Bactericidal Activity of Liposomal Form of Lytic Mycobacteriophage D29 in Cell Models of Tuberculosis Infection In Vitro.

The use of lytic mycobacteriophages to treat tuberculosis under conditions of acquired resistance to anti-tuberculosis drugs is one of the most practical ways to improve the effectiveness of therapy and reduce the spread of this disease. We studied the efficacy of antimycobacterial action of mycobacteriophage D29 encapsulated into 400-nm liposomes in cell models of tuberculosis infection in vitro. The antimycobacterial action of lytic mycobacteriophage D29 used in free or liposome-encapsulated forms was demonstrated on cell models of intracellularly infected RAW264.7 macrophages and tuberculous granuloma formed by human blood mononuclear cells. The experiments demonstrated pronounced advantage of liposomal form of mycobacteriophage according to the criteria of their penetration into macrophages and lysis of Mycobacterium tuberculosis in culture.

Evaluation of the Efficiency of Lytic Mycobacteriophage D29 on the Model of M. tuberculosis-Infected Macrophage RAW 264 Cell Line.

Culture of mouse macrophages (RAW 264.7 ATCC strain) in wells of a 6-well plate was infected with M. tuberculosis in proportion of 15 mycobacteria per one macrophage and then treated with a lytic strain of mycobacteriophage D29. Antibacterial efficacy of mycobacteriophages was studied using D29 phage (activity 108 plaque-forming units/ml) previously purified by ion exchange chromatography. After single and double 24-h treatment, the lysed cultures of macrophages were inoculated onto Middlebrook 7H10 agar medium. The number of mycobacterial colonies in control and test wells (at least 3 wells in each group) was 300.178±12.500 and 36.0±5.4, respectively (p<0.01).

[The application of lytic micro-bacteriophage В29 for accelerated phenotype detection of sensitivity mycobacteria of tuberculosis to anti-tuberculosis medications.]

In conditions of prevalence of medicine-resistant strains of mycobacteria of tuberculosis necessity in accelerated, including phenotype techniques of detection of sensitivity of mycobacteria to anti-microbial chemotherapeutic medications in clinical samples is an actual issue. The results of application of accelerated phenotype techniques of detection of sensitivity of clinical strains of mycobacteria of tuberculosis to anti-microbial chemotherapeutic medications on the basis application of lytic mycobacteriophage D29 are presented. The principle of technique is in evaluation of reproduction of mycobacteriophage in cells of mycobacteria of tuberculosis in presence of sensitive to them anti-bacterial medications. The reproduction of mycobacteriophage is evaluated by quantitative analysis of phage DNA in polymerase chain reaction in real-time. The study used 102 clinical strains of mycobacteria of tuberculosis obtained after primary cultivation or re-cultivation in tubes of MGIT system (Bactec). After positive results of growth of mycobacteria of tuberculosis were obtained, the samples were incubated during 48 hours in CO2 incubator in the presence of critical concentrations of 10 widely applied in case of treatment tuberculosis medicinal substances in liquid nutrient medium Middlebrook 7H9 enriched with components OADC, in format of 24 well cultural plate with volume of nutrient medium 1 ml per well. Whereupon, in plate wells deposited 2x103 plaque-forming units of mycobacteriophage D29. After 24 hours a qualitative detection of phage DNA was implemented with polymerase chain reaction in real-time using reagents phage D29 ("Syntol", Russia). The increasing of threshold level of fluorescence of Ct more than to 2 cycles in samples with antibiotic as compared with control testifies sensitivity of the analyzed strain of mycobacteria of tuberculosis to antibiotic. The level of coincidence made up to 91% in comparative study with inoculation in Lowenstein-Jensen nutrient medium. The level of coincidence made up to 96% in comparative study with Bactec test-system of limited number of strains with establishment of sensitivity for 10 medications. The data was confirmed concerning inverse relationship of value ∆Ct and minimal inhibiting concentration of medication. The supposed high efficiency of possible reagents' set on the basis of presented technique on cost/quality criterion.

Application of water-soluble nanofilters for collection of airborne Mycobacterium tuberculosis DNA in hospital wards.

A simple inexpensive technique for collection of airborne biomarkers of nosocomial infections is described. Biomarkers were collected on water-soluble electrospun nanofilters attached to a household vacuum cleaner from 6-10m(3) of air in 10-15min within several wards of a tuberculosis clinic. Filters were then dissolved in water and tested for the presence of the IS6110 and regX3 genes of Mycobacterium tuberculosis (MTB) using real-time polymerase chain reaction. It was demonstrated that trace amounts of airborne MTB DNA (>3gene copies/m(3)) were always present in the air and on the surfaces in the wards occupied with tuberculosis patients having positive smear tests.

Enzyme linked immunosorbent assay for identification of whole Mycobacterium bovis isolates with the monoclonal antibodies.

OBJECTIVE: The development of a test system for identification of Mycobacterium bovis with the use of monoclonal antibodies (Mabs). DESIGN: BALB/c mice were immunized with whole gamma-radiation-exposed M. bovis strains. The splenocytes of the immunized mice were used for producing hybridomes which generated Mabs specific to M. bovis. The specificity of the Mabs was determined with the ELISA procedure for the whole cells of the 11 mycobacteria of various species fixed in the wells of a microtitre plate. The optimal conditions for performing identification were selected. RESULTS: Three Mabs specific to M. bovis have been produced which identify various epitopes of the protein with a molecular weight of 31 kD. The ELISA tests with the 2A1 Mab have been conducted for 213 strains of 15 species of mycobacteria. the technique has been shown to be highly specific and reproducible. CONCLUSION: An ELISA test system with a Mab for identification of M. bovis has been developed.

The comparative pharmacokinetics of 3H-dihydrostreptomycin in solution and liposomal form in normal and Mycobacterium tuberculosis infected mice.

In intact and tuberculous mice a comparative pharmacokinetics of 3H-dihydrostreptomycin (DHS) encapsulated into the liposomes (LE) prepared from egg lecithin and soluble form of DHS (SOL) after their intravenous administration has been studied. The values of the total area under the serum concentration--time curve of DHS (AUC 0----infinity), administered in the LE form was 8.8 times higher in normal and 5.9 times higher in mice with advanced tuberculosis than in mice treated with soluble form of DHS (SOL). AUC 0----infinity values in spleen and liver were 2.2 and 4.5 times higher, respectively, in use of LE form, where as AUC 0----infinity values in lungs and kidneys were independent from the drug form of DHS in normal mice. DHS levels in lungs of tuberculous mice were 3 times higher 3 hours after administration of LE in comparison with those after SOL DHS. AUC 0----infinity values in spleen and liver of tuberculous mice were 9.2 and 7.3 times higher, respectively, in comparison with those after SOL DHS. AUC 0----infinity values in kidneys of tuberculous mice didn't differ significantly independently from the use of one or another form of the drug.

Antibacterial activity of liposome-entrapped streptomycin in mice infected with mycobacterium tuberculosis.

Streptomycin sulfate was entrapped in liposomes obtained from lecithin by a detergent method at the ratio of 250-300 mcg streptomycin-base per 1 mg of lecithin. Intravenous injection of liposome-entrapped streptomycin (LES) to the mice infected with Mycobacteria tuberculosis H37 Rv led to a statistically significant decrease in the spleen mycobacterium number, but not in the lungs, as compared to the buffered solution of streptomycin (SS). Intravenous treatment of the mice with LES resulted in prolonged mouse survival, as compared to that with SS. Acute streptomycin toxicity was also reduced by its intravenous administration in the form of LES.

In vitro restoration by thymosin of T lymphocytes from patients with lung tuberculosis.

The percentage of E-rosette forming T-lymphocytes is considerably decreased in a number of patients with severe form of lung tuberculosis, particularly in those with tuberculous empyema of the pleura (28.5% to 38%). After in vitro incubation of lymphocytes from those patients with thymosin V fraction T-lymphocytes restored normal.