Model Construction And Resuscitation Of Dormant M.tuberculosis

ObjectiveTo induce dormant cells of M.tuberculosis by potassium-deficient culture and investigate the potential of supernatant,mycobacteriophage TM4 and Guo1 in resuscitating dormant cells of M.tuberculosis.Methods1.Model construction of dormant M.tuberculosisThe potassium-deficient Sauton medium was used to culture M.tuberculosis at logarithmic phase and to induce it into a dormant state.Mycobacterium tuberculosis colony count,resazurin colorimetric assay and most probable number(MPN)method were used to estimate the viability rate of M.tuberculosis and the proportion of dormant cells.And transmission electron microscope(TEM)was adopted to observe whether the cell wall thickening.Auramine O-Nile red double fluorescent staining was employed to detect the characteristics of loss of acid fastness and lipid accumulation.The drug resistance rates to isoniazide and rifampicin were also tested.2.Resuscitation of dormant M.tuberculosisDormant M.tuberculosis was cultured with supernatant,mycobacteriophage TM4 and Guo1 respectively,and M.tuberculosis colony count was used to detect whether the dormant cells of M.tuberculosis had been resuscitated.Results1.Model construction of dormant M.tuberculosisAfter cultured under potassium-deficient condition for 30 days,M.tuberculosis turns spherical,experiences lipid accumulation and loss of acid fastness,becomes resistant to antituberculosis drugs and its cell wall thickening.2.Resuscitation of dormant M.tuberculosisThe concentration of M.tuberculosis in control,supernatant(SN),TM4 and Guo1 groups was(2.67±0.58)×102 CFU/mL,(3.00±1.00)×102 CFU/mL,(2.67±0.58)×102 CFU/mL,(2.33±0.58)×102 CFU/mL respectively after culturing 1d.Along with further culture,part of M.tuberculosis turned culturable,the concentration of M.tuberculosis increased to(7.33±1.53)×104 CFU/mL,(6.67±2.08)×1010 CFU/mL,(3.00±1.00)×1010 CFU/mL,(3.33±1.53)×1010 CFU/mL separately at the 15 th day.ConclusionsPotassium-deficient culture can induce M.tuberculosis at logarithmic phase into a dormant state.Dormant M.tuberculosis can be resuscitated by supernatant,mycobacteriophage TM4 and Guo1.And the resuscitation ability of supernatant is the strongest.But there is no statistical significance in the resuscitation ability between mycobacteriophage TM4 and Guo1.