Transcript Luciferase Reporter Mycobacteriophage (LRP) Assays for

Clare Bruggeman
(http://www.nih.gov/researchmatters/march2009/03092009tuberculosis.htm)
(http://www.biochem.wisc.edu/faculty/rayment/lab/gallery.aspx)
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WHO reported 9.9 million incident cases of TB
in 2008.
TB is treatable, yet it killed 2.3 million people
in 2008.
TB diagnostic tools are old-fashioned.
Goal is to develop an inexpensive, rapid and
accurate tool to diagnose TB, MDR TB and
XDR TB.
Primary Infection in the
Lungs
 Cough
 Coughing up blood
 Chest pain
http://www.path.cam.a
c.uk/partIB_pract/P09
/
TB in Other Organs
 Kidney
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Brain
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Blood in urine
Headache and fever
Spine
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Extreme Curvature of
the spine
http://www.merck.com/mmhe/sec17/ch193/ch193a.html
http://www.path.cam.ac.uk/partIB_pract/P09/
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Although TB is less common in developed
countries, there are a few highly burdened
countries that have 80% of the reported TB cases
(WHO 2009 Report).
DOTS-Directly Observed Therapy
TB can be treated if patients adhere to a strict,
multi-drug regimen.
6-12 months of antibiotic treatment
Attempting to prevent the spread of multi-drug
resistant strains.
Fails to identify 30-50% of active cases
BACTEC and MGIT
http://pathmicro.med.sc.edu/infectious%20disease/mycobacterial%20diseases.htm
1. Find a mycobacteriophage that infects M.
tuberculosis
2. Genetically engineer the mycobacteriophage to
contain luciferase.
3. Infect the cultured sputum sample with the phage.
4. If viable mycobacteria are present, light is produced
in the presence of D-luciferin.
5. If the light does not diminish when the
mycobacteria are treated with an antibiotic, then
the mycobacteria are resistant.
6. Measure Relative Light Units (RLU) with a
luminometer.
Banaiee et al. (2001)
http://www.lifesci.ucsb.edu/~biolum/ch
em/
Schematic of phAE39 and
phAE40 DNA. Constructed in
the E. coli cosmid pYUB216,
then inserted into TM4
mycobacteriophage.
ColE1 = ORI Ap = Amp
(selectable marker for E. coli).
Phsp60 = promoter Fflux =
firefly luciferase (Jacobs 1993)
NTM = Nontuberculosis
mycobacterium
MTC = M. tuberculosis complex
Only really changing the detection method and maybe the culture
media.
Conclusions:
 LRP has the potential to be the rapid and affordable method that
developing countries need to diagnose active TB, MDR TB and XDR TB.
 76% of positive smears were identified with LRP
 If contamination is minimized and both solid and liquid media
methods are used, LRP efficiency increases to 97%.
 94% sensitivity and 100% specificity to MTC(agreement between
BACTEC and LRP)
 LRP determination of drug resistance was possible within 2-4 days
Concern:
 Phage resistant TB strains or phages with too broad of a host range.
Had evidence that a
temperate phage (compared
to a lytic phage) might have
increased light output (i.e.
better sensitivity).
 Collected phage samples
near a tuberculosis
sanatorium in India
 Che12 was a likely candidate
(formed turbid plaques on a
lawn of M. smegmatis).
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M. smegmatis
M. tuberculosis
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Conclusions
 Che12 infects M. smegmatis and M. tuberculosis.
▪ In the lab you can work with the safer M. smegmatis
 Che12 integrates with the host genome.
 phAETRC16 had increased and sustained light output
 Che12 is the first true temperate phage that infects
M. tuberculosis.
 Hope that Che12 will be useful for assays and vaccine
construction because of the sustained light
production.
“The LRP assay appears to be the most
consistently accurate test” (Minion and Pai,
2010).
But the method is still slow, requires noncontaminated samples, and uses up
antibiotics during susceptibility tests.
Minion, J., and M. Pai. 2010. “Bacteriophage assays for rifampicin resistance detection in
Mycobacterium tuberculosis: updated meta-analysis.” Int J Tuberc Lung Dis 14 (8): 941-951.
WHO.int. 2010. The World Health Organization. 29 October 2010. <http://www.who.int/en >.
CDC.gov. 2010. The Center for Disease Control and Prevention. 29 October 2010.
<http://www.cdc.gov >.
“Hypersensitivity.” 2010. Practical Pathology Class Website. Department of Pathology,
University of Cambridge. 29 October 2010. <http://www.path.cam.ac.uk/partIB_pract/P09/ >.
Kumar, V., P. Loganathan, G. Sivaramakrishnan, J. Kriakov, A. Dusthakeer, B. Subramanyam, J.
Chan, W. Jacobs Jr. and N. Paranji Rama. 2008. “Characterization of temperate phage Che 12
and construction of a new tool for diagnosis of tuberculosis.” Tuberculosis 88, 616-623.
Banaiee, N., M. Bobadilla-del-Valle, S. Bardarov Jr., P. F. Riska, P. M. Small, A. Ponce-deLeon, W. Jacobs Jr., G. F Hatfull and J. Sifuentes-Osornio. 2001. “Luciferase Reporter
Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of
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Gali, N. J. Dominquez, S. Blanco, C. Prat, F. Alcaide, P. Coll, V. Ausina and the Mycobacteria
Research Group of Barcelona. 2006. “Use of a mycobacteriophage-based assay for rapid
assessment of susceptibilities of Mycobacterium tubersulosis isolates to isoniazid and
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Jacobs, W. R. Jr, R. G. Barletta, R. Udani, J. Chan, G. Kalkut, G. Sosne, T. Kieser, G. Sarkis,
G. Hatfull, B. Bloom. 1993. “Rapid Assessment of Drug Susceptibilites of Mycobacterium
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