RESUMEN

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.

Asunto(s)

Liposomas/química , Micobacteriófagos/efectos de los fármacos , Animales , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Micobacteriófagos/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Células RAW 264.7

RESUMEN

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).

Asunto(s)

Lisogenia/fisiología , Micobacteriófagos/patogenicidad , Mycobacterium tuberculosis/virología , Animales , Cromatografía por Intercambio Iónico , Ratones , Micobacteriófagos/fisiología , Células RAW 264.7 , Ensayo de Placa Viral

RESUMEN

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.