To Explore The Application Of Mycobacteriophages On Latent Tuberculosis Infection

ObjectiveTo make it clear that the genitic information and biological characteristics of mycobacteriophage Leo, select appropriate dormant models for mycobacterium tuberculosis and verify whether mycobacteriophage Leo and Guol could crack dormant mycobacterium tuberculosis, and explore their worth treating latent tuberculosis infection (LTBI).Methods1. The biological characteristics of mycobacteriophage LeoMycobacteriophage plaques were observed after amplifying phage by double-layer agar plate method. The frequency of Mycobacterium smegmatis mutation treated with mycobacteriophage Leo was inspected by the endpoint titration test. The effect of temperature and alcohol on Leo survival was surveyed. The ability of Leo to crack host bacteria at different pH values was examined. The effect of Leo on Mycobacterium tuberculosis was determined by bactericidal assay.2. The genitic information of mycobacteriophage LeoMycobacteriophage Leo DNA was extracted by lamda DNA extraction protocol and then digested by restriction enzyme to identify the nucleic acid type. Otherwise,Phred/PhraD/Consedh software packages were used to assemble the shutgun sequences,then the gaps were filled with PCR fragments.DNAStar software Packages, Glimmer3.0 and Promoter prediction/Prokaryotic were applied to predict the general characteristic of Mycobacteriophage Leo genome,the function of the coding gene and the promoter region. In addition,the colinearity analysis and phylogenetic tree were construted. The motif 3 was searched by ClustalX.3. To explore the bactericidal effect of mycobacteriophage on dormant mycobacterium tuberculosis3.1 A comparative study of two in vitro dormant model for Mycobacterium tuberculosisThe hypoxia model was constructed by applying gradually low oxygen. The multiple-stress model was developed by applying combined stress of low oxygen (5% O2), high carbon dioxide (10% CO2), faintly acid (pH 5.0) and lack of nutrient (10% 7H9 broth).After both the model were constructed for 20 days and 30 days, respectively, the sample was collected. Loss of acid fastness and accumulation of lipid bodies was observed through confocal microscopy after dual staining of mycobacterium tuberculosis (Mtb) with the combination Auramine-O and Nile Red. Cell wall thickness of Mtb was observed by electron microscope. And the drug-resistance rate to rifampicin (RFP) was also detected. At 30th day, equivalent Mtb of two models was subcultured into fresh 7H9 broth to observe the auto-resuscitation.3.2 The bactericidal effect of mycobacteriophage on dormant mycobacterium tuberculosisThe hypoxia model was constructed by applying gradually low oxygen for more than one year;The potassium-deficient model was construsted by potassium-deficient Sauton medium for 35 days. The culture of both models was divided into five groups randomly, and then exposed to isoniazide (5μg/mL), rifampicin (5μg/mL), phage Leo (2.6×108PFU/mL),phage Guol (3.26×108PFU/mL), equivalent ddH2O, respectively, for 7 days. Suspensions were employed in triplicate for MPN assays in 48-well Corning microplates at 37 ℃ for 15 days. Resazurin was used to color and the results was recorded.Results1. The biological characteristics of mycobacteriophage LeoThe Leo plaque was round and transparent with a diameter of 1.5 run. The mutation frequency was 10-7. Leo could not only crack host bacteria in solid medium at pH 7.4 but also at pH 5.0. After 72 hours, the amount of Mycobacterium tuberculosis in Leo group was less than that in control group (P<0.05).2. The genitic information of mycobacteriophage LeoThe genome of Leo could be digested by restriction enzyme of Hind III and Bgl I. The full length was 39981bp with a G+C content of 66.86%.59 predicted genes and 3 possible promoter region were contained. Neither tRNA nor tandem repeat sequences was discovered.Leo gene 32 encodes integrases.There were no genes encoding repressor and no toxin genes.Leo was highly similar with BPs and Angel. Motif 3 was contained in Tape measure protein.3. To explore the bactericidal effect of mycobacteriophage on dormant mycobacterium tuberculosis3.1 A comparative study of two in vitro dormant models for Mycobacterium tuberculosisAt 20th day, Mtb of two models showed loss of acid fastness and accumulation of lipid bodies but no transformation of cell wall. At 30th day, Mtb of multiple-stress model almost showed loss of acid fastness and accumulation of lipid bodies. However, portions of Mtb did not show loss of acid fastness and accumulation of lipid bodies in hypoxia model.At 30th day, cell wall thickness of Mtb was observed in multiple-stress model but did not in hypoxia model. The drug-resistance rate to rifampicin of multiple-stress model was obviously higher than that of hypoxia model, and the amount of Mtb in multiple-stress model was smaller than that of hypoxia model after auto-resuscitation in fresh 7H9 broth.3.2 The bactericidal effect of mycobacteriophage on dormant mycobacterium tuberculosisLeo had no bactericidal effect on dormant mycobacterium tuberculosis by hypoxia model.On the contrary, Guo1 had obvious bactericidal effect, and Colony-forming unit counts of the cultures are reduced by 3 logs. In contrast, Leo could completely kill dormant mycobacterium tuberculosis by potassium-deficient model; but Guol just kill a small portion of bacteria.ConclusionsMycobacteriophage Leo was a lytic phage containing motif 3 but no toxin genes. Leo was able to crack mycobacterium tuberculosis. Mycobacterium tuberculosis enter into dormant state faster and more steadily in multiple-stress model than those in hypoxia model. But futher experiment result shows that bacteria in potassium-deficient model suffer deepest dormancy. Mycobacteriophage Leo and Guo1 show potenial in cracking dormant mycobacterium tuberculosis. They could be applied to treat Latent tuberculosis infection.