[Functional characterization of Pseudomonas batumici 17/20 upon long-term storage].

Variability of properties and antibiotic activity, as well as cells survival of Pseudomonas batumici 17/20--the producer of batumin (antistaphylococcal antibiotic) after long-term storage under vaseline oil layer have been studied. The main culture-morphological and physiological biochemical properties of the mutant strain have been investigated. It has been shown that storage under vaseline oil allows to preserve high level of antibiotic activity: batumin synthesis by the producer was 150 mg/l. Therewith, the survival of cells decreases by two orders during 5 years of storage. The conditions of strain maintenance have been formulated.

[Algorithm of identification of atypical variants of Staphylococci, pathogens of chronic pyo-inflammatory processes in humans].

Staphylococcal pathogens of chronic relapsing infections, such as cystic pneumosclerosis and osteomyelitis are characterized by atypical morphology of the colonies (atypical variants of staphylococci) and present a subpopulation in clinically significant staphylococci. Since the loss of some phenotypic characteristics important for the genus Staphylococcus due to mutations, identification of such staphylococcal variants is difficult and sometimes impossible. An algorithm of identification of atypical variants of S.aureus (SSCVs) was developed. The advantages of the molecular methods and in particular the tRNA-PCR analysis, as well as the use of the diagnostic preparation Diastaph for correct identification of Staphylococcus atypical variants were shown.

[Morpho-functional changes of Staphylococcus aureus biofilms under the effect of batumin].

AIM: Study the effect of batumin isolated from metabolites of Pseudomonas batumici bacteria on the formation of biofilms by staphylococci under the control of atomic force microscopy. MATERIALS AND METHODS; S. aureus 25923 (ATCC) and S. aureus 104 were used as test cultures. Batumin with the degree of purification of 80% was used in the experiments. Microscopy of the preparations was carried out on atomic force microscope SMM-2000 (Proton-MIET Closed Joint-Stock Company, Russia) in contact mode in air environment. Biofilm formation (BFF) was studied by photometric method (O'Toole G.A., 2000). Dissociation of microbial population was detected during seeding of staphylococci into agarized LB medium. RESULTS: Changes of structural component of biofilm were noted visually under the effect of the preparation--exopolymeric matrix and reduction of quantity of adherent staphylococci in the form of separate islet formations. Similar pattern was detected during determination of staphylococci biofilm formation by photometric method. Redistribution of S. aureus clonal structure with the appearance of dissociants that do not possess the ability to form biofilms and reduction of quantity of clones with high values of BFF also occurred under the effect of batumin. CONCLUSION: The data obtained reveal one of the mechanisms of antimicrobial effect of batumin based on suppression of staphylococci biofilm formation by the preparation.

[Influence of antistaphylococcus antiobiotic batumin on microorganism biofilm formation].

AIM: Study the influence of batumin on microorganism biofilm formation. MATERIALS AND METHODS: Experimental data on the antimicrobial effect of batumin on microorganisms and biofilm formation (BFF) was obtained by studying 80 strains of bacteria and fungi isolated from microbial biocenosis of the nose of staphylococcus carriers and patients during examination for intestine dysbiosis. 80% pure batumin was used in the experiments. Antimicrobial activity of batumin was studied by serial dilutions method (CLSI, 2005), BFF--by photometry method (O'Toole G., 2000). The results were statistically treated by non-parametric method by using Mann-Whitney criterion. RESULTS: Minimal inhibitory concentration (MIC) of batumin varied from 0.25 mcg/ml to 64 mcg/ml depending on the species of the studied microorganisms. The most sensitive to batumin strains were Staphylococcus aureus when compared with escherichia and klebsiella. Batumin had no antimicrobial effect on the studied Candida albicans. Inhibitory effect of batumin against BFF of staphylococci, klebsiella and fungi that have an initial level of this property above 2.5 units was established. While in strains that have the initial BFF level of 2.5 units or less, batumin stimulated biofilm formation. Such a dependence was not detected in the studied escherichia coli cultures: batumin stimulated BFF of Escherichia in 80 - 90% of cases. CONCLUSION: The data obtained uncover one of the possible mechanisms of microsymbiocenosis formation in the human associative symbiosis and open the perspectives for further studies of batumin not only as an antimicrobial preparation but also a substance possessing anti-persistence effect against pathogens.

Pseudomonas batumici sp. nov., the antibiotic-producing bacteria isolated from soil of the Caucasus Black Sea coast.

Four novel strains of saprophytic bacteria were isolated from the soil samples collected in the moist subtropics region (the Black Sea coast of the Caucasus) and studied using methods of polyphasic taxonomic analysis. Microorganisms were Gram-negative, oxidase positive, aerobic, rod-shaped motile bacteria that produced antibiotic named batumin with high and selective activity against staphylococci; its total formula was C30H48N2O7 Phylogenetic analysis of 16S rRNA gene sequences (1376 bp, accession number in Genbank--JF306642) indicated that the isolates belonged to the gamma-Proteobacteria formed a separate branch within the genus Pseudomonas and had 98% 16S rRNA gene sequence similarity with Pseudomonas gingeri. The latter essentially differed from the studied strains in its phenotypic characteristics. The predominant cellular fatty acids of isolates were similar and included Cl6:0, C16:1, C18:1, and up to 22.9% of deltaC17:0; their DNA G+C content was 64.0 mol%. An analysis of taxonomic data indicated that the studied isolates represented a novel species, for which the name Pseudomonas batumici sp. nov. is proposed with the type strain UCM B-321 (Ukrainian Collection of Microorganisms).

[Characteristics of atypical forms of staphylococci (SCVs) isolated from patients with osteomyelitis].

Small-colony variants (SCVs), isolated from a population of the parental strains of Staphylococcus aureus, S. haemolyticus and S. epidermidis lost a number of features typical of the species and genus and were characterized by delayed growth, altered colony morphology, lack of pigmentation and changed carbohydrate consumption. Some SCVs of S. aureus had no plasmocoagulase and lecithinase activities. The analysis of 14 SCVs showed that they were auxotrophic for hemin and menadione and resistant to aminoglycoside antibiotics. Such aberrant phenotypic characteristics complicated or made it impossible their identification by the common clinical laboratory methods. The tRNA intergenic spacer length polymorphism analysis was used to identify the atypical forms of the staphylococci.

[Characterization of batumin resistant variants of Staphylococcus aureus clinical isolates].

Population analysis of 20 batumin susceptible staphylococcal strains, isolated from clinical materials allowed to detect resistant clones only in 3 strains. The frequency of batumin resistant variants isolation was low and depended on the antibiotic concentration. Resistance to batumin in 5 clinical strains and in 1 reference strain of Staphylococcus aureus ATCC 6538 P (209P) developed under laboratory conditions and induced significant phenotypic and chemotaxonomic changes in the pathogen. The variants, resistant to batumin, had the same degree of virulence for mice as the initial susceptible strain. The batumin resistant variants of S. aureus were unstable and on the medium without batumin completely restored their initial phenotype.

[Antimicrobial spectrum of antibiotic batumin].

Antibacterial activity of antibiotic batumin, isolated from Pseudomonas genus has been studied using CLSI standard methods. Batumin was highly active against all studied strains belonging to 10 species of Staphylococcus genus (minimal inhibitory concentration (MIC)--0.25-0.5 microg/ml); it has shown a moderate activity against enterobacteria of genera Salmonella, Bordetella, Escherichia, Klebsiella (MIC 8-64 microg/ml) and practically did not inhibit strains ofmicrococci, streptococci, sporeforming bacteria including Clostridium sporogenes (MIC 256 microg/ml or above). Strains of yeasts and microscopic fungi Candida tropicalis, C. utilis, C. albicans, Saccharomyces cerevisiae u Aspergillus niger were resistant to batumin. Antibiotic activity depended on test-culture cells concentration and medium pH and was greatest at pH 5.5 characteristic of human skin.

[Study on activity of antistaphylococcal antibiotic batumin against methicillin resistant strains of staphylococci].

A stable increase in the frequency of methicillin resistant clinical isolates of staphylococci was recorded. 258 strains of Staphylococcus aureus and S. epidermidis, including methicillin resistant ones, were susceptible to batumin. The minimum inhibitory concentration of batumin ranged within 0.04 and 0.5 mcg/ml and depended on the concentration of the microbial cells and the medium pH. The medium composition had limited influence on the batumin activity. No differences in the batumin activity against methicillin susceptible and methicillin resistant staphylococci were observed. The antibiotic had bactericidal action on the strains (2-4 mcg / ml). The results of the study showed that batumin ointment was prospective for eradication of S. aureus. including MRSA, in cases with the nasal carriage and for control of multiresistant strains during hospital outbreaks.

[Transposon mutagenesis in preparing mutants of a strain--a producer of a new antibiotic batumin]. / Transpozonovyi mutagenez pri poluchenii mutantov shtamma--produtsenta novogo antibiotika batumina.

Various plasmids carrying transposon Tn5 were used to generate insertion mutants synthesizing batumin, a unique antibiotic with a selective antistaphylococcal effect. One of the plasmids used provided a sufficient yield of the clones in question. An analysis of over 7000 clones allowed us to select the mutant clones with increased and decreased levels of batumin synthesis and the mutants that lost the ability to synthesize this antibiotic.

[Isolation of highly active strain producing the antistaphylococcal antibiotic batumin]. / Poluchenie vysokoaktivnogo shtamma--produtsenta antistafilokokkovogo antibiotika batumina.

The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin. The strain of Pseudomonas batumici N17 producing 87-100 mg batumin per liter culture liquid was selected. Its activity was 3.5-5 times higher than the activity of the most potent natural strain. P. batumici N17 was shown to be stable in relation to the synthesis of batumin.

[Ecological diagnosis of pyoseptic diseases in newborns using the "Diastaph" disc]. / Etiologicheskaia diagnostika gnoino-septicheskikh zabolevanii u novorozhdennykh s ispol'zovaniem diskov "Diastaf".

The etiological spectrum of pyoseptic diseases was studied in 113 newborns. It was established, that between 187 strains of microorganisms, belonging to 19 species, 73.3% were staphylococci and streptococci, 25.1%--Enterobacteriaceae, 1.6%--Candida albicans. Mixed infection, caused by association of 2-5 Gram-positive and (or) Gram-negative microorganisms, was marked in 51.3% of newborns. In cases of mono-infection (42.5%) staphylococci and streptococci were leading etiological agents. The possibility of generic identification of staphylococci using the diagnostic disks "Diastaph" which contain a new antibiotic batumin has been studied. All the isolated staphylococci strains were highly sensitive to batumin in contrast to other Gram-positive and Gram-negative microorganisms that caused pyodermia in newborns.

[The use of batumin-containing disks for the rapid identification of staphylococci]. / Ispol'zovanie diskov s batuminom dlia ékspress-identifikatsii stafilokokkov.

The study of 467 microbial strains obtained from collections and from clinical sources revealed that microorganisms of the genus Staphylococcus were highly sensitive to batumin, a new antibiotic obtained from bacteria of the genus Pseudomonas. 378 strains of 15 Staphylococcus species proved to be highly sensitive to the diagnostic preparation "Diastaph", developed on the basis of batumin (antibiotic-impregnated discs); After 18-hour incubation the diameter of the growth inhibition zones on agar-containing culture media was 18-38 mm. Strains belonging to the genera Micrococcus, Dermacoccus, Kocuria and Kytococcus, as well as the tested representatives of other taxa (Planococcus, Streptococcus, Corynebacterium, Acinetobacter, Pseudomonas, Neisseria, the representatives of all tested genera of the family Enterobacteriaceae, fungi of the genus Candida) were insensitive to the diagnosticum. "Diastaph" permits not only the rapid identification of staphylococci pure cultures, but also the determination of their presence in association with other microbial species directly in pathological material, which makes it possible to recommend this diagnostic preparation for use in medical, veterinary and sanitary microbiology.

[Biosynthesis of the new antistaphylococcal antibiotic batumin]. / Nekotorye osobennosti biosinteza novogo antistafilokokkovogo antibiotika batumina.

Levels of batumin biosynthesis by separate cells of the antibiotic-producing strain were investigated. The strain belongs to the genus Pseudomonas. It was found that the cell population was not homogeneous by that property. There were isolated highly active, active and low active clones. Specific selection of the most active clone made it possible to increase the number of the cells with high levels of the antibiotic biosynthesis in the population from 8.8 to 84.7 per cent. A clone was isolated which formed no zones of the growth inhibition of Staphylococcus aureus 209P. The data showed that the biosynthesis of batumin was genetically unstable which could be indicative of the plasmid localization of the genes controlling its production. However, with the basic methods of the plasmid DNA isolation it was demonstrated that the batumin-producing strain contained no extrachromosomal DNA which indicated to the chromosomal localization of the genetic locus responsible for the antibiotic synthesis.

[The effect of the antibiotic AL-87 on the amino acid yield from Staphylococcus aureus 209P cells]. / Vliianie antibiotika AL-87 na vykhod aminokislot iz kletok Staphylococcus aureus 209P.

Antibiotic AL-87 has been studied for its effect on the composition of intracellular free amino acids and of amino acids in culture fluid of Staphylococcus aureus 209 P. It is established that the content of amino acids in the culture fluid of S. aureus 209 P is doubled due to antibiotics, while the content of intracellular free amino acids considerably decreases. Spectrum of free amino acids of S. aureus 209 P is presented by 17 basic amino acids. When there is a sub-bacteriostatic concentration of the antibiotic in the medium all free amino acids tend to leave the cells, the content of aspartic acid, serine, threonine and leucine in the medium being increased. Data obtained when studying the effect of antibiotic AL-87 on the composition of free amino acids of Staphylococcus agree well with the previously obtained results from the study of the fatty acid composition of cells. In the light of these data it may be supposed that an increase of the membrane permeability and as a result of it an outlet of amino acids into the medium is one of constituents of the mechanism of antibiotic AL-87 action on Staphylococcus cells.

[Antibiotic AL-87 induction of changes in the fatty acid composition of phospholipids and neutral lipids in a sensitive strain of Staphylococcus aureus 209P]. / Induktsiia antibiotikom AL-87 izmenenii v zhirno-kislotnom sostave fosfolipidov i neitral'nykh lipidov chuvstvitel'nogo k nemu shtamma Staphylococcus aureus 209P.

Under the action of the sub-bacteriostatic concentration (0.02 microgram/ml) of antibiotic AL-87 there formed in all the fractions of phospholipids and neutral lipids of S. aureus 209P unsaturated branched fatty acids not detected in the control and the content of shorter chain saturated branched fatty acids increased. This means that there were changes leading to increased lipid fluidity. The findings showed that fatty acids of the neutral lipids and phospholipids were involved in regulation of the bacterial cell functional state and participated in this case in providing increased membrane fluidity and permeability.

[Effect of antibiotic AL-87 on the growth and ultrastructure of staphylococci]. / Vliianie antibiotika AL-87 na rost i ul'trastrukturu stafilokokkov.

The effect of antibiotic Al-87 on the ultrastructure of staphylococcal cells was studied. The cells of a control culture of a sensitive strain of S. aureus, 209P at the early exponential growth phase were characterized by thin walls (20-22 nm) and septa (30 nm). In the presence of the subbacteriostatic concentration of antibiotic AL-87 (0.02 microgram/ml) the thickness of the cell walls and septa increased up to 80-90 and 150 nm, respectively. Segregation of the septa was retarded and 4 cell conglomerates formed. The cell division appeared to be highly active: the septa were detected in 80-90 per cent of the sections against 40 per cent in the control. Therefore, antibiotic AL-87 induced significant thickening of the cell walls and impairment of the cell division regulation. Investigation of the staphylococcal variant resistant to the antibiotic showed that there were no significant differences between the cells grown in the absence and presence of antibiotic AL-87 (in a concentration of 200 micrograms/ml). In both the experiments there were detected cells in their majority with thinner walls, L-form-like structures, protoplasts and single conglomerates of the cells with thicker walls and anomalous division and the cells at the moment of lysis. It suggested that the effect of antibiotic AL-87 on the cell wall structure was not direct i. e. by inhibition of protein synthesis but mediated i. e. due to shifts in lipid synthesis inducing changes in lipid-dependent synthesis of the cell wall polymers.

[Fatty-acid composition of staphylococci induced by antibiotic AL-87 as a marker of their resistance or sensitivity to the given antibiotic]. / Indutsirovannyi antibiotikom AL-87 zhirnokislotnyi sostav stafilokokkov kak marker ikh ustoichivosti ili chuvstvitel'nosti k dannomu antibiotiku.

Three strains of S. aureus isolated from patients were studied and it was shown that their population was heterogenous with respect to antibiotic AL-87: it contained both the antibiotic sensitive and the antibiotic resistant species in different ratios. The fatty acid composition induced by low concentrations of the antibiotic (subbacteriostatic for the sensitive microorganism) characterized by predominance of branched chain fatty acids in the antibiotic sensitive microorganisms and straight chain fatty acids in the antibiotic resistant microorganisms may serve a marker of sensitivity or resistance. Therefore, the data are indicative of the presence of genetic information in staphylococci determining their resistance to antibiotic AL-87 not yet used in medical practice.