Preliminary Research About Mycobacteriophage Cocktail Therapy Against Drug-Resistance Tuberculosis

ObjectiveTuberculosis severely jeopardizes human health while the preventionand control of the drug-resistant TB is not optimistically. LyticMycobacteriophage is one of the therapeutic potential for it can infects anddestroy mycobacterium. We researched on its biological characteristics,complete genome sequence and annotation of phages, the reactionconstants between antiserum and phage to select the phage ofmycobacteriophage cocktail. We have observation on the effect ofmycobacteriophage cocktail on M. tuberculosis in vitro, on howmycobacteriophage cocktail affects the resistance mutation frequency of M.smegmatis and on the safety of mycobacteriophage cocktail for explorationits anti-drug-resistant tuberculosis potentials.Methods1. Research on the biological characteristics of mycobacteriophage,including size, ultrastructure, MOI, the replication cycle and the host range,to select the phases of strong lysis ability and broad host range phageamong D29,33D, TM4, Legendre, DNAIII, BO4, Leo, Clark and Sedeg forthe mycobacteriophage cocktail preparation. 2. The genome of the selected33D and Legendre were sequenced byshotgun, and the gene annotations of those genomes were completed so thatwe can exclude the lysogenic phage and the phage which has the virulencegenes.3. The reaction constants between antiserum and corresponding phagewere identified by neutralization test to antigenicity identification. Thecross reaction constants between antiserum and another phage wereidentified by cross neutralization test. The effect of mycobacteriophagecocktail to M.tuberculosis in vitro was investigated. The frequency ofM.smegmatis mutation treated with mycobacteriophage cocktail wasinspected by the endpoint titration test. The mice were randomly divided3groups: the control group, the phage cocktail-MS group, and the phagecocktail group. Mice exposed in different groups were executed separatelyat1h and4h after the exposure. The picked-off whole lung and tracheawere used to prepare the lung homogenate and marination respectivelyafter the anatomization. The phage concentration of those samples wasmeasured by the double-layer agar plate count method to determine theeffect of MS on the phage delivery efficiency and the survival rate in vivo.4. Evaluate the safety of mycobacteriophage cocktail by the Allergytest and the pyrogen detection. Observe the process of the phage clearanceby macrophages. Mycobacteriophage cocktail was delivered to the micethrough nasal drops and normal saline was used as the control. Exposure was performed twice in the first week, then once every Sunday since thesecond week. Mice were executed and anatomized after8-week treatment.Animal physiological and behavioral state was observed. The mouse organsat a gross and histological level were observed.Results1. The plaques of D29are transparent and the size is about5mm indiameter. D29has a long tail about129nm in length. Host bacteria infectedby10^-4MOI produced the highest phage offspring. The single step growthtest showed that, phage D29had a latent period of50minutes and a burstsize of10. The susceptibility to phage D29was also investigated with otherclinical strains of Mycobacterium except H37Rv. The plaques of33D aretransparent and the size is about2mm in diameter.33D has a long tailabout201nm in length. Host bacteria infected by10^-5MOI produced thehighest phage offspring. The single step growth test showed that, phage33D had a latent period of150minutes and a burst size of24. Thesusceptibility to phage33D was also investigated with other clinical strainsof Mycobacterium except H37Rv. The plaques of TM4are transparent andthe size is about1.5mm in diameter. TM4has a long tail about177nm inlength. Host bacteria infected by10^-4MOI produced the highest phageoffspring. The single step growth test showed that, phage TM4had a latentperiod of150minutes and a burst size of28. The susceptibility to phageTM4was also investigated with other clinical strains of Mycobacterium except H37Rv. The plaques of Legendre are transparent and the size isabout1mm in diameter. Legendre has a long tail about215nm in length.Host bacteria infected by10^-4MOI produced the highest phage offspring.The single step growth test showed that, phage Legendre had a latentperiod of180minutes and a burst size of13. The susceptibility to phageLegendre was also investigated with other clinical strains ofMycobacterium except H37Rv. The plaques of DNAIII are turbid and thesize is about0.5mm in diameter. DNAIII has a long tail about208nm inlength. Host bacteria infected by10^-4MOI produced the highest phageoffspring. The single step growth test showed that, phage DNAIII had alatent period of165minutes and a burst size of28. The susceptibility tophage DNAIII was also investigated with other clinical strains ofMycobacterium except H37Rv. The plaques of BO4are transparent and thesize is about1mm in diameter. BO4has a long tail about129nm in length.Host bacteria infected by10^-4MOI produced the highest phage offsprings.The single step growth test showed that, phage BO4had a latent period of120minutes and a burst size of37. The susceptibility to phage BO4wasalso investigated with other clinical strains of Mycobacterium exceptH37Rv. The plaques of Leo are transparent and the size is about1.5mm indiameter. Leo has a long tail about150nm in length. Host bacteria infectedby10^-5MOI produced the highest phage offspring. The single step growthtest showed that, phage Leo had a latent period of150minutes and a burst size of74. The susceptibility to phage Leo was also investigated with otherclinical strains of Mycobacterium except H37Rv. The plaques of Clark areturbid and the size is about0.5mm in diameter. Clark has a long tail about227nm in length. Host bacteria infected by10^-4MOI produced the highestphage offsprings. The single step growth test showed that, phage Clark hada latent period of150minutes and a burst size of26. The susceptibility tophage Clark was also investigated with other clinical strains ofMycobacterium except H37Rv. The plaques of Sedeg are turbid and thesize is about1mm in diameter. Sedeg has a long tail about201nm inlength. Host bacteria infected by10^-4MOI produced the highest phageoffspring. The single step growth test showed that, phage Sedeg had alatent period of120minutes and a burst size of39. The susceptibility tophage Sedeg was also investigated with other clinical strains ofMycobacterium except H37Rv. Based on the biological characteristics ofmycobacteriophage,the mycobacteriophage D29,33D,TM4,Legendrewere chosen as the phage of mycobacteriophage cocktail.2.33D was sequenced by shotgun and a dsDNA sequence of50036bpwas obtained. Sequence analysis was performed using Glimmer. Thegenome encodes84gene,only17have been identified to share significantsequence similarities to genes with known functions. The comparison ofgene order and the protein sequence similarities indicate the putativefunctions of the17gene in the genome of33D. We propose that gene6, gene7,gene8,gene9,gene14are related to DNA Replication. Wepropose that gene6(WhiB) is related to gene regulate. We propose thatgene47(Lysin B) and gene48(Lysin A) are related to bacteria lysis. Wepropose that gene57, Gene59,gene60,gene61,gene64,gene70,gene74,gene75,gene76are related to structure and assembly.33D islytic phage which have not virulence gene.Legendre was sequenced by shotgun and a dsDNA sequence of40733bp was obtained. Sequence analysis was performed using Glimmer. Thegenome encodes58gene,only19have been identified to share significantsequence similarities to genes with known functions. The comparison ofgene order and the protein sequence similarities indicate the putativefunctions of the19gene in the genome of Legendre. We propose gene25(RDF),gene26(Repressor) and gene27(Integrase) encodes lysogenymodule. We propose that gene31(Lysin B) and gene32(Lysin A) arerelated to bacteria lysis. We propose that gene39,gene41,gene42,gene43,gene44,gene45,gene46,gene52,gene53,gene56,gene57are related to structure and assembly. Legendre is lysogenic phagewhich have not virulence gene.3. This phage cocktail consisted of three phages (33D, D29and TM4)which have different but overlapping host strains.33D, D29and TM4weresensitive to temperature, UV ray, pH value and alcohol. Bacteria-lysisabilities of D29was stable,33D had decline, TM4was lost. Rate constant of reaction between antiserum and D29was1070. Rateconstant of reaction between antiserum and33D was703.80. Rate constantof reaction between antiserum and TM4was234. The lowest antigenicitywas TM4. Serological cross-neutralization test showed that there was littlecorrelation among D29,33D and TM4.Compared with any single phage (10^-6), the phage cocktail (10^-8)significantly reduced the mutation frequency of MS (P<0.05). Comparedwith single phage D29, the phage cocktail significantly decreased thesurvival rate of MS and MTB (P<0.05).The production of resistant variant following phage cocktail treatmentwas significantly less compared with monophage treatment.The detection result of double-layer agar plaque test on the PFU ofphage treated with different groups for1h,4h in Trachea and Lung ofmices at1h and4h after exposure were analyzed. The PFU of phage ofprotective agent group higher than the nude phage at1h or4h, and thedifference was significant (P<0.05).4.14days after sensitization and challenge with crude phagesuspension, there were anaphylactic reaction of guinea-pigs.14days and21days after sensitization and challenge with purified phage, there was noanaphylactic reaction of guinea-pigs. New Zealand rabbits of purifiedphage (D29,33D, TM4) treated, there was no obvious change in bodytemperature at the above given time by pyrogen reaction (P>0.05). The process of macrophages phagocytosis phage was observed.Double immunofluorescent data show that maccophage phagocyted a largenumber of phage,but no phagolysosome after10min interaction betweenmaccophage and phage. There was a little phage-phagolysosome after15min interaction between maccophage and phage. There was a large numberof phage-phagolysosome after20min interaction between maccophage andphage. Phage was eliminated completely after48h interaction betweenmaccophage and phage.When mice had been treated with phage cocktail or saline normal for8weeks, the food intake and active values were normal in each group.Summarizing the results of weight, pathologic reaction of gross organs andthe histological findings, the lung or liver or spleen of mice occurred nopathological changes.ConclusionsCompared with the single phage, mycobacteriophage cocktail therapyis more capable to kill the drug-resistant tuberculosis, with lower mutationfrequency for the host bacteria, and without obvious toxic side effect forthe test animals. Therefore mycobacteriophage cocktail therapy is safer toexplore the anti-drug-resistance tuberculosis potentials. This paperprovided the basic experiment data for developing mycobacteriophagecocktail therapy into a new anti-drug-resistance tuberculosis drug, but suchdevelopment still needs data support of animal experiment in vivo. It is also important to develop a phage torpent for the human being in the follow-upresearches.