Creating a new problem space: Genetic Diversity of Mycobacterium tuberculosis

1. Creating a new problem space: Genetic Diversity of Mycobacterium tuberculosis Robert Ketcham & Sue Katz

2. Current Lab Activity in General Biology • Models a multiple tb strain human infection • Students model selection of drug resistant strains • Based on a presumption that more than one genetic variant of mtb is present in an infected individual • implication - a mutation • BUT - is that really the case?

3. Natural History of Tuberculosis • Infection with M. tb can lead to a case of tuberculosis immediately or after a long delay (during which time the body defends itself against the bacterium) • Treatment of tuberculosis infection must often use multiple antibiotics and drug resistance is frequent

4. Traditional Thinking has been: • Infection with M. tb. is usually considered to be monoclonal • Recent advances beg to differ

5. Review Article: Gillespie: Evolution of Drug Resistance in Mycobacterium tuberculosis: Clinical and Molecular Perspective [2002] [1955] A series of animal and human trials has led to the concept that there are different populations of bacteria present within the host

6. Different strains identified through • Antibiotic Sensitivity differences • Not Genetic Differences (no techniques available at that time)

7. What Is the Current State of Knowledge? • Many reports on resistance in human isolates, mostly representing • single isolates from groups of infected individuals • sequential isolation studying evolution of resistance to antibiotic treatment

8. These Studies Don’t Address the Question of Clonal Diversity How many strains might an individual be infected with AND do those strains interact

9. Tyrolian Region, Austria, 1999 • Bartender acquired tuberculosis • Epidemiological investigation showed unusual genetic characteristics • Two clusters of tuberculosis tracked to two pubs in the town

10. Epidemiological Study of These Two Clusters of Cases The two clusters differed in antibiotic sensitivity • Some patrons visited both bars………

11. Bartender at Pub ‘Y’ Acquired TB • First characterized as a single strain, but was observed to yield incomplete restriction digestion • ??????? • Moment of Inspiration - perhaps more than one strain present?

12. Subcultured Original Isolation Tube • 28 colonies • 20 Strain P [Predominant in Pub X Cluster] • 6 Strain Oe [Bartender’s Pub Y] • 2 Mixed

15. The Unique Band in Strain from Bartender “This extra band is probably due to an IS6110 duplication event in one of the strains of the mixture” Pavlic et al. 1999

16. South Africa, Warren et al., 2004 • 192 patients • 19 % were infected with multiple strains • Beijing or non-Beijing evolutionary lineage

17. Warren et al. also noted • Genetic diversity not adequately detected by RFLP - the method most frequently used • PCR IS sensitive enough - using primers specific for Beijing type strains and non-Beijing type strains

18. de Viedma et al., 2004 “The assumption that Mycobacterium tuberculosis infections should be considered clonally homogeneous has been weakened in the last few years…”

19. de Viedma et al., 2004 Recent studies have shown • isolation of different strains from sequential episodes • mixed infections by two strains • genetic variations in subpopulations due to microevoution events

20. One Final Piece of Information • Mycobacterium tuberculosis has many ways to become drug resistant • several genes control antibiotic sensitivity/resistance

21. Guillespie, 2002

22. TABLE 1. Characteristics of PZA-R clinical isolates of M.tuberculosis Lemaitre et al 1999