robert ketcham & sue katz creating a new problem space: genetic diversity of mycobacterium...

Robert Ketcham & Sue Katz

Creating a new problem space: Genetic Diversity of Mycobacterium tuberculosis

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?

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

Traditional Thinking has been:

• Infection with M. tb. is usually considered to be monoclonal

• Recent advances beg to differ

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

Different strains identified through

• Antibiotic Sensitivity differences– Not Genetic Differences (no

techniques available at that time)

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

These Studies Don’t Address the Question of Clonal Diversity

How many strains might an individual be infected with AND do those strains interact

Tyrolian Region, Austria, 1999

Bartender acquired tuberculosis Epidemiological investigation

showed unusual genetic characteristics

Two clusters of tuberculosis tracked to two pubs in the town

Epidemiological Study of These Two Clusters of Cases

The two clusters differed in antibiotic sensitivity– Some patrons visited both

bars………

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?

Subcultured Original Isolation Tube

• 28 colonies – 20 Strain P

[Predominant in Pub X Cluster]

– 6 Strain Oe [Bartender’s Pub Y]

– 2 Mixed

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

South Africa, Warren et al., 2004

192 patients 19 % were infected with multiple

strains Beijing or non-Beijing evolutionary

lineage

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

de Viedma et al., 2004

“The assumption that Mycobacterium tuberculosis infections should be considered clonally homogeneous has been weakened in the last few years…”

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

One Final Piece of Information

• Mycobacterium tuberculosis has many ways to become drug resistant– several genes control antibiotic

sensitivity/resistance

Guillespie, 2002

TABLE 1. Characteristics of PZA-R clinical isolates of M.tuberculosis

Lemaitre et al 1999