title: Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.
abstract: TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.
authors.affiliation: Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.
authors.forename: C
authors.initials: C
authors.lastname: Carrière
authors.forename: P F
authors.initials: PF
authors.lastname: Riska
authors.forename: O
authors.initials: O
authors.lastname: Zimhony
authors.forename: J
authors.initials: J
authors.lastname: Kriakov
authors.forename: S
authors.initials: S
authors.lastname: Bardarov
authors.forename: J
authors.initials: J
authors.lastname: Burns
authors.forename: J
authors.initials: J
authors.lastname: Chan
authors.forename: W R
authors.initials: WR
authors.lastname: Jacobs
mesh_terms: D001431:Bacteriological Techniques / Q000706:statistics & numerical data; D004352:Drug Resistance, Microbial; D017930:Genes, Reporter; D016679:Genome, Viral; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009160:Mycobacteriophages / Q000235:genetics* / Q000502:physiology; D009163:Mycobacterium bovis / Q000821:virology; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification / Q000821:virology*; D012680:Sensitivity and Specificity; D013696:Temperature; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis; D014779:Virus Replication
chemical_list: D008156:Luciferases