The strategy of editing and modification of the genomes of bacterial viruses

In recent decades, a major problem for health systems around the world is the wide spread of bacterial pathogens that are resistant to various antimicrobial agents. A possible approach to solving this problem is the use of bacteriophages, viruses that specifically infect bacterial cells, as well as enzymes and proteins encoded in their genomes. The development of genomic editing technologies, including those based on CRISPR-Cas editing, makes it possible to create genetically engineered or recombinant phage particles with desired properties that are important for further practical application. In this review, we consider issues related to the characterization of bacteriophages as biological objects and as promising candidates for controlling the spread of antibioticresistant bacterial strains. We discuss modern approaches and strategies for modifying the phage genomes using various methods of genetic engineering and molecular biology to solve a variety of practical and research problems. Keywords: bacteriophages, phage genome editing, CRISPR-Cas system

Obtaining and determination of the concentration in the culture fluid of phaecin – peptide bacteriocin Enterococcus faecium

Phaecin, a peptide bacteriocin with a molecular mass of ~5 kDa, was obtained and purified to an electrophoretically pure state with a yield of ~70% of the total activity in the culture fluid. The method of time-of-flight mass spectrometry (MALDI) showed that bacteriocin consists of two components A and B with similar molecular mass. In previously published works, the yield of purified bacteriocins, usually did not exceed 4–5% of the total activity in the culture fluid. The authors believe that the increase in yield is due to the fact that not only the similarity of bacteriocins with high molecular mass proteins was taken into account, but also differences associated with low molecular mass, the ability to hydrophobic interaction at slightly alkaline pH values and resistance to denaturation. Using the Scopes method, it was possible to determine the concentration of phaecin in the final stage of purification and in the culture fluid in absolute units (mg/l). The methods of the purification with high yield described in this work are probably applicable to other bacteriocins due to the proximity of their physicochemical properties. Key words: Enterococcus faecium, Listeria monocytogenes, bacteriocin, phaecin, culture liquid, adsorption chromatography, Scopes equation

Ensuring biological safety requirements when carrying out biotechnological processes with microorganisms of I–IV pathogenicity groups

The article deals with the issue of ensuring and monitoring biological safety requirements in units performing work with microorganisms of I–IV pathogenicity/hazard groups using biotechnological equipment. It is shown that the specificity of the production of biotechnological products requires strict observance of the requirements and provisions of sanitary and epidemiological regulatory and methodological documents establishing requirements, including safety, at all stages of the technological process. Key words: biosafety, microorganisms, biotechnology, technogenic situations

Isolation of endogenous antimicrobial peptides from blood cells

A technique for isolating the endogenous antimicrobial peptides (AMP) has been developed. Leukocyte-erythrocyte-platelet blood mass of donors was used as the material for preparing endogenous AMP. The hydrolysate was obtained with the trypsin solution from this mass, and then the components were fractionated using the filter chromatography column (Simax CSN ISO 3585, Russia). For gel filtration, 1.5 g of Sephadex G-25 was applied to the filter surface followed by sterilizing filtration of the obtained substance through bactericidal filters having a pore diameter of 0.2 μm. The antimicrobial peptide fractions were isolated using high performance liquid chromatography. Subsequently, AMP were encapsulated into the organosilicon niosomes. Key words: antimicrobial peptides, defensins, niosomes, antibiotic-resistant microorganisms

CRISPR/Cas-Systems: characteristics and possibilities of use for editing bacterial genomes

CRISPR-Cas is the adaptive immune system of bacteria and archaea. Since 2012, when the first opportunity to use the CRISPR/Cas system for genome editing was realized, the number of studies in this area has been growing rapidly. Today, genomic editing to modify specific regions of the genomes of various organisms is considered one of the key methodologies of modern biology. This review is devoted to the history of discovery, classification, structure, operational mechanisms of CRISPRCas systems and strategies for editing the genomes of various bacterial species using this technology. Key words: genome editing, genome, CRISPR-Cas system, bacteria

Morphological aspects of microbial biofilm formation on atherosclerotic plaque fragments

Modern research indicates the presence of living bacteria in atherosclerotic plaques. However, the potential of microbes in the formation of biofilms in arterial plaques has not been studied. Purpose of the study. Identification of biofilms on fragments of arterial vessels with atherosclerotic plaques. Materials and methods. Fragments of atherosclerotic plaques isolated during coronary artery bypass grafting in patients with coronary artery disease were cultured in an exhaustive fluid system under anaerobic conditions in vitro for 1 or 14 days. The presence of biofilms was evaluated using scanning electron microscopy. Results. Mixed biofilms, represented by rod-shaped bacteria and cocci, were identified after in vitro cultivation in all fragments of atherosclerotic plaques. The characteristic ultrastructure revealed the main stages of the biofilm life cycle. Conclusions. Modeling biofilms on biotic surfaces in a system of exhausted fluids under anaerobic conditions will allow us to study in detail the phenotype of biofilms and provide a significant understanding of the pathophysiology of infectious processes in blood vessels and atherosclerotic plaques. Key words: atherosclerotic plaque, chronic periodontitis, biofilm-forming microbes, periodontal pathogens, causative agents of nosocomial infections, fluid technology

Comparative evaluation of nutrient media of Escherichia coli isolating for using in veterinary laboratories

The article is focus on comparative studies of modern differential diagnostic nutrient media for effective cultivation and identification of Escherichia coli. The assessment of the quality of media by biological properties was carried out in accordance with their purpose. Special attention is paid to the new generation of differential diagnostic nutrient media. It was found that the tested media have high productivity and inhibitory properties. New culture media of inland producers are not inferior in terms of the characteristics declared by the manufacturers. In case of methods validation the veterinarian laboratory will be able to use new nutrient media. Key words: nutrient media, Escherichia, productivity, selectivity and specificity

Phytoncides: history and application prospects

A brief informational material on the history of the use of phytoncides in medical practice over the past few centuries and the results of our own research on the study of the properties of new drugs based on phytoncides in liposomes is proposed. The effectiveness of phytoncides use for 20 years within the framework of the «Indoor Airspace Ecology» program indicates that this direction is promising. Key words: phytoncides, liposomes, antibacterial agents

Determination of sensitivity of microorganisms of the Klebsiella genus to antimicrobial drugs by the MALDI-TOF MS method

Actual microbiological diagnostics of infections caused by Klebsiella spp. should include isolation of the strain, its identification and fastest possible determination of the pathogen susceptibility to antimicrobial agents. We evaluated the prospects of using MALDI-TOF mass spectrometry to determine the susceptibility of Klebsiella spp. strains to antimicrobial agents. According to the results of mass spectrometry Klebsiella spp. strains analysis, we carried out cluster analysis by the UPGMA method based on mass spectra and data on the susceptibility of the studied strains to antimicrobial agents, and then studied the obtained dendrograms. We identified the areas with the highest probability of the location of antibiotic resistance markers by comparing the mass spectra of susceptible and resistant microorganisms at different concentrations of antimicrobial agents. Thus, using MALDI-TOF mass spectrometry, a new direction in assessing the susceptibility of Klebsiella spp. to antimicrobial agents can be formed. Key words: antibiotic resistance, Klebsiella spp., MALDI-TOF mass spectrometry

Biological properties and molecular and genetic characteristics of plague microbe strains circulating in sandy natural plague foci of the Republic of Kazakhstan

Since 2010, an active course of epizootics with the release of the plague pathogen, isolated from hosts and vectors has been established in 8 autonomous foci of the plague from 14 autonomous foci of the Central Asian plague focus in Kazakhstan. It was necessary to take into account the parameters of variability of the main component of the parasitic system – the plague microbe in the process of certification of landscape and epizootological zoning of natural foci of plague in Kazakhstan. The aim of the work was to study the phenotypic and genetic properties of strains of the plague microbe isolated in natural sandy plague foci of Kazakhstan. Materials and methods. The work used 1196 strains of Yersinia pestis isolated over the past 10 years (2010–2019) from natural sandy plague foci, strain passports, literature sources, data on certification of plague foci in Kazakhstan. The study of the strains was carried out by bacteriological, serological and molecular genetic methods. Results. Certification and typification of the territories of sandy plague foci were carried out, taking into account the phenotypic and molecular-genetic properties of Y. pestis strains isolated from 12 autonomous foci of the Central Asian plague focus of Kazakhstan in 2010–2019. According to the results of the study, 84 atypical strains were identified. As a result of the analysis, 18 genotypes were identified among the studied strains, of which 13 (72.2%) were unique and did not repeat in the sample. The remaining 5 genotypes formed 5 clusters, combining 20 strains (60.6%) and all strains were phylogenetically assigned to representatives of the Mediaevalis biovar. Key words: plague microbe, plague foci, phenotypic features, molecular genetic features