ABSTRACT
Objective·To construct recombinant mycobacteriophage TM4-RpfE to lay a foundation for experimental research about how to eradicate Mycobacterium tuberculosis in combination with anti-tuberculosis drugs,and how to shorten treatment for tuberculosis ultimately.Methods·Electrotransformation was used to introduce pJV53 plasmid into Mycobacterium smegmatis to prepare recombinant engineering bacteria.After amplification of hsp60-RpfE fusion gene by overlap PCR,a long gene fragment (homologous +hsp60-RpfE+homologous,HHRH) was amplified by multi-step overlap PCR.The DNA of mycobaeteriophage TM4 and HHRH fragment were cotransfected into the recombinant engineering bacteria by electrotransformation,then the recombinant phage from the single primary plaques were confirmed by PCR and sequencing.SDS-PAGE was used to analyze the protein expression in recombinant phage.Results·The hsp60-RpfE fusion gene at the length of 901 bp and HHRH fragment at the length of 1 873 bp were identified by overlap PCR.The PCR product produced 955 bp and 301 bp DNA bands in the first generation plaques colony.SDS-PAGE analysis showed a specific protein band at 21 000 in the recombinant phages.Conclusion·The recombinant mycobacterium phage TM4-RpfE was successfully constructed and the expression of target gene RpfE was initially verified.
ABSTRACT
Purpose: The aim of this study was to isolate a novel mycobacteriophage and then explore its anti‑tuberculosis (TB) potential. Materials and Methods: Phage was isolated from enriched soil sample. A total of 36 mycobacterial strains obtained from clinical specimens were subjected to investigate the host range of phage by the spot lysis assay. Biological characteristics were investigated through growth curve, host range and phage antimicrobial activity in vitro. Then, genome sequencing and further analysis were accomplished by using an ABI3730XL DNA sequencer and comparative genome, respectively. Results: A lytic mycobacteriophage (Chy1) was isolated and the plaque morphology was similar to D29. The genome of Chy1 was estimated to be about 47,198 base pair (bp) and strong similarity (97.4% identity) to D29, especially, the Chy1 gene 7 encoding holin which is considered as a clock controlling growth cycle of the corresponding phage, was identical (100% identity) to phage D29 gene 11, thus classifying Chy1 as a member of the cluster A2 family. However, to our surprise, Chy1 can infect a narrower range of host‑mycobacterial strains than that of D29. The latent period of Chy1 was quite longer compared to D29. Moreover, Chy1 has a weaker ability to lyse Mycobacterium smegmatis compared to D29. Conclusions: The sequence of Chy1 showed 97.4% homology with the genome sequence of D29, but there was a large difference in their biological characteristics. Overall, the results of this investigation indicate that Chy1 is not an ideal candidate for developing mycobacteriophage‑based anti‑TB therapies but for future researches to investigate the reason why biological characteristics of Chy1 and D29 were remarkably different.
ABSTRACT
To investigate the biological characteristics of mycobacteriophage Leo and its bactericidal effect on Mycobacterium tuberculosis for cocktail therapy ,we observed mycobacteriophage plaques after amplifying phage by double‐layer agar plate method .The morphological characteristics were observed by electron microscopy .Mycobacteriophage Leo DNA was extracted and then digested by restriction enzyme to identify the nucleic acid type .Leo was amplified in different multiplicity of infection (MOI) to find the optimal MOI and the minimum MOI .One step growth experiment was carried out to find the latent period and burst size of Leo .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 bacteri‐cidal assay .Results showed that the Leo plaque was round and transparent with a diameter of 1 .5 nm .Leo has an isometric head (70 ± 3 .0 nm in diameter) and a flexible tail (211 ± 31 .7 nm in length) .Its genome could be digested by restriction en‐zyme of Hind Ⅲ and Bgl Ⅰ .The optimal MOI and the minimum MOI of Leo were 0 .000 01 and 0 .000 1 ,respectively .The mutation frequency was 10-7 .The latent period was 150 min ,and the burst size was 74 .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) .In conclusion ,Leo has a bactericidal effect on Mycobacterium tuberculosis and could be a candidate of phage cocktails .
ABSTRACT
Background & objectives: Multiple drug resistance (MDR) among Mycobacterium tuberculosis poses a serious therapeutic problem. Early detection of MDR can be valuable but the conventional drug susceptibility tests take 4-6 wk time after the laboratory isolation of M. tuberculosis. The bacterial phage assay has been reported as a rapid tool for rifampicin susceptibility testing of tubercle bacilli using the suspension of isolated cultures. The present study was aimed to set up a phage assay for testing drug susceptibility to isoniazid (INH), rifampicin, ethambutol, streptomycin and ciprofloxacin in M. tuberculosis isolates. Methods: Mueller-Hinton broth instead of Middle Brook 7H9 broth was used to make it more economical. The phage assay was compared with the proportion method using 100 M. tuberculosis isolates from pulmonery TB cases. Phage assay results were available in 48 h for rifampicin and streptomycin while 72 h required for INH, ethambutol and ciprofloxacin. The assay was compared with gold standard proportion method. Interpretation of the results was easy and clear. Results: In the present study, sensitivity and specificity of the phage assay when compared to proportion method were in the range of 97 to 100 per cent for all the drugs except for ciprofloxacin for which it was 93 and 96 per cent, respectively. Interpretation & conclusions: The phage assay was economic, easy to perform and rapid for the detection of drug resistance in M. tuberculosis isolates with no requirement of expensive equipment. It is within the reach of microbiology laboratories in developing countries having high loads of tuberculosis.
Subject(s)
Drug Resistance, Multiple, Bacterial/drug effects , Ciprofloxacin , Ethambutol , Humans , Isoniazid , Mycobacteriophages/drug effects , Mycobacterium tuberculosis/drug effects , Rifampin , Streptomycin , Tuberculosis/drug therapyABSTRACT
Eighteen temperature-sensitive mutants of mycobacteriophage I3 have been isolated and partially characterized. All the mutants were defective in vegetative replication. Based on temperature shift experiments with the temperature sensitive mutants, the thermosensitive phase of the phage development period has been characterized for each mutant. The genes have been mapped by recombination analysis. The early, continuous and middle genes seem to cluster on the genetic map.
ABSTRACT
A significant positive correlation was observed between multiplicity of infection and burst size of mycobacteriophage I3. During multiple infections, the average contribution of each infecting phage to the burst size was inversely correlated with multiplicity of infection even when bacterial resources were not limiting. We conclude that the efficiency of phage-coded functions rather than the extent of bacterial resources determines the burst size.