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.

[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.

[Infectious genesis of nephroliths (electron-microscopic study)].

Scanning electron microscopy and x-ray microstructural analysis were employed in the study of nephroliths from patients suffering from nephrolithiasis. Bacterial biofilms, urease producing microorganisms, alkaline reaction of the urine are basic factors for local urine crystallization, formation of the base of the nephroliths and its rigid fixation to the pelvic mucosa. Mechanic trauma of the pelvic tissues by the concrement results in destruction of the pelvic mucosa epithelium at the site of the nephrolith. Subsequent inflammation in the underlying connective tissue contributes to formation of connective tissue commissures fixing the conrement in the kidney. It is shown that bacteria as a part of a biofilm are capable to persist in nephroliths for a long time. Destruction of the stones during operation or lithotripsy can trigger activation of growth of bacteria integrated in the biofilm and cause septic complications. Preservation of commissures with elements of the destroyed stone after lithotripsy or surgical removal is one of the leading causes of recurrent nephrolithiasis.

[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.

[Biofilms of pathogenic bacteria and their role in chronization of infectious process. The search for the means to control biofilms].

The role of biofilms in the environment and in the hosts of pathogenic bacteria has recently attracted much attention of researchers. Microorganisms form biofilms at any biotic and abiotic surfaces and thereby cause serious problems in medical practice and other areas of human activity. Biofilms have been shown to be pathogenetic factors responsible for chronization of infectious process. The data are presented illustrating ubiquitous nature of biofilms, their structural and functional characteristics, and modern methods for the study of microbial communities. The discussion is focused on the role of biofilms in chronization of infectious process, enhanced resistance of biofilm organisms to antibiotics and its underlying mechanisms. Approaches to the search for new means for biofilm control during chronic infections are considered.