Download Creating a new problem space: Genetic Diversity of Mycobacterium

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