Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli.

Dusthackeer, A.; Kumar, V.; Subbian, S.; Sivaramakrishnan, G.; Zhu, G.; Subramanyam, B.; Hassan, S.; Nagamaiah, S.; Chan, J.; Narayanan, P.R.

Journal of Microbioligcal Methods; 2008; 73; 18-25.

Abstract: The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity.We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis . Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenicmycobacteria switch theirmetabolisminvolving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis . These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis . The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.

Keywords: Dormancy; Mycobacteriophage; Luciferase reporter phage assay

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