Impact of molecular methods on taxonomy and diagnostics of mycobacteria

1. Impact of molecular methods on taxonomy and diagnostics of mycobacteria Enrico Tortoli Regional Reference Center for Mycobacteria Florence - Italy Buenos Aires -International symposium on tuberculosis and non tuberculous mycobacteria, September 26, 2005

2. The taxonomy of mycobacteria • Until 1980s • based on phenotypic features • In the last 15 years • shift to genotype

3. Phenotypic features • Biochemical tests • Cultural tests • Growth inhibition tests • Because of the rise of the number of mycobacterial species a proportionally large number of tests would be required for their differentiation • unbearable workload • lack of phenotype databases

4. The genotypic approach • Various regions of the genome are differently affected by mutations • Regions encoding for essential life-function are little prone to changes • Non encoding regions vary widely • Halfway are placed the regions encoding for non essential functions and, among the non encoding ones, those which are transcribed

5. Taxonomically important genetic regions • 16S rDNA • ITS-1 • hsp65 • 23S rDNA

6. The 16S rRNA gene • 1,500 bases long • highly conserved • hypervariable regions • AE. coli pos. 120 • helix 10 • BE. coli pos. 430 • helix 18

7. Helix 18 M. tuberculosis 5’ CCA TCG ACG AAG GTC CGG GTT CTC TCG GAT TGA CGG TAG GTG GAG AAG AAG CAC M. chelonae 5’ GTA GGG ACG AAG C-- --- --- --- -GAAAG TGA CGG TACCTACAG AAG AAG GAC M. simiae 5’ GCA GGG ACG AAG C-- --- --- --- -GC AAG TGA CGG TACCTG CAG AAG AAG CAC M. terrae 5’ GTA TCG GCG AAG CTC CGTGG TTT TCTGCG GGG TGA CGG TAACTG GAG AAG AAG CAC

8. Helix 18 M. tuberculosis 5’ CCA TCG ACG AAG G-- TC CGG GTT CTC TCG GAT TGA CGG TAG GTG GAG AAG AAG CAC M. chelonae 5’ GTA GGG ACG AAG C-- -- --- --- --- -GAAAG TGA CGG TACCTACAG AAG AAG GAC M. simiae 5’ GCA GGG ACG AAG C-- -- --- --- --- -GC AAG TGA CGG TACCTG CAG AAG AAG CAC M. terrae 5’ GTA TCG GCG AAG CTC CGTGG TTT TCTGCG GGG TGA CGG TAACTG GAG AAG AAG CAC

9. Helix 10 M. tuberculosis 5’ CAC TTC GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA GGA CCA CGG GAT GCA TGT CT M. chelonae 5’ CAC TCT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA TGA CCA CACACT TCA TGGTG M. simiae 5’ CAC TTC GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA TGA CCA CGG AAC GCA TGT TT M. terrae 5’ CAC TCT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA GGA CCG CGC GCT TCA TGGTG M. smegmatis 5’ CAC TTT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGA ATA GACC CTT CTG ATC CGA TGGTC

10. Helix 10 M. tuberculosis 5’ CAC TTC GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA GG-A CCA CGG GAT GCA TGT CT M. chelonae 5’ CAC TCT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA TG-A CCA CACACT TCA TGGTG M. simiae 5’ CAC TTC GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA TG-A CCA CGG AAC GCA TGT TT M. terrae 5’ CAC TCT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGG ATA GG-A CCG CGC GCT TCA TGGTG M. confluentis 5’ CAC TTT GGG ATA AGC CTG GGA AAC TGG GTC TAA TAC CGA ATA GACC CTT CTG ATC CGA TGGTC

11. M.simiae-related slow growers slow growers M.terrae-related slow growers thermotolerant rapid growers CCGTGGTTTTCTG TCCGTGGTTTTCTG rapid grovers CCGTGGTTTTCTG C Evolution of mycobacteria

12. Phylogenetic studies • The type and the position of mutations provide important phylogenetic information • Phylogenetic trees provide graphic representation of the evolutionary pathway • Different algorithms • Neighbor joining, maximum likelihood, . . . • Different graphic representations • Phenogram, cladogram, curvogram, . . . • Possible confusing results

13. Pathologies due to “new” NTM • Nonchromogenic are more frequently pathogenic in comparison to chromogenic; this is particularly true for rapid-growers • Slow-growers are frequently responsible of pulmonary infections and of disseminated disease in immuno-compromised patients • A number of slowly growing new species have been isolated from cervical lymphadenitis of children. In the last years the isolations of M. scrofulaceum in the same pathology has collapsed • The commonly believed little significance of rapid growers has revealed groundless. Many such new species are very frequently involved in post-traumatic and post-surgical wounds infections or isolated in catheter-related sepsis • M. immunogenum is commonly isolated from aerosols of industries using metal working abrasives known to be associated with hypersensitivity pneumonitis

14. Sp. nov. description: hot topics • Role of biochemical and cultural tests • Lipid investigations • Sequencing of conserved genetic regions • Minimum mismatch number • DNA/DNA hybridization • homology < 70%, divergence > 5% • Guanosine + cytosine content • 61-71 mol% • Minimum strains’ number • Phylogenetic analysis • Clinical information • Names of sp. nov.

15. Diagnostic problems • With conventional tests a correct identification is achievable for “classical” mycobacteria only. Selected tests are however useful to resolve ambiguities due to overlapping of genetic characteristics • DNA probes are highly specific but are not available for new species • TLC or GLC may be useful complementary tests but are clearly inadequate when used alone • HPLC has been considered for years the only phenotypic analysis which could be used alone. In the last years problems with many new species, in particular of rapid growers, have emerged • PCR Restriction Analysis is very easy and chip, unfortunately the number PRA-patterns shared by two or more species and that of the species with multiple PRA-patterns is steadily increasing • Sequencing of 5’-end (about 500 bp) of 16S rDNA is the most reliable standalone approach. A few species remain however undistinguishable. The number of species with multiple sequevars is on the rise

16. Cultural features + HPLC Cultural features + PRA + + identification DNA probe + Genetic sequencing identification + new sequence Tentative identification protocol identification