Next-Generation Antibiotics, Bacteriophage Endolysins, and Nanomaterials for Combating Pathogens.

This review presents various strategies to fight causative agents of infectious diseases. Species-specific programmable RNA-containing antibiotics open up new possibilities for creating next-generation of personalized drugs based on microbiome editing and can serve as a new tool for selective elimination of pathogenic bacterial species while keeping intact the rest of microbiota. Another promising approach in combating bacterial infections is genome editing using the CRISPR-Cas systems. Expanding knowledge on the molecular mechanisms of innate immunity has been actively used for developing new antimicrobials. However, obvious risks of using antibiotic adjuvants aimed at activation of the host immune system include development of the autoimmune response with subsequent organ damage. To avoid these risks, it is essential to elucidate action mechanisms of the specific ligands and signal molecules used as components of the hybrid antibiotics. Bacteriophage endolysins are also considered as effective antimicrobials against antibiotic-resistant bacteria, metabolically inactive persisters, and microbial biofilms. Despite significant advances in the design of implants with antibacterial properties, the problem of postoperative infections still remains. Different nanomodifications of the implant surface have been designed to reduce bacterial contamination. Here, we review bactericidal, fungicidal, and immunomodulating properties of compounds used for the implant surface nanomodifications, such as silver, boron nitride nanomaterials, nanofibers, and nanogalvanic materials.

[The use of synthetic glycoconjugates as components of the immunochromatographic test for rapid serological diagnosis of leprosy.]

The glycoconjugates with BSA (bovine serum albumin) were synthesized using a next saccharide: disaccharide derivative M.leprae PGL-1 (phenolic glycolipid-1); a complex of the disaccharide fragment and the branched hexasaccharide fragment LAM (lipoarabinomannan); diarabinofuranose fragment LAM. These glycoconjugates were used as antigenic components for leprosy rapid serotest construction in immunochromatographic format (leprosy LF serotest). The data obtained with sera of leprosy patients, patients who have been in contact with leprosy, and healthy donors indicate that the most promising antigenic component is a BSA conjugate with two synthetic epitopes - a disaccharide derivative of PGL-1 and a branched hexasaccharide fragment of LAM. The leprosy LF serotest with such glycoconjugate demonstrated the greatest diagnostic sensitivity for main forms of leprosy - paucibacillary (PB) and multibacillary (MB).

Structure and antibacterial properties of Ag-doped micropattern surfaces produced by photolithography method.

Photolithography methods offer ample opportunities for creating biological surface patterns over large areas. Herein, samples with patterned surface having the same Ag total coverage area and content, but different surface topography made of periodically spaced Ag/Si pillars with a diameter of 10 and 50 µm and a height of 3, 1, and 0.2 µm were produced by photolithography technique and studied to uncover the dependences of bactericide ion release on surface topography and antibacterial effect on Ag+ ion concentration. Reactive ion etching of Si wafers in areas unprotected by Ag capping layer was accompanied by a number of competing processes: (i) formation of Ag particles on the tops of pillars due to temperature-activated diffusion and coalescence, (ii) sputtering of Ag from the pillar to surface and redeposition into the etching cavities, resulting in the formation of small Ag nanoparticles located in areas between pillars, (iii) precipitation of AgSix phase as a result of chemical interaction of sputtered Si ions with Ag ions and atoms in surrounding plasma. Samples with the largest pillar heights which had also Ag particles formed between pillars demonstrated the fastest Ag+ ion release and, correspondingly, a noticeable antibacterial effect toward antibiotic-resistant hospital Escherichia coli K-261 strains already after 3 h. All samples showed 100% antibacterial effect after 24 h. Thus our results open up new possibilities for the production of scalable micropattern surfaces with controlled bactericide ion release and pronounced antibacterial characteristics for future applications in the orthopedic field.

Antibacterial Performance of TiCaPCON Films Incorporated with Ag, Pt, and Zn: Bactericidal Ions Versus Surface Microgalvanic Interactions.

It is very important to prevent bacterial colonization at the early postoperative stages. There are four major strategies and their corresponding types of antibacterial surfaces specifically designed to fight infection: bactericide release, anti-adhesion, pH-sensitive, and contact-killing. Herein, we aimed at determining the antibacterial efficiency of different types of bactericidal ions and revealing the possible contribution of surface microgalvanic effects arising from a potential difference on heterogeneous surfaces. We considered five types of TiCaPCON films, with Ag, Zn, Pt, Ag + Zn, and Pt + Zn nanoparticles (NPs) on their surface. The Ag-modified film demonstrated a pronounced antibacterial effect at a very low Ag ion concentration of 0.11 ppb in physiological solution that was achieved already after 3 h of immersion in Escherichia coli ( E. coli) bacterial culture. The Zn-containing sample also showed a noticeable antibacterial effect against E. coli and Staphylococcus aureus ( S. aureus) strains, wherein the concentration of Zn ions was 2 orders of magnitude higher (15 ppb) compared with the Ag ions. The presence of Ag NPs accelerated the leaching of Zn ion out of the TiCaPCON-Ag-Zn film, but no synergistic effect of the simultaneous presence of the two bactericidal components was observed. After the incubation of the samples with Ag, Zn, and Ag + Zn NPs in E. coli and S. aureus suspensions for 24 and 8 h, respectively, all bacterial cells were completely inactivated. The Pt-containing film showed a very low Pt ion release, and therefore the contribution of this type of ions to the total bactericidal effect could be neglected. The results of the electrochemical studies and Kelvin probe force microscopy indicated that microgalvanic couples were formed between the Pt NPs and the TiCaPCON film, but no noticeable antibacterial effect against either E. coli or S. aureus strains was observed. All ion-modified samples provided good osteoblastic cell attachment, spreading, and proliferation and therefore were concluded to be nontoxic for cells. In addition, the TiCaPCON films with Ag, Pt, and Zn NPs on their surface demonstrated good osteoconductive characteristics.

Synergistic and long-lasting antibacterial effect of antibiotic-loaded TiCaPCON-Ag films against pathogenic bacteria and fungi.

Implant-related bacterial infections remain a serious problem that is not solved yet. Herein we combined several antibacterial agents to achieve synergistic effects and broader protection of widely used metallic implants. Titanium samples with microcontainers for drug, produced by selective laser sintering, were coated with Ag-doped biocompatible and bioactive TiCaPCON film and loaded with an antibiotic (gentamicin or a mixture of gentamicin and amphotericin B). Bactericide release tests demonstrated that the release rate of one bactericide agent (Ag+ ions or gentamicin) depended on the presence of the other antibacterial component. The antibacterial activity of the biocide-doped samples was evaluated against clinically isolated Escherichia coli O78 (E. coli), Staphylococcus aureus (S. aureus) bacteria, and Neurospora crassa wt-987 (N. crassa) spores. It was found that samples loaded with low gentamicin concentration (0.2 and 0.02 mg/cm2), i.e. 10 and 100 times less than the standard gentamicin concentration (2 mg/cm2), demonstrated a superb antibacterial activity against E. coli bacteria. We showed that a crosslinking reaction between gentamicin and TiCaPCON film proceeded either through the formation of amide bonds or via the electrostatic interaction between amine groups of gentamicin and COOH groups of TiCaPCON and led to the formation of relatively stable drug/film conjugates that prevented a rapid dissolution of gentamicin and ensured its long-term (for 72 h) antibacterial protection. Leaching of silver ions provided an effective antibacterial protection after the depletion of the drug reservoirs. The obtained results clearly show a synergistic antibacterial action of Ag+ ions and gentamicin against S. aureus bacteria. In addition, in the presence of Ag+ ions, the antifungal activity of samples loaded with a mixture of gentamicin and amphotericin B against N. crassa fungus was observed to increase. Thus, it is demonstrated that silver can be successfully coupled with different types of antibiotics to provide innovative hybrid metal-ceramic bioconstructions that are able to deliver precise doses of bactericide agents within a certain period of time and are equally effective against Gram-negative E. coli bacteria, Gram-positive S. aureus, and N. crassa fungus.

Approaches for Controlled Ag+ Ion Release: Influence of Surface Topography, Roughness, and Bactericide Content.

Silver is the most famous bactericidal element known from ancient times. Its antibacterial and antifungal effects are typically associated with the Ag ionization and concentration of Ag+ ions in a bacterial culture. Herein we thoroughly studied the influence of surface topography and roughness on the rate of Ag+ ion release. We considered two types of biocompatible and bioactive TiCaPCON-Ag films with 1 and 2 at. % of Ag and nine types of Ti surfaces with an average roughness varying in the range from 5.4 × 10-2 to 12.6 µm and different topographic features obtained through polishing, sandblasting, laser treatment, and pulsed electrospark deposition. It is demonstrated that the Ag+ ion release rates do not depend on the Ag content in the films as the main parameter, and it is other factors, such as the state of Ag agglomeration, surface topography and roughness, as well as kinetics of surface oxidation, that play a critical role. The obtained results clearly show a synergistic effect of the Ag content in the film and surface topography and roughness on Ag+ ion release. By changing the surface topographical features at a constant content of bactericidal element, we showed that the Ag+ ion release can be either accelerated by 2.5 times or almost completely suppressed. Despite low Ag+ ion concentration in physiological solution (<40 ppb), samples with specially fabricated surface reliefs (flakes or holes) showed a pronounced antibacterial effect already after 3 h of immersion in E. coli bacterial culture. Thus, our results open up new possibilities for the production of cost-effective, scalable, and biologically safe implants with pronounced antibacterial characteristics for future applications in the orthopedic field.

Toward bioactive yet antibacterial surfaces.

The fabrication of antibacterial yet biocompatible and bioactive surfaces is a challenge that biological and biomedical community has faced for many years, while no "dream material" has been developed so far. The primary goal of this study was to establish an optimal range of Ag concentration and its state of agglomeration in bioactive nanocomposite TiCaPCON films which would provide a strong bactericidal effect without compromising the material biocompatibility and bioactivity. To obtain samples with different Ag content and redistribution, two different methods were employed: (i) TiCaPCON films deposition by magnetron sputtering of composite TiС0.5-Ca3(РО4)2 target followed by Ag(+) ion implantation and (ii) Ag-doped TiCaPCON films obtained by co-sputtering of composite TiС0.5-Ca3(РО4)2 and Ag targets. In order to reveal the antibacterial role of Ag nanoparticles and Ag(+) ions, both separate and in synergy, part of the samples from the first and second groups was subjected to additional ion etching to remove an Ag rich surface layer heavily populated with Ag nanoparticles. All resultant films were characterized with respect to surface morphology, chemical composition, surface roughness, wettability, and Ag(+) ion release. The antibacterial and antifungal effects of the Ag-doped TiCaPCON films were evaluated against clinically isolated Escherichia coli O78 (E. coli) and Neurospora crassa wt-987 spores. The influence of the surface chemistry on spreading, proliferation, and early stages of MC3T3-E1 osteoblastic cell differentiation was also studied. Our data demonstrated that under optimal conditions in terms of Ag content and agglomeration, the Ag-doped TiCaPCON films are highly efficient against E. coli bacteria and, at the same time, provide good adhesion, spreading, proliferation and differentiation of osteoblastic cells which reflect high level of biocompatibility and bioactivity of the films. The influence of Ag(+) ions and nanoparticles on the MC3T3-E1 osteoblastic cells and E. coli bacteria is also discussed.

[Development and testing of an enzyme immunoassay-based monoclonal test system for the detection of the Yersinia pestis V antigen].

An enzyme immunoassay-based test system for Y. pestis V antigen detection was developed. The specificity and sensitivity of this system met the requirements for medical immunobiological preparations for the identification of causative agents of highly fatal diseases. The sensitivity of the test system was assessed, and its high specificity was also demonstrated: the test system did not detect bacterial cells of closely related (four Y. pseudotuberculosis strains) and heterologous microorganism strains. The test system developed was able to detect the V antigen at concentrations as low as 2.0 ng/mL in cells of nine experimental Y. pestis cultures. The obtained preparation can be recommended for use in laboratory diagnostics of plaque.

[Spectrophometric analysis of volatile compounds in microorganisms].

A simple modification of a spectrophometric method was proposed for the rapid detection of microorganisms based on their ability either to excrete or to absorb volatile compounds. The method provides the possibility of contactless control for bacterial growth at a concentration above 10(7) cells/ml. In addition, the method allows discriminating mutants of the fungus Neurospora crassa defective in the nitrogen metabolism from the wild type strains. It is likely that nitrite reductase and nitrate reductase enzymes regulated by the nit-2 and nit-6 genes are involved in formation of the water soluble volatile compounds of this organism.

A fiber-optic lactate sensor based on bacterial cytoplasmic membranes.

A new type of fiber-optic biosensor based on bacterial cytoplasmic membranes (CPM) as the biological recognition element and an oxygen sensitive dye layer as the transducer is described for the detection of lactate. CPMs from bacteria with an induced lactate oxidase system are adsorbed onto a cellulose disk. The disk is fixed mechanically over an oxygen sensitive siloxane layer on the distal end of an optical fiber. This system detects lactate with no interference from glucose, fructose or glutamic acid.

[Repair of membrane damage caused by low temperature freezing of E. coli cells]. / Izuchenie reparatsii povrezhdenii membrannogo apparata, vyzvannykh nizkotemperaturnym zamorazhivaniem kletok E. coli.

Low temperature freezing of E. coli cells causes an almost complete cell damage. A transfer of the frozen cells to nutritional media results in a repair of some of the damages, i.e. in reconstitution of the barrier stability of the E. coli outer membrane detected by a decrease in sensitivity of the frozen cells to the detergent and lysozyme action and in a change of the cell membrane potential measured by the penetrating ion method. The repair of the cytoplasmic membrane damage is followed by the changes in the permeability barrier for H+ and endogenous substrates, which results in restoration of ATP synthesis as a response to the artificial proton motive force and in an induction of beta-galactosidase synthesis. At the same time the synthesis of the periplasmic protein, alkaline phosphatase, in the cells after repair remains suppressed. An analysis of various biosynthetic processes demonstrated that the inhibition of lipid synthesis completely suppresses the reduction processes, while protein synthesis is not necessary for the repair. The importance of the transmembrane electrochemical proton gradient for the repair processed in E. coli cells was established; the ATP biosynthesis essential for the repair occurs, in all probability, via the glycolytic pathway and not via oxidative phosphorylation.

[Functional and structural changes of E. coli membranes induced by low temperature freezing]. / Issledobanie funktsional'nykh i strukturnykh izmenenii membrannogo apparata Escherichia coli posle nizkotemperaturnogo zamorazhivaniia.

Low temperature freezing of E. coli cells causes a fall in endogenous respiration and stimulation of respiration by the non-penetrant substrate NADH. This decrease is not due to disturbances in the function of electron transport chain, since the dehydrogenase and oxidase activities and cytochrome content in the membranes of intact and frozen cells are practically the same. Frozen E. coli cells are incapable of ATP synthesis by artificial proton motive force, although the ATPase activity of isolated membranes is not changed. The disturbances in the penetrability barrier of protons after freezing can be revealed from changes in pH of cell suspensions after rapid acidification. It is assumed that the cell penetrability barrier undergoes alterations causing a loss of respiration substrates and, probably, oxidative phosphorylation uncoupling. A correlation between constant damages of the membrane penetrability barrier and cell survival was established. Using spin labelling of different localization, the changes in the state of membrane surface at the intact hydrophobic lipid zone were demonstrated. It was found that freezing does not induce lipid peroxidation.

[Biosynthesis of L-asparaginase-2 by cultures of Bacillus polymyxa var. Ross]. / Biosintez L-asparaginazy-2 kul'turoi Bacillus polymyxa var Ross.

Cell extracts of Bacillus polymyxa var. Ross.--producer of the polypeptide antibiotic polymyxin M. showed activity of L-asparaginase-2 (L-asparagine aminohydrolase EC 3.5.1.1). The enzyme activity in the growing culture increased with the biomass. The highest specific activity was detected in the cells at the onset of the stationary stage. The synthesis of L-asparaginase-2 was subjected to glucose catabolite repression in response to its addition to the culture at the logarithmic stage. After purification L-asparaginase-2 was obtained that was 350 times more active than the initial preparation. The enzyme properties were examined.