Isolation and Characterization of Salmonella Bacteriophages as Potential Agents for Phage Therapy of Antibiotic-Resistant Intestinal Infections.

Two bacteriophages specifically active against to pathogenic strains of the Salmonella genus were isolated. The morphology of phage colonies (size, transparency, and shape of the plaque edge, and halo) and the spectrum of their lytic activity and interaction with microbial cells (adsorption rate, duration of the latency, and reproductive efficiency) were examined. Using genome-wide sequencing, we determined the taxonomic position of bacteriophages and verified the absence of unwanted genes encoding toxins, adhesins, and invasins, as well as pathogenicity islands responsible for antibiotic resistance. In addition, phage stability under different physical conditions and their productivity were studied.

Assessment of the Safety of Anti-Salmonella Disinfectant for Veterinary Use Based on a Cocktail of Bacteriophages.

The toxicity and safety of a veterinary anti-salmonella disinfectant based on three highly virulent bacteriophage strains (titers 1010 PFU/ml) were studied. Acute, chronic, and inhalation toxicity, as well as local irritancy of the disinfectant were evaluated on outbred white mice CD1 (n=65), Soviet chinchilla rabbits (n=20), and rats (n=20). No toxic effects of the disinfectant was observed after its intraperitoneal or intragastric administration to mice and intragastric administration to rats; in rabbits, application on the skin and eyes produced no local irritation effect. Inhalation of 10% of the disinfectant did not cause any pathologies in mice. Thus, the tests confirmed the high level of safety of the disinfectant based on a mixture of bacteriophages for use as an additional specific disinfection agent against Salmonella in veterinary and livestock facilities.

[Antiviral and virucidal activity of sodium deoxyribonucleate and its complex with iron against viruses of different kingdoms and families].

INTRODUCTION: The urgent problem of modern medicine is the fight against acute respiratory viral infections (ARVI). To combat ARVI, drugs of wide antiviral potency are needed, as well as immunomodulating drugs. Such antiviral and immunomodulatory effects has sodium deoxyribonucleate (DNA-Na) and its complex with iron (DNA-Na-Fe) developed on the basis of double-stranded DNA of natural origin. AIM OF THE STUDY: To assess antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against viruses of different kingdoms and families. MATERIALS AND METHODS: Antiviral and virucidal activity of DNA-Na and DNA-Na-Fe was assessed in cell cultures infected with viruses. RESULTS AND DISCUSSION: DNA-Na and DNA-Na-Fe had antiviral activity against adenovirus at concentrations of 2501000 mcg/ml. Antiviral effect of both drugs was not detected in case of poliovirus. DNA-Na and DNA-Na-Fe had antiviral activity against coronavirus in all administration schemes. EC50 for DNA-Na ~ 2500 mcg/ml, for DNA-Na-Fe ~ 1000 mcg/ml. In cells treated with DNA-Na-Fe, secretion of following proinflammatory cytokines was detected: Interleukin (IL) 1, IL-2, IL-6, IL-18, interferon- (IFN-), IFN-, as well as anti-inflammatory cytokines: IL-4, IL-10, antagonist of IL-1 receptor. Evidently, DNA-Na and DNA-Na-Fe have antiviral effect, but mechanism of action does not seem to be associated with specific effect on viral replication. Presence of virucidal activity of drugs against representatives of Coronaviridae, Adenoviridae, Picornaviridae, Retroviridae, Herpesviridae in vitro test in range of 1.03.0 lg TCID50 was identified. CONCLUSION: Presence of simultaneous antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against adeno- and coronaviruses shows their prospects for prevention and treatment of ARVI.

Determination of Bactericidal Activity Spectrum of Recombinant Endolysins of ECD7, Am24, Ap22, Si3, and St11 Bacteriophages.

The bactericidal activity of recombinant endolysins LysECD7, LysAm24, LysAp22, LysSi3 and LysSt11 was assayed in multidrug resistant strains (n=120) of Salmonella enterica, E. coli, Acinetobacter baumannii, Enterobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Campylobacter jejuni. The assay showed that the recombinant endolysins had a wide spectrum of bactericidal activity compared to endolysins of their progenitor phages. Among examined endolysins, we selected the active pharmaceutical substances with broad spectrum of bactericidal activity. Most strains were sensitive to LysECD7 (70.7%), LysAm24 (65%), and LysAp22 (58.6%), which seems to be promising causative agents for the development of finished dosage form.

Analysis of the Pharmacokinetics of Suppository Forms of Bacteriophages.

Pharmacokinetics of suppository forms of bacteriophages was studied on male Chinchilla rabbits. Suppositories with various composition of bacteriophages were administered once per rectum to rabbits, and the presence of phage particles was estimated in the blood, urine, and feces over 24 h. Pharmacokinetic study showed that the phages were detected in the blood, urine, and feces at various terms of the experiment irrespective of the size of viral particles, which confirmed the possibility of their systemic effects after rectal administration. Thus, the use of suppository form of bacteriophages can ensure the presence of phage particles even in infection foci that cannot directly contact with the preparation.

[Antiviral activity of extracts of basidiomycetes and humic compounds substances against Human Immunodeficiency Virus (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus 1) and Herpes Simplex Virus (Herpesviridae: Simplexvirus: Human alphaherpesvirus 1)].

INTRODUCTION: One of the most urgent problem of modern medicine is the fight against the disease caused by the Human Immunodeficiency Virus (HIV) - HIV infection. The chemical compounds have improved the situation for infected people, but they are toxic, disrupt the metabolism and cannot eliminate the integrated virus from the body. The emergence of resistant HIV strains makes these treatments ineffective. Often, the death of HIV-infected people occurs as a result of the development of opportunistic infections caused by viruses of the Herpesviridae family. Therefore, the search for new therapeutic and preventive drugs that are less toxic and active against several viruses at the same time is relevant. Basidiomycetes, higher fungi, are a source of medicinal compounds that have antimicrobial properties, as well as antiviral ones. Humic compounds (HS) of various nature also have antiviral activity.The aim of the study was to obtain nontoxic compounds from the basidiomycete Inonotus obliquus and humic compounds from brown coals and to test their activity against viruses that are pathogenic to humans: HIV and Herpes Simplex Virus (HSV). MATERIAL AND METHODS: The antiviral activity of melanin extracts obtained from the culture of the chaga fungus Inonotus obliquus and HS from the brown coal of the Kansko-Achinsk Deposit was studied using a model of MT-4 lymphoblastoid cells infected with HIV type 1 (HIV-1) strains and a monolayer culture of Vero cells infected with HSV type 1 (HSV-1) using virological and statistical research methods. RESULTS AND DISCUSSION: It was found that all the studied compounds did not have a cytotoxic effect on cells at a concentration of 100 mcg/ml. It was shown that extracts of basidiomycetes and HS have antiviral activity against HIV-1 and HSV-1. EC 50 (50%-effective concentration) for HIV-1 was 3.7-5.0 mcg/ml, selectivity index 28-35. Antiherpetic activity was detected at a dose of 50-100 mcg/ml. The antiviral effectiveness of melanin compounds was established both in the «preventive¼ (2 hours before cell infection) and in the «therapeutic¼ regimen of drug administration, both for HIV-1 and HSV-1. The presence of antiviral activity of melanin and HS in relation to the RNA-containing HIV-1 virus and DNA-containing HSV-1 virus in our study coincides with the results of a number of authors in relation to influenza viruses, herpes virus, HIV, hepatitis B virus, Coxsackievirus, smallpox vaccine virus, which suggests that the type of nucleic acid in the virus does not play a fundamental role in the antiviral action of these drugs. It is also clear that HS is effective against both enveloped and non-enveloped viruses. CONCLUSION: In general, it can be concluded that melanin and humic compounds are characterized by low toxicity in the presence of both virucidal and antiviral activity. This allows us to consider the studied compounds as the basis for creating safe medicines that are effective against pathogens of various viral infections.

Bacteriophage Cocktail Effectively Prolonging the Shelf-Life of Chilled Fish.

We developed a technological accessory bacteriophage-based preparation and a method for phage-mediated bioprocessing for elimination of pathogenic microorganisms from the surface of fresh fish and for prolongation of the shelf-life of chilled hydrobionts. Specimens of rainbow trout (Salmo irideus) served as the objects of the study carried out at a fish-processing plant in the Republic of Karelia. The specimens were decontaminated by a bacteriophage cocktail containing six original virulent phage strains characterized by their pheno- and genotypical properties. A new method of biodecontamination (plunging the rainbow trout for 30 sec into a solution of bacteriophage cocktail (bacteriophage titers ≥108 PFU/ml) delayed bacterial degradation of hydrobionts by 3 days. The use of the new method for decontamination of food half-products - phage-mediated bioprocessing - promoted preservation of the initial ecological purity, nutritive value, and taste of the products and prolonged their shelf-life in comparison with the actual standards.

Development of a Bacteriophage Complex with Superhydrophilic and Superhydrophobic Nanotextured Surfaces of Metals Preventing Healthcare-Associated Infections (HAI).

We studied antibacterial properties of organo-inorganic hybrid coatings on the AMg2 aluminum alloy including superhydrophilic and superhydrophobic nanotextured metal substrates with applied bacteriophage particles. Bactericidal activity of surfaces after artificial contamination with a bacterial suspension was evaluated. To increase bactericidal effect of the plates, bacteriophage was sorbed on their surface. In the experiments simulating possible spreading of HAI pathogens, higher bactericidal activity of superhydrophilic surfaces in comparison with superhydrophobic ones. Application of bacteriophage particles did not prevent primary colonization of textured metal surfaces by strains used in the experiment, but in some cases increases their bactericidal activity.

[Diagnostic bacteriophage V32 as a tool for the rapid identification of Escherichia coli serogroup O157.]

Bacteriophage V32, a representative of bacterial viruses of the Myoviridae family Ounavirinae subfamily, is proposed for search and identification of E. coli O157 serogroup, including Shiga-toxin producing E. coli O157:H7 (STEC O157:H7), among cultures of enterobacteria from the primary seeding of the material studied. Phage genome containes a linear double-stranded DNA of 87875 base pairs with G/C-content of 38.9% and includes 132 open reading frames (ORF). In the genome, there are no determinants of antibiotic resistance, virulence genes of STEC and other well-known pathogroups of E. coli. It has been established that phage V32 has lytic activity against all studied cultures of E. coli O157 serogroup (n=183) isolated from people and farm animals in various regions of the Russian Federation, as well as in Japan and Italy. At the same time, the phage lyses only 6 of 182 strains (3.3%) of E. coli not belonging to the O157 serogroup and is not active against strains of other enterobacteria. That is, the phage has a high specificity. The use of bacteriophage V32 as a diagnostic tool is a highly efficient, fast, cheap and simple method for identifying E. coli serogroup O157, including the serotype E. coli O157: H7, in any bacteriological laboratory without special equipment and special training of performers.

Experimental Application of Organic-Inorganic Hybrid Coatings with Adsorbed Bacteriophages for Reducing the Risk of STEC Infections.

A new approach to prevention of STEC infection is based on the synergic effect of combined bactericidal activity of bacteriophages and organic-inorganic hybrid coating of metals. The coatings are characterized by superhydrophilic or superhydrophobic properties and multimodal granularity with the texture incorporating metal oxide nanoparticles and bacteriophages. Superhydrophilic surfaces are characterized by significantly higher bactericidal activity than superhydrophobic ones. The cytotoxicity of superhydrophobic surfaces can be increased due to antibacterial activity of bacteriophages combined with the superhydrophobic characteristics of the materials.

A small-scale experiment of using phage-based probiotic dietary supplement for prevention of E. coli traveler's diarrhea.

Traveler's diarrhea (TD) is caused by Escherichia coli in 30% of cases. We have developed a phage cocktail for prophylaxis of TD caused by E.coli, Shigella flexneri, Shigella sonnei, Salmonella enterica, Listeria monocytogenes or Staphylococcus aureus, and investigated its effectiveness against infection caused by the non-pathogenic Lac (-) strain of E.coli K12 C600 in animal and human trials. On the 6th day of both animal and human trials E. coli K12 C600 strain was detected in titer of 104 CFU/g of mice feces and 106 CFU/g of human feces in the control (untreated) groups, while it was not detected in the samples of either of the study (phage-treated) groups. These results have great significance because the original coliphages included in the cocktail have a broad host-range including ETEC, EAEC and EHEC strains which cause severe cases of TD.

[A panel of the drug-resistance HIV-1 clinical isolates].

A panel of 16 HIV-1 isolates was designed. Those isolates were isolated from patients undergoing HAART and developing resistance to the antiretroviral drugs. It was shown that the isolates were resistant to nucleoside RT inhibitors (retrovir, epivir) and non-nucleoside inhibitors (viramun). Isolates had stable replication activity. Average rate of cells expressing viral Ag was 14-20%. The infectious titer was 2.4 Ig TCID50. The sequencing showed that all isolates were of the subtype A dominating in the major part of Russian Federation. This panel could be used as the biotechnological base for studying antiretroviral drugs of new generation and for the design of experimental vaccines.

[Clinical application of Potentilla alba for prevention and treatment of thyroid gland pathologies].

Available data on the effect of various trace elements on iodine metabolism and function of thyroid gland (TG) were systematized in this article. Subsequently, promising possibilities of herbal therapy in treatment and prevention of TG diseases were considered. Systemic analyis of the results of 3 independent open-label clinical studies performed in accordance with requirements of evidence-based medicine that involved herbal medicinal product Alba® (root extract of white cinquefoil) was carried out. Therapeutic options of Alba® use in various TG pathologies were presented.

[Application of herbal medicine alba in treatment of patients with the pathology of thyroid].

High prevalence of hyperplastic and autoimmune diseases of thyroid in Ukrainian population is determined by endemic deficit of iodine and selenium. The aim of this research was to assess the place of biologically-active additions on the basis of herbal material containing an iodine and selenium in prophylaxis and treatment of thyroid pathology. During the six month period 55 patients received herbal preparation Alba twice a day. The levels of TSH, volume of thyroid, the sizes of nodular goiter (ultrasound investigation) were measured before and at the end of the investigation. The levels of thyroid stimulating antibodies to TSH receptor (AB-r TSH) were evaluated in patients with hyperthyroidism. The results of Alba application showed that in patients with thyroid pathology (diffuse nontoxic goiter, hyperthyroidism and chronic thyroiditis) it was possible to reduce the volume of thyroid, normalize its function, and decrease the level of AB-r TSH in diffuse toxic goiter. We also found approximately 20 % shortening of the time needed to get target level of TSH and finally the duration of treatment of thyrotoxicosis.

[Activity of "Ferrovir" preparation towards RNA and DNA viruses]. / Aktivnost' preparata "Ferrovir" v otnoshenii RNK- i DNK- soderzhashchikh virusov.

Ferrovir (trivalent iron in complex with native sturgeon milt DNA) is nontoxic, its 50% inhibiting concentration (IC50) is at least 4000 micrograms/ml, 90% effective concentration (EC90) towards HIV-1 is 800 micrograms/ml. These effects do not depend on the cell culture or individual biological characteristics and subtypes of 7 strains of HIV-1 used in our study. The chemotherapeutic index of the drug is more than 20. Combined therapy with ferrovir and retrovir had an additive antiviral effect. Ferrovir reduced the titer of human CMV in fibroblast culture by 1-2 Ig TCD50. Ferrovir protected mice after intracerebral inoculation with lethal herpes simplex virus (type 1) (survival 33.7%, protection 27.1%, which is close to the reference group treated with zovirax). These facts evidence antiviral activity of ferrovir towards RNA and DNA viruses and prompt further study of this drug with the aim of its clinical application.

[Morphological analysis of a cell system, infected with different strains of the human immunodeficiency virus]. / Morfologicheskii analiz kletochnykh sistem, infitsirovannykh razlichnymi shtammami virusa immunodefitsita cheloveka.

Cell systems infected with 63 strains of types 1 and 2 HIV virus (HIV-1 and HIV-2) were examined under electron microscope. HIV virions were most frequently detected near the cell membrane or budding from it. In the cytoplasm HIV occurred only in vacuole-like formations. Accumulations of mature virions were seen in the cell-to-cell space. Mature particles of HIV-1 and HIV-2 differed by their morphology from oncoviral C particles and were similar rater to the Visna/Medi type Lentiviruses. Morphological analysis of HIV strains isolated in Russia demonstrated their similarity to be foreign HIV strains.

[The biological properties of Bifidobacterium]. / O nekotorykh biologicheskikh svoistvakh bifidobakterii.

The possibility of the formation of exoenzymes, such as DNAase, RNAase and hemolysin, by bifidobacteria was studied in comparison with their acid-forming and adhesive activity. Bifidobacterium reference strains, originally isolated from healthy adults and children, were studied. The study involved altogether 73 strains of bifidobacteria, including 24 B. bifidum strains, 13 B. adolescentis strains, 7 B. infantis strains, 10 B. breve strains and 19 B. longum strains. The bifidobacteria under study were shown to differ not only in the presence and activity of properties useful for macroorganisms, but also in the presence of enzymes having depolymerizing activity (DNAase, hemolysin). Thus, out of 73 strains under study 9 proved to be DNAase-positive and 6, hemolysin positive. At the same time a specific feature of bifidobacteria was their high acid-forming activity with the complete absence of RNAase activity and insignificant DNAase- and hemolysin-forming activity.

[The characteristics of the HIV isolated from HIV-infected persons and AIDS patients on the territory of the CIS]. / Kharakteristika VICh, vydelennykh ot VICh-infitsirovannykh lits i bol'nykh SPIDom na territorii SNG.

HIV strains were isolated from HIV-infected patients and AIDS patients in CIS. A total of 81 HIV isolates were obtained. The isolates were identified by using immunofluorescence and enzyme immunoassay, by determining the activity of reverse transcriptase, immunoblot, electron microscopy, polymerase chain reaction. Of the 81 isolates 79 were HIV-1 and 2 HIV-2. The strains differed in their infectivity, the kinetics of virus antigen accumulation, and the spectrum of susceptible cell lines. The viruses isolated may be assigned as two groups: high and low infective. The biological properties of the national HIV isolates were shown to be similar to the prototype HIV strains isolated elsewhere.

[The role of echography in the complex dynamic examination of patients with retardation of sexual development]. / Rol' ékhografii v kompleksnom dinamicheskom nabliudenii za bol'nymi s zaderzhkoi polovogo sozrevaniia.

Seventy-nine patients with sexual development retardation were examined, 24 of these suffered from ovarian genesis condition and 55 from central genesis condition. The findings evidence that detection of the uterus and gonads presenting as cords is one of the diagnostic criteria indicating gonadal dysgenesis. Echographic examinations carried out over the course of therapy yield a more accurate picture of ovarian function. No increase in uterine size on the echogram after discontinuation of hormonal therapy and the appearance of follicles in the ovaries after treatment point to normally functioning ovaries and helps specify the origin of sexual development retardation. In sexual development retardation of a central origin ultrasonic scanning helps assess the therapy efficacy and predict its outcome.