Novel Polycationic Photosensitizers for Antibacterial Photodynamic Therapy.

Antibacterial photodynamic therapy (APDT) is a promising method of treating local infected foci, in particular, surgical and burn wounds, trophic and diabetic ulcers. Photodynamic inactivation (PDI) is able to effectively destroy bacterial cells without them developing resistance in response to treatment.This work was dedicated to the study of photophysical and antibacterial properties of new photosensitizers (PS) based on polycationic phthalocyanines and synthetic bacteriochlorins for photodynamic inactivation of P. aeruginosa bacteria and their biofilms. Gram-negative bacteria P. aeruginosa are often found in infected wounds, presumably in biofilm state and are characterized by rather low susceptibility to APDT, which is a problem. PS were studied for possible aggregation at various concentrations by means of absorption and fluorescence spectroscopy. The results of studies of the ZnPcChol8, (3-PyHp)4BCBr4 and (3-PyEBr)4BCBr4 in water and serum confirm the assumption of a low degree of their aggregation at high concentrations.Consequently, their photodynamic efficiency is high enabling to use these PS at high concentrations to sensitize pathological foci for APDT.It was shown that all the investigated PS had a high efficiency of photodynamic inactivation of Gram-negative bacteria P. aeruginosa, as well as their biofilms. Tetracationic hydrophilic near-infrared photosensitizer (3-PyEBr)4BCBr4 with reduced molecule size had significantly higher efficacy of photodynamic inactivation of P. aeruginosa biofilms compared with other studied photosensitizers.

[STUDY OF COLONIZATION PROCESSES AND PERSISTENCE OF MICROORGANISMS IN ARTIFICIAL MATERIALS FOR MEDICAL USE].

AIM: Study processes of microbial colonization and persistence of microorganisms in polymer materials for medical use. MATERIALS AND METHODS: Samples (1 x 1 cm plates) of polymer plastics for production of removable dental prosthesis based on polyurethane and acryl were used, that were incubated with clinical isolates of Pseudomonas aeuruginosa, Staphylococcus aureus in Luria-Bertani broth nutrient media for 24, 48 hours and 7, 14 days and for 1, 5 and 3 months at a temperature of 37 degrees C. Dynamics of interaction process of microorganisms with polymer materials were studied using scanning electron microscope Quanta 200 3D (FEI Company, USA). The samples were fixated after incubation with 10% of neutral formaldehyde, dehydration with alcohols or acetone, typical for SEM, was not carried out, that allowed to conserve the native structure of the samples, including exo-cell matrix of biofilms. RESULTS: Electron-microscopical data on stages of interaction of bacteria with the surface of medical plastics were obtained. Biofilms were shown to be formed on abiotic surfaces and biodestructive changes of plastics appeared. A question on the possibility of prolonged persistence of pathogenic for human microorganisms in artificial prosthesis is discussed. CONCLUSION: The developed experimental model of formation of biofilm on abiotic surfaces could be the basis for carrying out studies directed on the fight with biofilms, by using SEM.

[Double-component bacterial regulation systems as a target for searching new antimicrobial agents].

A review of the literature data concerning double-component bacterial regulation systems was given. The observed data concern: a) structural and functional organization based on the example of systems of the family OmpR (EnvZ/OmpR, PhoQ/PhoP), which regulate a number of processes providing adaptation to stress conditions in the environment and host body providing the virulence and biofilm formation, the cause of different human chronic infections; b) the genes and functions regulated by the double-components systems, based on the example of EnvZ/OmpR system, especially OmpR protein, and PhoQ/PhoP system. The possibilities for the searching of the double-component system protein inhibitors and their role in depressing pathogenic bacteria virulence were discussed.

[Renal calculus microflora in urolithiasis and search for agents of control of biofilms formed by uropathogenic bacteria].

AIM: Study bacterial biofilms in native material (renal calculus) by electron microscopy method and developmeit of biofilm model by isolates in vitro on sterile calculi of various chemical composition. MATERIALS AND METHODS: Bacterial spectra of microflora of renal calculus lavages were studied, isolated pure cultures were identified up to species. Comparisons of urine microflora obtained before operation in patients with urolithiasis with microflora of removed renal calculi were carried out. RESULTS: Urease activity and genes coding pathogenicity factors were detected, and the ability to form biofilms by isolates was studied. Model of formation of biofilms in vitro on sterile renal calculi was developed and candidate agents reducing the biofilm forming ability were tested. CONCLUSION: Uropathogenic microorganisms infecting renal calculi and forming biofilms on them not only support chronic infection by increased resistance to therapy but also facilitate novel lithogenesis.

[The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections].

The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections is discussed. Recent research showed that most of severe chronic somatic diseases are derived from chronic infection induced in the first place by infectious agents. The role of the T3SS of different species in transition from an acute infection to persistence is reviewed. Clinical and bacteriological research showed that microorganisms are persistent in the form resistant to antibiotics. That is why one of the promising targets for the development of antibacterial new-generation treatment is T3SS that conducts transport of bacteria pathogenicity factors into eukaryotic cell. The presence of this structure is necessary for the development of an acute infectious process and chronization of an infection is essential for its functioning.

[Role of salmonella in biliary lithogenesis].

By the methods of light microscopy and immunocytochemistry studies of interaction between S. typhimurium and corpuscular biliary components was investigated in experimental model "bile-bacteria" It was shown that the results of this interaction was bacterial-biliary sludge formation. Bacterial extracellular mucopolysaccharides matrix and flagella's play crucial role in mechanism of sludge formation.

[SOS-induction in the presence of the plasmid pKM101 in the bacterial cells Escherichia coli K12].

Induction of transcription by the plasmid pKM101 (mutability mediating derivate of the plasmid R46) of the sfiA gene controlling cell division and of the fruA gene encoding the fructose specific enzyme II of the phosphoenolpyruvate-phosphotransferase system in intact cultures of Escherichia coli was studied. The genes under study were fused to the bacteriophage Mu dl (Ap lac). Activation of the sfiA gene, a typical member of the SOS-regulon, was demonstrated to depend on the key genes of the SOS-system-recA and lexA. In contrast, the fruA gene that is non-inducible by the UV-light, a classical SOS-inducing agent, is not activated by the presence of the plasmid pKMIO1 in the bacterial cells. The data obtained suggest that the presence of pKMIO1 plasmid in the Escherichia coli cells induces a SOS-signal as a consequence of the plasmid DNA replication or its conjugative transfer.

[Effects of plasmid pKM101 on the expression of Escherichia coli and Salmonella typhimurium genes under ultraviolet irradiation]. / Deistvie plazmidy pKM101 na ékspessiiu genov pri UF-obluchenii bakterii Escherichia coli i Salmonella typhimurium.

The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity. We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes. The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes. An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e. of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E. col controlling the cell division.

[Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism]. / Deistvie plazmidy pKM101 na ékspressiiu bakterial'nykh genov, ne imeiushchikh otnosheniia k metabolizmu DNK.

An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

[Relationship between the UV-induction of exact exclusion of transposons and the function of umuDC, lexA, recA genes and plasmid pkM101]. / Zavisimost' UF-induktsii tochnogo iskliucheniia transpozonov ot funktsii genov umuDC, lexA, recA i plazmidy pKM101.

A pair of isogenic strains-E. coli K-12 and E. coli B/r differing by the status of umuDC genes and presence of pKM101 plasmid-were constructed and the relationship between UV induction of transposons Tn5 and Tn 10 and the gene umuDC function shown. This relationship is not absolute, in contrast to that of point mutations. Induction of precise excision of these transposons can be inhibited by pKM101 plasmid. Induction of precise excision of Tn5 and Tn 10 from the sites under study is absolutely lexA- and recA- dependent.

[A comparative analysis of UMUDC-dependent induction of tandem duplications, point and insertional reversions in Salmonella]. / Sravnitel'nyi analiz UMUDC-zavisimosti induktsii tandemnykh duplikatsii, tochkovykh i insertsionnykh reversii u sal'monell.

Comparative analysis of UV induction of tandem chromosome (Mtc+) duplications and reversions of point mutations (base pair substitutions) and of insertion mutations (precise excision of Tn10) was carried out. Salmonella (umuST) genes deficiency preventing dose-dependent UV induction of point mutation reversion were shown to have a less pronounced effect on the induction of tandem chromosome duplications. UV induction of tandem chromosome duplications is similar to UV induction of insertion mutation reversion: dose-dependent UV induction of both occurs in umuST Salmonella strains. E. coli umuDC+ genes included in Salmonella genome appreciably enhance the UV induction of both tandem duplications and insertion mutation reversion. The presence of umuDC+ genes ensures an expressed dose-dependent UV induction of point mutation reversion.

[UV-induction of the LT-toxin operon depending on genes lexA, recA, and umuD]. / UF-induktsiia operona LT-toksina v zavisimosti ot genov lexA, recA, i umuD.

UV induction of the elt operon (the LT-toxin operon in Escherichia coli) was demonstrated in experiments using fusion of elt::lac operons with the help of Mud1(Ap lac) phage. UV induction of the elt operon is lexA-dependent; thus, the possibility of SOS regulation of this process may be assumed. However, UV induction of the elt operon turned out to be recA-independent, which makes it impossible to consider this induction as a typical SOS response. UV induction of the elt operon is also observed in Salmonella typhimurium, which differs from E. coli in the product of umuD, which suggests that the UV induction of the elt operon is umuD independent.

[UV-inducibility of the LT-toxin operon]. / UF-indutsibel'nost' operona LT-toksina.

The plasmid elt-operon pVZ14 was constructed by fusing of the eltoperon of the plasmid pVZ357 with the lac-gene of the bacteriophage Mud1 (Amp, Lac). lacZ gene has been proven to be fused with an elt-promoter by the loss of toxin production coded by pVZ357 and acquiring of Lac+ phenotype by pVZ14 containing cells, as well as by HindIII fragments hybridization of pVZ357 and pVZ14 with the labelled elt-probe. The kinetics of beta-galactosidase synthesis in E. coli cells harboring pVZ14 shows an elt-operon promoter to have expressed constitutive activity and to be activated by a SOS-inducing agent, UV-light.

[SOS-induction of the RP4 plasmid tet-determinant]. / SOS-indutsibel'nost' tet-determinanty plazmidy RP4.

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.

[Sensitivity of Vibrio cholerae to the lethal and mutagenic effects of UV-irradiation mediated by the presence of plasmids]. / Chuvstvitel'nost' kholernykh vibrionov k letal'nomu i mutagennomu deistviiu UF-sveta, zavisiashchaia ot nalichiia plazmid.

The effect of UV-irradiation on Vibrio cholerae cells and its changes mediated by the plasmid R245 have been studied. Vibrio cholerae strains 569B and RV31 have been shown to be considerably more sensitive to lethal effect of UV-irradiation as compared with Escherichia coli and Salmonella typhimurium cells. Highly toxigenic strain 569B and practically atoxigenic strain RV31 have the same UV-sensitivity. Lethal effect of UV-irradiation on Vibrio cholerae cells is increased when the irradiated cells are plated on enriched media. UV-induction of mutations was not registered in plasmidless strains of Vibrio cholerae. Plasmid R245 increases UV-resistance of vibrio cells and makes them UV-mutable.

[Isolation of the R'his plasmids of Vibrio cholerae]. / Poluchenie plazmid R'his Vibrio cholerae.

V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

[Mobilization of Vibrio cholerae chromosomal genes by plasmid RP4::Mu cts62]. / Mobilizatsiia khromosomnykh genov Vibrio cholerae plazmidoi RP4::Mu cts62.

The RP4::Mu cts62 plasmid was constructed in Escherichia coli cells and subsequently transferred by conjugation into the cells of Vibrio cholerae. This plasmid had been shown to mobilize chromosomal genes of V. cholerae during intrageneric matings. The frequency of mobilization is higher in matings at 37 degrees C, the temperature which is semipermissive for temperature sensitive Mu cts62 phage. This is only true for strains harbouring RP4::Mu cts62, but not for strains containing the RP4 plasmid. Frequencies of mobilization per transferred plasmid exceeded conspicuously the known frequency for the mobilizing activity of the P-factor of V. cholerae. Transconjugants selected for chromosomal markers carried no traces of RP4 plasmid or Mu cts62 markers. This feature of their use in the genetical analysis of V. cholerae. Reasons for the absence of plasmid markers in recombinants are being discussed.

[Lethal and mutagenic action of UV light on salmonellae carrying wild or mutant alleles of the Escherichia coli lexA-gene]. / Letal'noe i mutagennoe deistvie UF-sveta na sal'monelly, nesushchie dikii ili mutantnyi alleli lexA-gena kishechnoi palochki.

To elucidate the reasons for the absence of UV-mutability in Salmonella typhimurium, the lexA gene of Escherichia coli has been transduced by phage P1 into S. typhimurium. The functioning of lexA+ allele of E. coli in the chromosome of Salmonella failed to cause UV-mutability of the hybrid. The transfer of pKM101 plasmid into the LexA+ hybrid mediates the expressed UV mutability and UV-protective plasmid effect. This plasmid harboured by the LexA hybrid fails to increase UV-resistance and mutability, thus showing the dependence of plasmid mediated effect on LexA+ phenotype.

[Intergeneric crossing: P1 phage transduction of the malB region in crosses between E. coli and S. typhimurium]. / Mezhrodovoe skreshchivanie: transduktsiia fagom P1 malB oblasti v skreshchivaniiakh E. coli i S. typhimurium.

In the intergeneric crossing of E. coli and S. typhimurium, no effective transduction of the malB gene was observed. The absence of effective transduction suggests the low homology of the malB chromosomal areas in E. coli and S. typhimurium. To carry out the transduction of the MalB gene together with the lexA gene from E. coli to S. typhimurium, a hybrid having no restriction of phage P1 and incorporating the malB area of E. coli should be previously created.