MYCOBACTERIOLOGY 2004MYCOBACTERIOLOGY 2004 What laboratories are doing What laboratories are doing and where they are headedand where they are headed

Current identification Current identification techniques techniques – Molecular Molecular

E. TortoliE. Tortoli

ASM 2004 – New Orleans, May 26

Conserved genetic Conserved genetic regions, the Rosetta regions, the Rosetta stone of molecular stone of molecular

identificationidentification

5’ 3’

Conserved genetic Conserved genetic regions, the Rosetta regions, the Rosetta stone of molecular stone of molecular

identificationidentification

variable genus-specific region

5’ 3’

Conserved genetic Conserved genetic regions, the Rosetta regions, the Rosetta stone of molecular stone of molecular

identificationidentification

variable genus-specific regionhypervariable species-specific region

5’ 3’

Conserved genetic Conserved genetic regions, the Rosetta regions, the Rosetta stone of molecular stone of molecular

identificationidentification

variable genus-specific regionhypervariable species-specific regionhypervariable intraspecies-specific region

5’ 3’

DNA probesDNA probes

Liquid-phase hybridizationLiquid-phase hybridization Solid-phase reverse Solid-phase reverse

hybridizationhybridization

Liquid-phase hybridizationLiquid-phase hybridization

AccuProbeAccuProbe Target: 16S rRNATarget: 16S rRNA Selection: hybridization Selection: hybridization

protection assayprotection assay Detection: chemiluminescenceDetection: chemiluminescence Available for: Available for:

2 complexes2 complexes 4 species4 species

Solid-phase reverse Solid-phase reverse hybridizationhybridization

Solid-phase reverse Solid-phase reverse hybridization 1hybridization 1

INNO LiPAINNO LiPA Target: ITSTarget: ITS Probes for: Probes for:

MycobacteriumMycobacterium genus genus 2 complexes2 complexes 15 (+1) species15 (+1) species Intraspecific Intraspecific

differentiation of 3 differentiation of 3 speciesspecies

GenoTypeGenoType Target: 23S rDNATarget: 23S rDNA Probes for:Probes for:

Mycobacterium Mycobacterium genus ???genus ??? 1 complex1 complex 11 species11 species Intraspecific Intraspecific

differentiation of 1 differentiation of 1 speciesspecies

New version: double New version: double strip with 13+12 speciesstrip with 13+12 species

Solid-phase reverse Solid-phase reverse hybridization 2hybridization 2

GenoType MTBCGenoType MTBC Targets: Targets:

23S rDNA23S rDNA M. tuberculosisM. tuberculosis complex-specific region complex-specific region

gyrBgyrB 4 regions containing base-substitutions specific for4 regions containing base-substitutions specific for M. M.

tuberculosis tuberculosis ((M. africanum M. africanum II,II, M. canettii M. canettii),), M. M. africanum africanum I,I, M. microti M. microti, BCG,, BCG, M. caprae M. caprae

RD1RD1 9,500 bp region present in all members of 9,500 bp region present in all members of M. M.

tuberculosistuberculosis complex other than BCG complex other than BCG

DNA-probes: discrepant DNA-probes: discrepant resultsresults

M. kansasiiM. kansasii types iii-iv: AccuProbe-MK NEG types iii-iv: AccuProbe-MK NEG M. palustreM. palustre: AccuProbe-MAC pos: AccuProbe-MAC pos ““M. paraffinicumM. paraffinicum”: LiPA-MAIS pos”: LiPA-MAIS pos M. thermoresistibileM. thermoresistibile, , M. agriM. agri, , M. mageritenseM. mageritense, ,

M. senegalenseM. senegalense, , M. alveiM. alvei: LiPA-MFO pos: LiPA-MFO pos MAC non MAC non aviumavium, non , non intracellulareintracellulare: GenoType-: GenoType-

MI pos or GenoType NEGMI pos or GenoType NEG M. interjectumM. interjectum: GenoType-MSC pos: GenoType-MSC pos M. saskatchewanenseM. saskatchewanense: AccuProbe-MAC pos: AccuProbe-MAC pos M. chimaeraM. chimaera: AccuProbe-MI pos; GenoType-MI : AccuProbe-MI pos; GenoType-MI

pos; LiPA-MIN2 pospos; LiPA-MIN2 pos

PCR-RFLP analysisPCR-RFLP analysis

Target amplification Target amplification Restriction enzymes digestionRestriction enzymes digestion ElectrophoresisElectrophoresis

Determination of size of Determination of size of fragments fragments ≥40 bp≥40 bp

hsp65 hsp65 PRAPRA

Bst Bst EIIEII no digestion (440 bp)no digestion (440 bp) 2-3 major fragments production (320-80 2-3 major fragments production (320-80

bp)bp) HaeHae III III

2-5 major fragments production (265-40 2-5 major fragments production (265-40 bp)bp)

hsp65 hsp65 PRA algorithm

many species with multiple patterns (up to 9)many species with multiple patterns (up to 9) few patterns shared by several speciesfew patterns shared by several species

320 bp

130 bp

120 bp

200/60/50 bp M. chelonae150/105 bp M. haemophilum

185/130 bp M. terrae145/130/50 bp M. simiae130/115/60 bp M. gordonae130/95/75/60 bp M. kansasii. . . . . .

Bst EII Hae III

a proper algorithm allows the differentiation of species, within a proper algorithm allows the differentiation of species, within each pattern of each pattern of Bst Bst EII fragments, on the basis EII fragments, on the basis Hae Hae III patternIII pattern

Genetic sequencingGenetic sequencing

16S rDNA16S rDNA Internal transcribed Internal transcribed

spacerspacer 23S rDNA23S rDNA hsp65hsp65 sodsod

16S rDNA16S rDNA About 1,500 bpAbout 1,500 bp Hypervariable regions A and BHypervariable regions A and B

Species-specificitySpecies-specificity Several sequevarsSeveral sequevars

Phylogenetic markersPhylogenetic markers Short helix 18 characterizing ancestral, Short helix 18 characterizing ancestral,

rapidly growing, mycobacteriarapidly growing, mycobacteria Evolution of slow growers associated with a Evolution of slow growers associated with a

12 nucleotide insertion in helix 1812 nucleotide insertion in helix 18 Development of two new branches, among Development of two new branches, among

slow growers, characterized by:slow growers, characterized by: further insertion of 2 nucleotides in helix 18further insertion of 2 nucleotides in helix 18 lost of the acquired 12 nucleotide insertion lost of the acquired 12 nucleotide insertion

Development of a new branch, among rapid Development of a new branch, among rapid growers, characterized by a cytosine growers, characterized by a cytosine insertion in helix 10insertion in helix 10

5 '

3 'A

B

1 8

1 0

ITSITS About 300 bpAbout 300 bp No hypervariable region recognizableNo hypervariable region recognizable Many mycobacteria characterized by Many mycobacteria characterized by

a high number of sequevars:a high number of sequevars: MACMAC M. fortuitumM. fortuitum complex complex M. gordonaeM. gordonae M. kansasiiM. kansasii

23S rDNA23S rDNA

About 3,000 bpAbout 3,000 bp 250 bp variable region250 bp variable region Species-specificity, several species with Species-specificity, several species with

overlapping sequence, no intraspecific overlapping sequence, no intraspecific variabilityvariability

Sequencing limitationsSequencing limitations

Limited portion of genome surveyedLimited portion of genome surveyed Investigation of highly conserved genes make Investigation of highly conserved genes make

species variability less evident than in the DNA-DNA species variability less evident than in the DNA-DNA homology investigations homology investigations

Species with overlapping sequenceSpecies with overlapping sequence Species with multiple sequevarsSpecies with multiple sequevars Lack of quality control of sequence entries in public Lack of quality control of sequence entries in public

databasesdatabases best match sometimes favoring ragged or outdated entriesbest match sometimes favoring ragged or outdated entries

Microarray-microchip Microarray-microchip technologytechnology

Microarray: miniaturized multiprobe Microarray: miniaturized multiprobe systemsystem electronically controlledelectronically controlled non electronicnon electronic

Multiple step identification of many Multiple step identification of many strains (amplicon down)strains (amplicon down)

Single step identification of one strains Single step identification of one strains (capture down)(capture down)

Amplicon downAmplicon down

PCR-products from various PCR-products from various unidentified mycobacteria are unidentified mycobacteria are bound to different pads of a bound to different pads of a micro-arraymicro-array In subsequent steps, one or more In subsequent steps, one or more (differently marked) probes are (differently marked) probes are addedadded At each step the monitoring of pads At each step the monitoring of pads presenting hybridization allows, on presenting hybridization allows, on the basis of the specificity of the the basis of the specificity of the probe used, the identification of the probe used, the identification of the corresponding ampliconcorresponding amplicon

Capture downCapture down

Different species-specific probes Different species-specific probes are bound to the various pads of a are bound to the various pads of a micro-arraymicro-array

Identification on the basis of the Identification on the basis of the specificity of the probe bound to the specificity of the probe bound to the pad presenting the hybridizationpad presenting the hybridization

Addition of PCR-product from an Addition of PCR-product from an unidentified strainunidentified strain

Revelation of the hybridization by Revelation of the hybridization by adding a reporter (marked probe) adding a reporter (marked probe) aiming to a non species-specific trait of aiming to a non species-specific trait of the amplified regionthe amplified region

Future prospectsFuture prospects

Use of microchip technology Use of microchip technology forfor IdentificationIdentification Genotypic detection of Genotypic detection of

resistancesresistances Molecular epidemiologyMolecular epidemiology