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Active and passive hybrid ways are described for controlling biofilms and the planktonic form of pathogenic microorganisms based on different bactericidal nanomaterials obtained with modern laser technologies. An innovative mobile laser application of the complete suppression of biofilms in situ is described.

Antibacterial photodynamic therapy (APDT) is a promising method of treatment of local infections, based on photochemical reaction of photosensitizer (PS) under irradiation with visible or near infra-red light of appropriate wavelength. A molecule of PS is activated by light energy and as a result singlet oxygen and other reactive oxygen species, which are toxic for microbes, are generated. Antibacterial action of new PS (synthetic polycationic bacteriochlorin derivatives with spectral maximum of absorbance at 760 nm) with improved features was investigated in vitro in this work. Tetra- and octacationic bacteriochlorins with lower molecular mass, as compared with ones previously proposed, turned out to be highly effective against Pseudomonas aeruginosa and Staphylococcus aureus in plankton and biofilm state: reduction of viable bacteria in biofilms reached 5log10. The dependence of the effect on PS concentration, the time of incubation with PS and light dose was ascertained. Light microscopy of biofilms after photodynamic treatment and Live/Dead staining confirmed the death of bacteria or damage of their membranes after exposure to PS and irradiation.

This overview discusses the literature data on the use of bacteriophages in the treatment of both acute and chronic infectious diseases caused by antibiotic-resistant pathogens. Traditionally, phage therapy is based on the use of naturally occurring phages for infection and lysis of bacteria at the site of infection. It has fundamental advantages over antibiotic therapy. At the same time, it has some disadvantages. Currently, the application of biotechnological methods, such as the development of the recombinant bacteriophages, makes it possible to eliminate the shortcomings of antimicrobial phage therapy and to expand its opportunities due to the use of lytic proteins of phages and their modified derivatives.

Making antibacterial PDT more effective is a task that calls for the development of photosensitizers (PS) based on polycationic synthetic bacteriochlorins and subsequent analysis of properties of such photosensitizers. This study aimed to explore photophysical and antibacterial properties of the nanostructured PS based on 3-Py4BSHp4Br4, tetracationic amphiphilic derivative of synthetic bacteriochlorin. The PS was solubilized in a 4% Kolliphor ELP to obtain its nanostructured dispersion. We researched the absorption and fluorescence spectra intensity and profiles, studying concentrations from 0.001 to 0.2 mM, and found that the aggregation level of the PS in question is low throughout the range investigated while the S. aureus (gram-positive) and P. aeruginosa and K. pneumoniae (gram-negative) PD inactivation effectiveness is high.

The method of application laser transfer of silver and copper nanoparticles for the first time has been shown to be effective against biofilms formed on a solid substrate. It has been experimentally confirmed that this effect is not associated with the influence of the laser itself. The proposed method allows one to increase the locality, availability, and efficiency of biofilm destruction due to the bactericidal effect of metal nanoparticles with a slight direct laser effect on the biofilm.

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 ZnPcChol

Bacteriochlorins as the antimicrobial photosensitizers have a promising future in the face of the unrelenting increase in antimicrobial resistance. The goal of this study was to investigate the infl uence of lipophilicity and number of positively charged substituents in these molecules on the photodynamic inactivation (PDI) of biofi lm bacteria in vitro. Testing how bacteriochlorin derivatives with different properties affect microbes will allow to determine the optimal ratio of these parameters within a single molecule. We have investigated 4 bacteriochlorin derivatives, all of which were synthesized in Organic Intermediates and Dyes Institute. These were: hydrophobic neutral meso-tetra(3-pyridyl) bacteriochlorin (ВС1), amphyphilic tetracationic meso-tetra(1- undecyl-3-pyridyl)bacteriochlorin tetrabromide (ВС2), hydrophilic tetracationic meso-tetra[1-(4'-bromobutyl)-3-pyridyl]bacteriochlorin tetrabromide (ВС3) and octacationic meso-tetra[1-(4'-pyridiniobutyl) -3-pyridyl]bacteriochlorin octabromide (ВС4) . The water-soluble cationic bacteriochlorin derivatives showed the most effective PDI of bacteria in biofi lms. While tetracationic BC3 caused total inactivation of S. aureus 15, octacationic BC4 was bactericidal for P. aeruginosa 32 to the same degree (>99.999%). Interestingly, increasing the number of cationic substituents from 4 to 8 in bacteriochlorin molecules enhances bactericidal action against gram-negative bacteria in biofi lms. The lack of charge-carrying groups and high degree of lipophilicity of PS have negative impact on PDI of biofi lm bacteria. Bacterial membrane damage as a result of PDI can be one of the causes of cell death.

Antibacterial nanostructures were written on the surface of a silver (Ag) film and titanium (Ti) wafer using femtosecond laser ablation. The bactericidal properties were tested on S. aureus and P. aeruginosa bacterial strains. The resulting nanostructures were characterized by scanning electron microscopy (SEM). A flow-through filter, based on Ag plates with nanospikes, used as a sterilization of the liquid, led to the almost complete death of bacteria. It is assumed that the antibacterial properties are due to mechanical damage caused by the sharp nanoscale relief.

Cationic bacteriochlorin derivatives which molecules differing by lipophilicity and positive charge degree have been investigated in vitro as photosensitizers for photodynamic inactivation of biofilm bacteria.

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.

Antibacterial photodynamic therapy is a promising method of treating local infected foci, especially surgical and burn wounds, trophic and diabetic ulcers. This work explores the photophysical and antibacterial properties of novel phthalocyanine- and synthetic-bacteriochlorin-based octacationic photosensitizers (PS). The results of the study confirm their low degree of aggregation at high concentrations, as well as high efficiency of photodynamic treatment of Gram-negative bacterial biofilms.

A review of the literature presents data on two-component bacterial regulatory systems that may be possible targets in the search of new antibacterial drugs because of their universality and the role in regulation of pathogenicity. We discuss (a) the structural-functional organization of two-component systems taking as example OmpR family (EnvZ/OmpR and PhoQ, PhoP) regulating a number of processes in bacteria faciliting adaptation to stress from changes in the environment and human host, providing virulence and formation of biofilms, these being a cause of many human chronic infections, and (b) the genes and functions regulated by two-component systems by EnvZ/OmpR system, OmpR protein in particular, and PhoQ/PhoP. The review speculates on possibilities of searching for inhibitors of the proteins of two-component systems and their possible role in virulence attenuation of pathogenic bacteria.

Polycationic derivatives of synthetic bacteriochlorin with absorption in the near infrared range are promising for the creation of photosensitizers (PS) for antimicrobial photodynamic therapy. In the present work, the photophysical and antibacterial properties of PS based on tetracationic derivatives of synthetic bacteriochlorins: hydrophilic 3-Py4BC(EtBr)4Br4 in aqueous solution and amphiphilic 3-Py4BCHP4Br4 in the dispersion of Kolliphor ELP were studied. Analysis of absorption and fluorescence in a wide range of concentrations has demonstrated low aggregation of the PS over the entire range. A high efficiency of photodynamic inactivation of Gram-positive Staphylococcus aureus and Gramnegative Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii bacteria was observed.

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding Lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

The bacteria Burkholderia cenocepacia, which cause pulmonary infections in people with low immunity and in children with inherited diseases such as cystic fibrosis, were studied. The virulence of these bacteria is related with their ability to form viscous biofilms in the lung bronchi and alveoli of patients. Data on the possibility of producing mutants with an altered changed biofilm formation ability using insertion mutagenesis and their characteristics in vivo and in vitro are presented. Three genes involved in the genetic regulation of biofilm formation were identified by cloning and sequencing fragments of mutant genes. Two of the identified genes are the global regulatory genes ompR and tetR, and their mutations result in a decrease or higher ability to form biofilms of B. cenocepacia. Proteomic analysis identified the proteins in mutant strains with a different expression level than in the initial strains. The adhesive properties of the biofilms formed by initial and mutant strains of Burkholderia were evaluated using light and scanning microscopy in an analysis of the interaction between these strains and abiotic surfaces and cell cultures of pulmonary epithelium HL. It was shown that mutant strains with a low ability to form biofilms were characterized by a disturbed adhesion process.

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. Susceptibility does not depend upon the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by a few interacting QTL mapped to chromosomes 3, 9 and 17. To find out whether tuberculosissusceptible I/St mice are susceptible to other intracellular bacteria, we investigated taxonomically distant pathogen, Chlamydia pneumoniae. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former are more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St 9.2 ± 1.2 days, A/Sn − 22.0 ± 2.0 days (p < 0.001). To estimate the degree of chlamydial multiplication in the lungs, we established a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite’s genome equivalents in infected tissue from 1 to 16 days after challenge. Interstrain difference of chlamydia burden in lungs we obtained after 24 hours after infection only. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection, and the numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-α and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

The antibacterial properties of silicon nanoparticles with Gram-positive and Gram-negative bacteria were demonstrated. The nanoparticles were prepared with laser ablation in various liquids (distilled water, isopropyl alcohol) using a nanosecond laser. Optical and scanning electron microscopy methods were used for the sample characterization. The presence of singlet oxygen and other reactive oxygen species on the surface of the silicon nanoparticles, leading to oxidative damage to bacterial membranes and further death of the bacteria, was considered a possible mechanism of this antibacterial action.

Data on the structural and functional traits of bacterial biofilms are presented. The structures involved in different stages of biofilm formation are discussed in detail. Techniques for biofilm modeling and visualization are considered. Results of light and electron microscopic investigations are analyzed. Original data on microscopy of biofilms are presented. The possible formation of dormant and nonculturable forms in the biofilm is discussed.

The goal of this work was to elucidate the mechanism of the direct interaction of bacteria cells with tumor necrosis factor (TNF-α, cytokine). It was shown previously that this interaction facilitated the activation of the bacterial growth and recultivation of noncultivated forms in vitro and in vivo. Mice deficient in genes that encode eukaryotic TNF-α receptors were infected with salmonella cells. When exogenous TNF-α was introduced into the suspension of the infected cells, the infection process (seeding of bacteria from spleens) started one day earlier in both the knockout and control mice, in comparison with that in the absence of cytokine. Bacteria are therefore able to interact with cytokine, even in the absence of eukaryotic receptors. The specificity of the bacteria-cytokine interaction was demonstrated and bacterial protein EF-Tu, which mediates the direct interaction of bacteria with cytokine, was identified using the method of immobilization of the recombinant protein TNF-α-spacer-CBD on cellulose.

Using the methods of transmission electron microscopy, the structure of the biofilms formed by the bacterium Burkholderia cepacia (clinical isolate and mutants with an increased and decreased ability to produce biofilm) were investigated. The biofilms were obtained on a liquid nutrient medium or on an abiotic surface (polystyrene). It has been demonstrated that the cultures of the studied strains differ in some morphological and functional characteristics. In biofilms, changes in the size and submicroscopic organization of all the components of bacterial cells occur. Staining biofilms with ruthenium red revealed the presence of exopolysaccharides in the intercellular space. The differences in the ultrastructure of bacterial films formed on nutrient medium and abiotic surfaces were demonstrated.

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.

Study processes of microbial colonization and persistence of microorganisms in polymer materials for medical use.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.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.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.

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.

This paper is a brief review of data on bacterial biofilms that occur inside and outside of host organisms. Such biofilms are of great ecological and clinical importance. The role of interspecies communications in the development of bacterial biofilms and infectious diseases is particularly emphasized. Considerable attention is given to the electron microscopic study of biofilms formed by Salmonella typhimurium cells incubated as a broth culture in microtubes without aeration. Bacterial samples taken from the biofilm and planktonic culture grown in the same microtube were comparatively investigated by transmission electron microscopy.

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are long DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA gene loci, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.

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.

Five mutants (called tnm) of Escherichia coli with impaired ability for transposition of Tn9 were isolated after treatment with ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NG). The map locations of the tnm mutations were determined by a combination of Hfr matings, F' episome complementation and P1 transductional mapping. The data obtained show that the five tnm mutations are located near 91 min on the Escherichia coli linkage map and are cotransducible with the metA marker with a frequency of 3%-4%. Introduction of F' plasmids containing this region complements the Tnm- phenotype for the two mutants tested i.e. tnm-1 and tnm-2 are recessive in tnm+/tnm- merodiploids.

The effects of different tnm mutations on the transposition of Tn3, Tn5, Tn10, Tn601, and bacteriophage Mu were studied. Five tnm mutations were placed into three phenotypic classes. The representatives of the first class, tnm-1 and tnm-2, caused a complete block of transposition of all Tn-elements studied; the representatives of the second class, tnm-3 and tnm-4, specifically affected Tn9 transposition while the tnm-5 mutation attributed to the third class caused formation of cointegrates between the donor genome of phage lambda carrying Tn-elements Tn3 or Tn5 and the recipient genome (bacterial chromosome).