Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis

Abstract

SUMMARYA temperate phage, Che12, able to infect Mycobacterium tuberculosis, was isolated from soil samples taken from tuberculosis sanatorium area in Chennai, India. The plaque morphology of this phage showed varying grades of turbidity on lawns of M. tuberculosis. The temperate nature of Che12 was established by super infection immunity. Phage integration into the host genomic DNA was confirmed by Southern hybridization using Che12 DNA as a probe. PCR amplification and sequencing of a part of the integrated phage genome in a M. tuberculosis lysogen also confirmed the temperate nature of Che12. The morphology of the phage particles was observed by electron microscopy, revealing similarities to other mycobacteriophages like L5, D29 and TM4. A luciferase reporter phage, phAETRC16, was constructed by cloning firefly luciferase gene into Che12. Infection of viable M. tuberculosis cells by phAETRC16 resulted in expression of luciferase leading to sustained light output. Che12, a true temperate phage infecting M. tuberculosis, is thus ideally suited for developing a diagnostic tool facilitating rapid diagnosis of M. tuberculosis.

Mycobacteriophages BPs, Angel and Halo: comparative genomics reveals a novel class of ultra-small mobile genetic elements

“…The genomes are not homogeneously diverse and can be assorted into clusters of phages that are more closely related to each other than to other mycobacteriophages (Hatfull et al ., 2006), although these clusters likely reflect the variation associated with a small sample size rather than a true representation of the larger population structure. Relatively few of these sequenced phages have been shown to infect Mycobacterium tuberculosis , with the notable exceptions of D29 (Ford et al ., 1998a), TM4 (Ford et al ., 1998b), L5 (Fullner & Hatfull, 1997) and Che12 (Hatfull et al ., 2006; Kumar et al ., 2008). …”

Genomic and proteomic features of mycobacteriophage SWU1 isolated from China soil

“…Plaque morphology of the temperate mycobacteriophage varies significantly (Kumar et al, 2008). The mycobacteriophage SWU1 exhibits characteristic alternative clear–turbid–clear–turbid circles on M. smegmatis mc 2 155 lawn.…”

“…Phage-based research tools such as integrative vector and phage display have contributed tremendously to biomedicine and biotechnology. This holds true for mycobacteriophages, a versatile toolkit for Mycobacterium tuberculosis molecular manipulation and tuberculosis control (Lee et al, 1991; Van Kessel and Hatfull, 2006; Kumar et al, 2008; Marinelli et al, 2008; Van Kessel et al, 2008; Piuri et al, 2009), such as the fluoromycobacteriophages for rapid, specific, and sensitive M. tuberculosis antibiotic susceptibility test, and more recently, the mycobacterial recombineering system enabling more simpler and more efficient gene mutagenesis in Mycobacteria .…”

Pathogen detection using engineered bacteriophages

“…Reviewing 31 studies consisting of a total of 3,085 specimens, Minion and Pai [89] estimated the sensitivity (i.e., the proportion of rifampicin-resistant strains that are correctly identified as being resistant) of the FASTPlaqueResponse method to range from 81 to 100% and its specificity (i.e., the proportion of rifampicin-sensitive strains that are correctly identified as being susceptible) to range from 73 to 100%. Homemade phage amplification kits based on the specificity of phages such as D29 are also used for M. tuberculosis drug resistance profiling [90], as is the luc-based bioluminescent luciferase reporter phage assay, which has been designed around phages such as L5, Che12, phAE40, D29, and TM4 [27,91]. A more recent reporter phage assay used TM4 phage carrying an enhanced GFP gene, but has yet to be tested clinically [92].…”