[The effects of alkylhydroxybenzenes on homoserine lactone-induced manifestations of quorum sensing in bacteria].

The effects of four alkylhydroxybenzene (AHB) homologs of different hydrocarbon chain lengths on synthesis of the pigment violacein induced by C6-homoserine lactone (HSL) and biofilm formation by Chromobacterium violaceum NCTC 13274 and on Escherichia coli pAL103 bioluminescence in the presence of C6-oxo-HSL were studied. It was shown that the inhibitory effect of alkylhydroxybenzenes on the growth of C. violaceum increased in the C5-AHB --> C12-AHB series in the absence of this activity in C1-AHB. Sub-inhibitory AHB concentrations reduced violacein production and suppressed biofilm formation. These effects were presented as individual and group regression dependencies between the analyzed parameters. It was shown using the bioluminescent model that the regulatory effects of AHBs are not associated with their direct competition with HSL and that they develop as a result of changes in the sensitivity of bacterial cells to the respective quorum sensing inducer.

[Investigation of ampicillin effect on morphological and mechanical properties of Escherichia coli and Bacillus cereus cells with atomic force microscopy].

The effect of subbacteriostatic concentrations of ampicillin on morphological and mechanical properties of gramnegative and grampositive cells of Escherichia coli K12 TG1 and Bacillus cereus IP 5832 respectively was studied with atomic force microscopy. Significant heterogeneity of the bacterial populations was shown by the character of the response to the antibiotic effect. The common feature was increase of the cell size likely due to the effect of the inner osmotic pressure on the lowered cell wall strength. In the E. coli population there were besides observed anomalous elongated cells with signs of septation disorder, as well as their structurs, lacking the cytoplasmic liquid fraction. In the B. cereus the inner osmotic pressure mainly enlarged the cell cross section, changing the cell shape from rod to sphere, that was accompanied by significant impairment of the surface structure with liberation of the peptidoglycane fragments to the medium. The particular features of the E. coli K12 TG1 and B. cereus IP 5832 respond to the ampicillin effect were attributed to the differences in the structure of their cell wall, also due to specific properties of the peptidoglycane synthesis and three-dimensional organization.

[Evaluation of the phagocytic activity of human peripheral blood neutrophils, by employing recombinant luminescent bacteria].

The authors show it possible to evaluate the phagocytic activity of human peripheral blood neutrophils from their impact on the fluorescence intensity of recombinant luminescent bacteria. They have established the preferred use of recombinant luminescent Escherichia coli bacteria with cloned luxCDABE genes OF Photobacterium leiognathii as phagocyted objects and substantiated the optimal method of their opsonization by normal human immunoglobulin. The variants of separate consideration of the luminal-dependent chemiluminescence of phagocytes and the bioluminescence of bacterial target in the phagocytic system, which are based on the study of two tests of different component composition or one test at two wavelengths of less than 420 nm for the assessment of chemiluminescence and more than 540 nm for the assessment of bioluminescence.

[Characteristics of the response of natural and recombinant luminescent microorganisms in the presence of Fe2+ ions].

It was found that divalent iron ions have alternative effects on the bioluminescence of the natural marine microorganism Photobacterium phosphoreum and the recombinant Escherichia coli strain with a cloned lux operon of P. leiognathi. In the presence of 0.25-5.0 mM FeSO4, the bioluminescence intensity of the former and the latter increased and decreased, respectively. To establish the causes of these differences, we studied the characteristics of the fatty acid composition of the compared microorganisms. The fatty acid profile of E. coli was characterized by a high proportion of unsaturated 11-octadecenoic (vaccenic) acid. A study of this acid in a cell-free enzyme system used for bioluminescence generation showed that it is a potent inhibitor of bacterial bioluminescence. It was found that such effects are enhanced if 11-octadecenoid acid is preincubated with Fe2+.

[Adaptation of bioluminescence technique for the evaluation of the toxicity of mineral waters].

Mineral waters have been ascertained to exert a pronounced inhibiting effect, that is in proportion in their mineralization and pH values, on the fluorescence of microbial luminescent biosensors. To prevent such adverse reactions masking the effects of chemical pollutants, the authors propose an adjusted procedure for preparing the samples of mineral waters for luminescence biotesting. The first approach is to differentially insert additional quantities of NaCl into the test samples depending on the level of their baseline mineralization to the baseline value 3 g/l, which is provided in the use of the natural sea luminescent bacterium Photobacterium phosporeum as a test object (Microbiosensor B-17 677f). The second approach is to normalize pH to values of not more than 7.5, which are identical in the use of both the Microbiosensor B-17 677f and a recombinant Escherichia coli strain with the cloned lux-operon P. leiognathi (Ecolum-9). This sample preparation maintaining the baseline fluorescence of bacterial biosensors is shown to permit bioluminescence detection of exogenously inserted toxicants in the test samples.

[Biotests for mineral waters with natural and recombinant luminescent microorganisms].

We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).

[Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors].

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.

[IR spectroscopic research on the impact of chemical analogues of autoregulatory d1 factors of microorganisms on structural changes in DNA].

Using IR spectroscopy, we investigated the impact of chemical analogues of autoregulatory d1 factors of microorganisms (methylresorcinol, hexylresorcinol, and tyrosol) on the conformational changes in DNA in films upon altering (decreasing) the relative humidity. We analyzed the appearance/disappearance of characteristic absorption bands of A and B DNA forms and determined D1080/D1224, the ratio between the band intensities of symmetrical and asymmetrical oscillations in their phosphate groups. The data obtained suggest the slowing down of the B-->A structural transition in DNA in the presence of methylresorcinol and its speeding up in the presence of tyrosol. We discuss the mechanisms of this phenomenon in relation to the chemical composition of d1 factors and their biological function.

[Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators].

The fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d1 factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.

[Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation].

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d1 factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the lambda phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (lambda = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear Hind III restricts of the lambda phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.

[Mechanisms of interaction between DNA and chemical analogues of microbial anabiosis autoinducers].

The alkylhydroxybenzene (AHB) autoregulatory factors d1 (fd1) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifesed themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd1 analogues tested. Using atomic force microscopy, we visualized the micelle-like DNA nanostructures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.

[Species diversity of staphylococci in the air and the organism of carriers upon technogenic chemical exposure].

The authors analyzed the biological diversity of sanitary indicative microorganisms of the genus Staphylococcus in man and environmental objects in an area adjacent to a gas industrial enterprise. The species diversity of staphylococci was found to increase upon technogenic chemical exposure, which was recorded in both ambient air and bacterial carriers. The rise in the biological diversity was shown to occur with the increased number of rare species which adequate assessment required the use of the Shennon index. Whether the index of biological species diversity of staphylococci during microbiological monitoring of natural ecological systems is discussed in the paper.

[Conditions that influence bacterial luminescence in the presence of blood serum].

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.

[Determination of the bactericidal activity of blood serum by recombinant luminescence strains of Escherichia coli].

It has been experimentally proven as possible to use recombinant strains of Escherichia coli carrying the genes of luminescence of natural fluorescent bacteria in evaluating the bactericidal activity of blood serum (BABS). The shaping fluorescence inhibition was shown to be in proportion to the developing bactericidal effect in respect to the luminescent strains of Escherichia coli, which depended on a used concentration of blood serum and time of contact. The list of E. coli recombinant strains, which fit in most of all with the evaluation of BABS, was drawn. The values of the bactericidal activity of blood serum determined by the routine bacteriological and nephelometric methods were demonstrated to coincide with those determined by the worked-out bio-luminescent method.

[Comparison of efficacy of three test-systems for identification of Staphylococcus: PBDS (Russia), "STAPHYtest-16" (Check Republic), "API-STAPH" (France)]. / Sravnenie éffektivnosti trekh test-sistem dlia identifikatsii stafilokokkov: PBDS (Rossiia), "STAPHYtest-16" (Chekhiia), "API-STAPH" (Frantsiia).

The diagnostic efficiency of three test-systems designed for staphylococci identification including the definition of opportunistic errors was comparatively studied. The identification results coincidence (IRC) made, versus the reference method, 57 +/- 14% for Staphylococci Differentiation Biochemical Plate (SDBP--Russia) 73.83 +/- 3.6% for STAPHYtest-16 (Check Republic) and 58.33 +/- 5.03% for API-Staph (France). The diagnostic efficiency of the studied test-systems was found to be in proportion with the total information density of the identification tests belonging to each of them, which could be used for determining an optimal test list that would ensure the maximum diagnostic effect with minimal cost.

[Biological characterization of Bifidobacterium and Lactobacillus preparations made with the use of hydrolysate-milk and hydrolysate-soybean media]. / Biologicheskie kharakteristiki preparatov Bifidobacterium i Lactobacillus, poluchennykh s ispol'zovaniem gidrolizatno-molochnoi i gidrolizatno-soevoi sred.

A complex of biological characteristics important for design of the therapeutic and prophylactic activities of Bifidobacterium and Lactobacillus preparations made with the use of hydrolysate-milk and hydrolysate-soybean media was comparatively estimated. The use of the hydrolysate-soybean medium resulted in an increase of the antagonistic activity of the preparations against a number of opportunistic pathogens that was not connected with changing of their acidity and the content of lysozyme, a bacteriolytic enzyme in the culture fluid. The change in the cultivation conditions due to substitution of the hydrolysate-milk medium for the hydrolysate-soybean medium stimulated the adhesive capacity of the production strains used in the study, which was especially evident with respect to the representatives of the genus Bifidobacterium.