1. The Search for Hypermutability: Determining Frequency of Mutation in Mycobacterium smegmatis mc2155 ___________
Susan Puckett
Mentors: Dr. Digby Warner, Dr. Valerie Mizrahi
MRC/NHLS/WITS Molecular Mycobacteriology Research Unit,
School of Pathology, University of the Witwatersrand and National Health Laboratory Service,
Johannesburg, South Africa
Funded by the Howard Hughes Medical Institute

2. Background: Tuberculosis (TB)
Contagious, airborne disease caused by Mycobacterium tuberculosis (MTB)
Coughing, weakness, weight loss, fever, death
Over 1/3 of the world’s population infected, with 5-10% projected to become infectious at some point
Estimated Death Toll in 2005
USA
1,345 people
South Africa
101,989 people
World
1,600,000 people
Source: World Health Organization (WHO) website

3. TB Treatment and Drug Resistance
BCG Vaccine, Antibiotics
MDR-TB
Isoniazid, rifampicin resistant
XDR-TB
Fluoroquinolone resistant
Resistant to at least one of three second-line drugs
capreomycin, kanamycin, and amikacin
All drug resistance is chromosomally encoded

4. Understanding Mutation
Problem of drug resistance caused by mutations in genes required for antibiotic effectiveness
Link between mutagenesis and drug resistance:
Boshoff, H., Reed, M., Barry, C., V. Mizrahi DnaE2 Polymerase Contributes to In Vivo Survival and the Emergence of Drug Resistance in Mycobacterium tuberculosis. Cell 113:

5. Questions to Answer
Do mutations occur more frequently under certain conditions?
Is there a hypermutable state in Mycobacterium?
Mycobacterium smegmatis (MSM)
Not pathogenic, genetically similar, faster grower
Hypothesis: Stress-induced hypermutability
Targeting mutation during a hypermutable state could be an effective way of combating TB

6. Karunakaran, P. , and J. Davies. 2000
Karunakaran, P., and J. Davies Genetic Antagonism and Hypermutability in Mycobacterium smegmatis. J. Bacteriol. 182:
FIG Growth phase-dependent hypermutability in M. smegmatis, shown as frequencies of the appearance of resistant mutants

7. Experiment
Test MSM strains mc2155 and mc26 using method outlined in the paper, with different types of media
Incubate cultures and plate on rifampicin plates during log phase and stationary phase over a period of several days
Expectations:
-- More RifR mutants in stationary phase than in log phase -- mc2155 and mc26 generate similar numbers of RifR mutants
-- Process is growth medium independent

8. Results with 7H9, 7H10
First tested with 7H9 and 7H10, media commonly used to grow MSM and MTB. With and without TW80.
Only a few RifR colonies per plate after plating 0.4 ml undiluted culture
NO increase in frequencies of mutation in stationary phase vs. log phase (mutation frequency ~10-8)

9. Results with TSB and TSA
Later tested with tryptic soy broth (TSB) and tryptic soy agar (TSA), the media used in the paper. No TW80.
Log phase: a few mutants and a haze of smaller colonies on rifampicin plates
Stationary phase: a lawn of growth on rifampicin plates
Seems to correlate with paper

10. Final TSB experiment mc2155 and mc26 grown in TSB at 30ºC with TW80
7H10 and TSA used for plating
Simultaneous experiment: sample from starting culture diluted with new media every day and incubated at same conditions as main culture

11. Results
Contamination issues, plates reflect trends as seen in last experiment with TSB
Simultaneous experiment: contaminated
However, certain plates show interesting results

12. mc2155 grows differently on TSA
Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80
mc2155 on TSA
mc2155 on 7H10
mc26 on TSA
mc26 on 7H10
Colonies are smaller, grow more slowly
Consistently different than mc26 results on TSA

13. mc2155 grows differently on TSA
Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80
mc2155 on TSA
mc2155 on 7H10
mc26 on TSA
mc26 on 7H10
Sometimes, colonies do not appear at all. Perhaps longer incubation is needed.

14. mc2155 RIFR mutants grow on TSA
Before plating, these were incubated in TSB at 30ºC, 100RPM, with TW80
mc2155 on TSA (RIF)
mc2155 on 7H10 (RIF)
Stationary phase cultures
Contamination problems with mc26

15. TW80-less TSB encourages growth on TSA RIF plates
Before plating, these were incubated in TSB at 30ºC, 100RPM, without TW80
CONTAMINATED
mc2155 on TSA (RIF)
mc2155 on 7H10 (RIF)
mc26 on TSA (RIF)
mc26 on 7H10 (RIF)
Cultures under similar log phase conditions with TW80 had 0-5 mutants per TSA plate and 0-1 mutants per 7H10 plate

16. Conclusion MSM behaves differently under different conditions
Media: TSB vs. 7H9, TSA vs. 7H10
Presence or lack of TW80
Greater number of RIFR mutants with TSB, TSA
mc2155 behaves differently than mc26 on TSA but not on 7H10
Frequency of mutation: more testing needed
Comparable results to Karunakaran and Davies paper

17. Analysis
If stationary phase hypermutability occurs, it is only under certain growth conditions
Other explanations: competitive advantage
Clustering of cells in TW80-less media could act like biofilm, allowing RIF susceptible cells to survive
Perkins, A., W. Nicholson Uncovering New Metabolic Capabilities of Bacillus sublitis Using Phenotype Profiling of Rifampicin-Resistant rpoB Mutants. J. Bact. 190:
Wrande, M., Roth, J., D. Hughes Accumulation of Mutants in “Aging” Bacterial Colonies is Due to Growth Under Selection, Not Stress-Induced Mutagenesis. Proc. Natl. Acad. Sci. USA . 105:

18. Significance Hypermutability is questionable due to error catastrophe
Significance: cannot assume hypermutability in MTB from in vitro studies, since there is so much variability
Emphasizes need for better understanding of in vivo conditions in order to mimic them in vitro

19. Acknowledgements Dr. Digby Warner Dr. Valerie Mizrahi Dr. Kevin Ahern
Mizrahi lab
University of the Witwatersrand
Howard Hughes Medical Institute (HHMI)