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.

The effect of mutation in the clpX gene on the synthesis of N-acyl-homoserine lactones and other properties of Burkholderia cenocepacia 370.

In order to study the regulation of N-acyl-homoserine lactones synthesis (AHLs, the signal molecules of Quorum Sensing regulation) in Burkholderia cenocepacia strain 370 we obtained mutants with increased AHL production. One of the mutants, named BC-B6, was obtained by TnMod-RKm(r) plasposon mutagenesis. The plasposon insertion was located within the clpX gene encoding the ATPase subunit ClpX of the ClpXP protease. The mutation reduced bacterial virulence in mice intranasal infection. The results of proteomic analysis demonstrated that the expression of at least 19 proteins differed not less than 2-fold between the parental and mutant strains. 18 of the proteins were upregulated in the mutant, and one protein was downregulated. The proteins included those that involved in protein synthesis and modification, in energy production, in general metabolism, in transport and regulation. To check the effect of the clpX mutation on the AHL synthesis, a mutant with inactivated clpX gene (BC-clpX:Km(r)) was constructed by gene replacement method. This mutant also exhibited increased AHLs production. A swarming motility of both mutants was reduced compared to the original strain. Thus, the obtained results show that the clpX gene was involved in the regulation of AHL production and a number of cellular processes in B. cenocepacia 370.

[PERSISTENCE OF MYCOPLASMA DURING UROLITHIASIS].

AIM: Study the frequency of detection of mycoplasma and ureaplasma in clinical material from urolithiasis patients. MATERIALS AND METHODS: Clinical material samples (blood sera, urine, uroliths) from 31 urolithiasis patients were obtained during operations of urolith-removal. Cultural method, LAR and PCR were used in the study. RESULTS: The study of clinical material from 31 patients by PCR has shown, that in 25 individuals. (80.6%) DNA of mycoplasma and ureaplasma was detected, and mycoplasma DNA was more frequently detected in uroliths and less--in-blood sera. Mycoplasma hominis DNA was detected in clinical material of a significantly largerninmber of patients. 23 cultures were isolated from 8 patients by a cultural method, that were identified by PCR as M. hominis. All the isolates have grown as "mini colonies". Even after multiple passages in agar medium, reversion of "mini-colonies" into colonies with a classic morphology was not obtained. CONCLUSION: A high frequency of detection of mycoplasma and ureaplasma in clinical material of patients with urolithiasis was established. The isolated M. hominis cultures have only grown as "mini-colonies". The phenomenon discovered could give evidence on high variability of mycoplasma and a possibility of existence of previously unknown form of their persistence in human organism.

[MICROBIAL COMMUNITIES ON KIDNEY STONES].

The clinical material obtained surgically in patients with kidney stone disease (KSD) was tested for content of the stone microflora using PCR and standard microbiological methods. It was demonstrated that about 50% of stones in patients with KSD were infected with various infection agents as observed using standard microbiological and molecular genetic methods. The percentage of detection of the Mycoplasma hominis using cultural method is lower than the percentage detected using PCR, which is due to difficult isolation and cultivation, as well as DNA fragments of mycoplasma observed after antibiotic therapy. Studies based on modern microscopy methods showed that microorganisms on the surface of the kidney stone formed multispecies biofilms.

[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.