MYCOBACTERIOLOGY 2004 What laboratories are doing and where they are headed Current identification techniques Molecular Current identification techniques – Molecular E. Tortoli ASM 2004 – New Orleans, May 26

Conserved genetic regions, the Rosetta stone of molecular identification 53

variable genus-specific region 53

Conserved genetic regions, the Rosetta stone of molecular identification variable genus-specific region hypervariable species-specific region 53

Conserved genetic regions, the Rosetta stone of molecular identification variable genus-specific region hypervariable species-specific region hypervariable intraspecies-specific region 53

DNA probes Liquid-phase hybridization Liquid-phase hybridization Solid-phase reverse hybridization Solid-phase reverse hybridization

Liquid-phase hybridization AccuProbe AccuProbe Target: 16S rRNA Target: 16S rRNA Selection: hybridization protection assay Selection: hybridization protection assay Detection: chemiluminescence Detection: chemiluminescence Available for: Available for: 2 complexes 2 complexes 4 species 4 species

Solid-phase reverse hybridization

Solid-phase reverse hybridization 1 INNO LiPA INNO LiPA Target: ITS Target: ITS Probes for: Probes for: Mycobacterium genus Mycobacterium genus 2 complexes 2 complexes 15 (+1) species 15 (+1) species Intraspecific differentiation of 3 species Intraspecific differentiation of 3 species GenoType Target: 23S rDNA Probes for: Mycobacterium genus ??? 1 complex 11 species Intraspecific differentiation of 1 species New version: double strip with species

Solid-phase reverse hybridization 2 GenoType MTBC GenoType MTBC Targets: Targets: 23S rDNA 23S rDNA M. tuberculosis complex-specific region M. tuberculosis complex-specific region gyrB gyrB 4 regions containing base-substitutions specific for M. tuberculosis (M. africanum II, M. canettii), M. africanum I, M. microti, BCG, M. caprae 4 regions containing base-substitutions specific for M. tuberculosis (M. africanum II, M. canettii), M. africanum I, M. microti, BCG, M. caprae RD1 RD1 9,500 bp region present in all members of M. tuberculosis complex other than BCG 9,500 bp region present in all members of M. tuberculosis complex other than BCG

DNA-probes: discrepant results M. kansasii types iii-iv: AccuProbe-MK NEG M. kansasii types iii-iv: AccuProbe-MK NEG M. palustre: AccuProbe-MAC pos M. palustre: AccuProbe-MAC pos M. paraffinicum: LiPA-MAIS posM. paraffinicum: LiPA-MAIS pos M. thermoresistibile, M. agri, M. mageritense, M. senegalense, M. alvei: LiPA-MFO pos M. thermoresistibile, M. agri, M. mageritense, M. senegalense, M. alvei: LiPA-MFO pos MAC non avium, non intracellulare: GenoType-MI pos or GenoType NEG MAC non avium, non intracellulare: GenoType-MI pos or GenoType NEG M. interjectum: GenoType-MSC pos M. interjectum: GenoType-MSC pos M. saskatchewanense: AccuProbe-MAC pos M. saskatchewanense: AccuProbe-MAC pos M. chimaera: AccuProbe-MI pos; GenoType-MI pos; LiPA- MIN2 pos M. chimaera: AccuProbe-MI pos; GenoType-MI pos; LiPA- MIN2 pos

PCR-RFLP analysis Target amplification Target amplification Restriction enzymes digestion Restriction enzymes digestion Electrophoresis Electrophoresis Determination of size of fragments 40 bp Determination of size of fragments 40 bp

hsp65 PRA Bst EII Bst EII no digestion (440 bp) no digestion (440 bp) 2-3 major fragments production ( bp) 2-3 major fragments production ( bp) Hae III Hae III 2-5 major fragments production ( bp) 2-5 major fragments production ( 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 species few patterns shared by several species 320 bp 130 bp 120 bp 200/60/50 bp M. chelonae 150/105 bp M. haemophilum 185/130 bp M. terrae 145/130/50 bp M. simiae 130/115/60 bp M. gordonae 130/95/75/60 bp M. kansasii... Bst EII Hae III a proper algorithm allows the differentiation of species, within each pattern of Bst EII fragments, on the basis Hae III pattern a proper algorithm allows the differentiation of species, within each pattern of Bst EII fragments, on the basis Hae III pattern

Genetic sequencing 16S rDNA 16S rDNA Internal transcribed spacer Internal transcribed spacer 23S rDNA 23S rDNA hsp65 hsp65 sod sod

16S rDNA About 1,500 bp About 1,500 bp Hypervariable regions A and B Hypervariable regions A and B Species-specificity Species-specificity Several sequevars Several sequevars Phylogenetic markers Phylogenetic markers Short helix 18 characterizing ancestral, rapidly growing, mycobacteria Short helix 18 characterizing ancestral, rapidly growing, mycobacteria Evolution of slow growers associated with a 12 nucleotide insertion in helix 18 Evolution of slow growers associated with a 12 nucleotide insertion in helix 18 Development of two new branches, among slow growers, characterized by: Development of two new branches, among slow growers, characterized by: further insertion of 2 nucleotides in helix 18 further 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 growers, characterized by a cytosine insertion in helix 10 Development of a new branch, among rapid growers, characterized by a cytosine insertion in helix 10

ITS About 300 bp About 300 bp No hypervariable region recognizable No hypervariable region recognizable Many mycobacteria characterized by a high number of sequevars: Many mycobacteria characterized by a high number of sequevars: MAC MAC M. fortuitum complex M. fortuitum complex M. gordonae M. gordonae M. kansasii M. kansasii

23S rDNA About 3,000 bp About 3,000 bp 250 bp variable region 250 bp variable region Species-specificity, several species with overlapping sequence, no intraspecific variability Species-specificity, several species with overlapping sequence, no intraspecific variability

Sequencing limitations Limited portion of genome surveyed Limited portion of genome surveyed Investigation of highly conserved genes make species variability less evident than in the DNA-DNA homology investigations Investigation of highly conserved genes make species variability less evident than in the DNA-DNA homology investigations Species with overlapping sequence Species with overlapping sequence Species with multiple sequevars Species with multiple sequevars Lack of quality control of sequence entries in public databases Lack of quality control of sequence entries in public databases best match sometimes favoring ragged or outdated entries best match sometimes favoring ragged or outdated entries

Microarray-microchip technology Microarray: miniaturized multiprobe system Microarray: miniaturized multiprobe system electronically controlled electronically controlled non electronic non electronic Multiple step identification of many strains (amplicon down) Multiple step identification of many strains (amplicon down) Single step identification of one strains (capture down) Single step identification of one strains (capture down)

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

Capture down Different species-specific probes are bound to the various pads of a micro-array Different species-specific probes are bound to the various pads of a micro-array Identification on the basis of the specificity of the probe bound to the pad presenting the hybridization Identification on the basis of the specificity of the probe bound to the pad presenting the hybridization Addition of PCR-product from an unidentified strain Addition of PCR-product from an unidentified strain Revelation of the hybridization by adding a reporter (marked probe) aiming to a non species-specific trait of the amplified region Revelation of the hybridization by adding a reporter (marked probe) aiming to a non species-specific trait of the amplified region

Future prospects Use of microchip technology for Use of microchip technology for Identification Identification Genotypic detection of resistances Genotypic detection of resistances Molecular epidemiology Molecular epidemiology