-acids. By the activity levels of the decarboxylases the low productive strain did not differ from the highly productive one. Propanol did not influence the growth of the low productive strain and the synthesis of erythromycin by it. However, it stimulated accumulation of keto-acids and especially pyruvate in both the mycelium and fermentation broth. Relation between the intensity of keto-acid metabolism and erythromycin biosynthesis is discussed.

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Nisin synthesis by Streptococcus lactis, strain MGU, grown as a combined culture together with Proteus vulgaris and Bacillus mesentericus under stationary conditions or with stirring does not depend on the quantity of inoculated associated cells. Nisin synthesis in the combined culture drops down by 10-20% at the initial pH 7.5 of the growth medium which is unfavourable for S. lactis producing nisin. The level of nisin biosynthesis does not rise when the pH of the medium is adjusted (either naturally or artificially) to 6.6-6.8 in the presence of glucose and yeast autolysate. S. lactis inhibits the growth of B. mesentericus when grown together with it whereas P. vulgaris inhibits the growth of S. lactis in their combined culture.

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Nisin sorption on some silica adsorbents in columns was studied. Silica gels, silochromes and sintered glass with various pore diameters, various specific surfaces, etc. were tested. Correlation between the adsorbent adsorption capacity with respect to nisin and the adsorbent pore diameter was observed. The tested silochromes, silica gels with the pore diameters of 70-80 nm and sintered glass with the pore diameters of 65-112 nm had the highest adsorption capacity with respect to nisin.

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The results of the study on optimization of the process of nisin isolation from the cultural liquid are presented. Different adsorbents, streptococcal cells, medium solid particles (biomass fermentalysates) and silica gel, were used for antibiotic adsorption.

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It has been shown that silica adsorbents can be used for adsorption of nizin from solutions obtained after centrifugation of the fermentation broth. The optimal structure of the adsorbent pores has been determined. Silica with pores 50 to 75 nm in size provided the highest adsorption rates. The value of silica adsorption of nizin depended on the medium pH. The maximum adsorption rates were observed at pH 6.5--7. At pH 3.5 the level of nizin adsorption was low.

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The intensive biosynthesis of nizin on the glucose-yeast medium is observed during the logarithmic and early lag phases of the staphylococcal growth. The ratio of nizin in the fermentation broth (free nazin) and that bound with the cells depended on pH of the medium. When pH was maintained at 6.6-6.8, the amount of nazin in the cells during and growth logarithmic phase was equal to its amount in the fermentation broth filtrate. During the lag phase marked inactivation of nizin was noted. periodical feeding of casein prevented the nizin inactivation. The preliminary data are indicative of the enzymatic nature of the antibiotic.

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Some conditions for absorption of nisin, a polypeptide antibiotic by the cells of Str. lactis were studied. The amounts of nisin adsorbed by the cells depended on the culture age: at the late stationary phase the adsorption level was 2 times higher than that at the logarithmic phase. The cells grown on a "poor" medium adsorbed 85-90 per cent of nisin added to the solution, while the cells grown on the "rich" medium adsorbed 50 per cent of the antibiotic. The adsorption level of nisin by the cells subjected to a thermal shock was higher than that by the live cells. Desorption of nisin from the cells with acid ethanol and bivalent cation solutions was insignificant. Nisin is adsorbed by the cells of other microorganisms, the adsorption levels by the cells of Bac. brevis being the same as those by the streptococcal cells, while the levels adsorbed by Bac. polymyxa being 4 times lower.

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Nizin is produced by Str. lactis, strain MSU. During biosynthesis it is excreted into the fermentation broth and gradually adsorbed on the organism cells. This was confirmed by experiments with an inactive variant of Str. lactis IIa. The cells of this culture adsorbed nizin from "active" fermentation broth. Adsorption of nizin depended on pH of the medium; at pH 2,3 the cells did not adsorbe the antibiotic and at pH 6.6 the amount of the antibiotic adsorbed by the cells was maximum.

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To accelerate determination of nizin concentrations by the agar-diffusion method it is suggested that Twin-80 be added to the agar. For making the media composition more simple, agarized phosphate buffer, pH 5.0, or agarized water, pH 7.0, is recommended to be used for the lower layer.

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