A Next Gold Standard for Genotyping of Mycobacterium tunerculosis complex isolates

1. By:
RishendraVerma &
Sangram Ramane
Mycobacteria Laboratory
WELCOME TO PRESENTATION
ON
DIVISION OF BACTERIOLOGY AND MYCOLOGY, IVRI, IZATNAGAR

2. CONTENTS
 Background
 MTBC
 Genome and genomic markers
 Typing of Mycobacteria
 What isVNTR and MIRU?
 MIRU-VNTR typing
 Advantages
 Conclusion

3. BACKGROUND
Tuberculosis (TB) - Most widespread infectious disease caused by
Mycobacterium tuberculosis complex (MTBC).
A leading cause of death due to a single infectious agent in the world.
Worldwide over 2 billion people carry theTB bacterium, with
approximately 15 million people suffering from an active infection at any
one time.
TB kills 1.6 million people every year;
4,400 people every day;
One person every 20 seconds (tballiance.org).

4. Mycobacterium tuberculosis complex
(MTBC)
M.tuberculosis,M.bovis,M.africanum,M.microti and M.canettii.
Other members: M.caprae (Aranaz et al.,2003)
M.pinnipedii (Cousins et al.,2003).
Natural habitat: M.tuberculosis and M.africanum: Human
M.microti:Small rodents.
M.bovis is having broadest host range among the all members of MTBC.

5. GENOME
M.bovis genome:
A single circular chromosome,
4,345,492 bp in length,
An avg. G + C content of 65.63%,
Includes a prophage and
42 IS elements
>99.95% identical to that of M.tuberculosis at the nucleotide level
(Garnier et al., 2003).

6. GENOMIC MARKERS
M.tuberculosis strain specific genomic markers (Thierry et al., 1990;
Frothingham et al., 1998):
Chromosomal locations,
Copy numbers and
Genetic recombination associated with IS and repetitive
elements
Genomes of the members of MTBC are highly conserved and lack
interstrain genetic diversity (Sreevatsan et al., 1997).

7. INSERTION SEQUENCES
IS6110
1361 bp long
Copy no.: M.tuberculosis - 8 to 15; M.bovis - 1 to 5; M.bovis BCG - 1
IS1081
1324-bp
Low polymorphism
REPETITIVE ELEMENTS
PGRS
Present in at least 26 loci and consisted of many tandem repeats of the
consensus sequence (~10bp length) of CGGCGGCAA
MPTR
Tandem repeats of a 10 bp consensus sequence separated by 5 bp highly
heterogeneous spacers.
DR
Multiple 36-bp DR copies, which are interspersed by non
repetitive short sequences of about equal length.
VNTR
The repetition in tandem of a short (>16 bp repetitive sequences) motif
spanning 0.5 kb to several kb; include MIRUs
Genomic markers contd……

8. Genomic markers contd……

9. TYPING OF MYCOBACTERIA
Non-DNA
typing
Based on
Biochemical
features
Sensitivity to
antibiotics
Phage typing
Genotyping
IS6110-RFPL Deligotyping Spoligotyping
MIRU-VNTR
typing

10. VARIABLE NUMBER TANDEM REPEAT
(VNTR)
Tandemly repeated sequences are dispersed by thousands of copies in
virtually all higher eukaryote genomes (Cox and Mirkin, 1997).
Microsatellites: Loci with short sequence repeats (SSRs) of 1-13 bp
Minisatellites: Those with 10-100 bp sequence repeats (Tautz and Renz,
1984;Tautz and Schlotterer, 1994).
The loci which shows hyper-variability in their repeat are called asVariable
NumberTandem Repeat (VNTR) loci (Nakamura et al., 1987; 1988).

11. VNTR contd……
VNTRs are also characterized in various
prokaryote genomes which shows
extensive polymorphisms (Field and
Wills, 1996; Hood et al., 1996; Peak et
al., 1996; van Belkum et al., 1997).
Valuable tools for studying pedigree &
evolutionary distant phylogenetic
relationships (Jeffreys et al., 1991;
Sutherland and Richards, 1994; Epplen et
al., 1997).

12. Mycobacterial Interspersed Repetitive
Units (MIRU)
In 1997 Supply et al. found minisatellite-like structures in the M.
tuberculosis genome.
Composed of 40-100 bp repetitive sequences called Mycobacterial
Interspersed Repetitive Units (MIRUs).
Scattered in 41 locations throughout the genome
12 of these loci display polymorphisms in MIRU copy numbers among non-
related M.tuberculosis isolates (Supply et al., 1997).

13. MIRU Types
TYPE 1: Consist of 77 nucleotide repeat units
TYPE 2: Has 24 bp gap in the 3’ portion of type 1 sequence
TYPE 3: It has 15 bp gap in 5’ portion of the type 1 sequence
MIXED type 2 and type 3 MIRUs have also been discovered, which
contain gaps in both 3’ and 5’ regions
MIRU contd……

14. MIRU contd……
41 MIRU loci
Bulleted 12 polymorphic loci

15. MIRU-VNTR Typing
VNTR typing is valuable for genotyping of genetically homogeneous
pathogens:
• Bacillus anthracis (Keim et al. 1999, Le Fleche et al. 2001),
• Yersinia pestis (Le Fleche et al. 2001, Klevytska et al. 2001),
• Streptococcus pneumonia (Koeck et al., 2005),
• Bordetella pertussis (Schouls et al., 2004),
• Legionella pneumophila (Pourcel et al., 2003),
• Pseudomonas aeruginosa (Onteniente et al., 2003),
• Lactococcus lactis (Quenee et al., 2005).
VNTR typing systems made for MTBC strains initially made use of very
limited sets of polymorphic loci (Frothingham et al. 1998; Goyal et al.
1994; Magdalena et al. 1998; Namwat et al. 1998), which were not be
sufficiently discriminatory (Kremer et al., 1999).

16. Subsequently, more extensive sets ofVNTR loci have been described
for MTBC genotyping:
• Exact tandem repeat (ETR)A, B, C, D, E
• Queens University Belfast (QUB)11a, 11b, 18, 26, 1895, 3232, 3336, 4156, 1451, 4052
• VNTR0424, 1955
• Mtub04, 30, 39, 29, 34, 21
• 12 loci MIRU: MIRU2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40
• 15 loci MIRU-VNTR: MIRU4, 10, 16, 26, 31, 40, Mtub04, Mtub30, Mtub39, Mtub21,
ETR-C, ETR-A, QUB4156, QUB11b, QUB26
• 24 loci MIRU-VNTR: MIRU2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40, Mtub04, 21, 30, 39,
29, 34, QUB11b, 26, 4156, ETRA, B, C
MIRU-VNTR typing contd……

17. In MIRU-VNTR typing mycobacterial isolates are typed by determining the
number of copies of specific repeat units at various MIRU-VNTR loci
scattered throughout genome.
After calculating number of repeats at all selected loci, obtained data is
analysed by using different softwares like BioNumerics (Supply et al.,2006;
Ali et al.,2007).
The number of repeat units at specific locus is determined by PCR
amplification of entire MIRU locus, followed by gel electrophoresis, where
the number is determined by amplicon size.
MIRU-VNTR typing contd……

18. MIRU-VNTR typing contd……

19. MIRU 02 04 10 16 20 23 24 26 27 31 39 40
Size
(bp)
283 332 201 168 365 287 414 303 226 264 226 220
No. of
copies
2 3 2 2 3 4 2 5 3 3 2 2
MIRU pattern: 232234253322
DIGITAL RESULT
MIRU-VNTR typing contd……

20. MIRU 4
DNA
MIRU 2
PCR
amplification
Strain 1
3 repeats 2 repeats
MIRU 4
DNA
MIRU 2
PCR
amplification
Strain 2
1 repeat2 repeats
MIRU-VNTR typing contd……

21. PROFILING OF MIRU LOCI
Loci MIRU
1
2
3
4
5
1042 2624232016 3127 39 40
6
7
8
M.tuberculosis H37Rv 2 33 2 2 5 1 3 3 3 2 1
MIRU-VNTR typing contd……

22. ANALYSIS OF MIRU
2 4 10 16 20 23 24 26 27 31 39 40
A B A B A B A B A B A B A B A B A B A B A B A B
Strains 2 4 10 16 20 23 24 26 27 31 39 40
A 2 3 3 2 2 5 1 4 3 3 2 6
B 2 3 3 3 2 5 1 5 3 2 2 5
Number of repeat units at each loci in above example
MIRU-VNTR typing contd……

23. ADVANTAGES
PCR-based, rapid, high-throughput technique and technically simpler
MIRU analysis can be automated and can thus be used to evaluate large numbers of
strains, yielding intrinsically digital results that can be easily catalogued on a computer
data base.
AWeb site has been set up so that a worldwide data base of MIRU patterns can be
created.
Can be applied directly to bacterial cell lysates without DNA purification.
Better resolution than spoligotyping. Evaluation of additional loci increases the
discriminatory power than IS6110 typing.
Automated analysis with fluorescence-tagged PCR primers and capillary separation.
MIRU-VNTR typing contd……

24. VNTR typing is now ready to become the “next gold standard” for typing of
MTBC isolates (Supply et al.,2006).
Various combinations of MIRUs, ETR andVNTRs are proven to be useful for
typing of M.bovis strains.
Recently, MIRU-VNTR (using 6- loci) with spoligotyping applied to characterize
97 M.tuberculosis isolates from rural area of Kanpur, North India (Sharma et al.,
2008).
Other than M.tuberculosis and M.bovis, MIRU-VNTR typing is also used for
characterization of M.ulcerans isolates fromAfrica. (Stragier et al., 2006).
MIRU-VNTR typing contd……

25. CONCLUSION
 The use of molecular methods, coupled with classical epidemiologic
approaches, has afforded greater resolution and accuracy in describing both
the local and global epidemiology ofTB, including the detection of
unsuspected transmission events and direct evidence for exogenous
reinfection in recurrent disease.
 The ability to discern the molecular “fingerprint” (genotype) of
Mycobacterium tuberculosis isolates has revolutionized our understanding of the
transmission of tuberculosis.
 MIRU-VNTR typing have added further to population genetic approaches
which have suggested biomedically and epidemiologically relevant
characteristics specific to phylogenetic lineages or strain families.

27. VALUE OF GENOTYPING
Identify and prevent transmission
 Enhance contact investigations
 Identify nontraditional settings of transmission
 Facilitate identification of clusters and outbreaks
Improve clinical management
 More readily identify false-positive cultures
 Help distinguish between relapse and reinfection
Enhance surveillance
 Evaluate prevalence of M.tuberculosis genotypes
 Monitor trends in recent transmission
Evaluate prevention activities
 Completeness of contact investigations
 Progress towardTB elimination
Other
 Local outbreak investigation
(confirm suspected epidemiological
links)
 Detect unknown links – targeted
interventions
 Exclude lab cross contamination
 Detect emergence of new strains
 Detect globally prevalent strains
(?inform vaccine
strategies/treatment protocols)

28. DEFINITIONS
 Genotype:The designation that results from one or more of the three
genotyping techniques used for M tuberculosis: Spoligotyping, MIRU analysis,
and IS6110-based RFLP
 Genotyping:Also referred to as DNA fingerprinting. A laboratory
approach that provides a description of the genetic makeup and relatedness
of M.tuberculosis isolates
 Cluster:A genotyping cluster is two or more M tuberculosis isolates that
share matching genotypes
An epidemiologic cluster is two or more persons withTB who share
epidemiologic links