2. Role of phenotypic and genotypic investigations for the identification of non-tuberculous mycobacteria Enrico Tortoli Florence – Italy Presented at 34° ICAAC – Chicago, September 14, 2003

3. The rise of mycobacterial species 1990 1995 2000

4. 94 officially recognized nontuberculous mycobacterial (NTM) species On average 3 new species/yr described in the last 14 years A large number of “new” mycobacterial sequences are piling up in GenBank The rise of mycobacterial species

5. Slow growers 10

6. Slow growers 10

7. Slow growers 10

8. Rapid growers 10

9. Rapid growers 10

10. NTM, frequently raised criticisms NTN differentiation is unnecessary, only their distinction from M. tuberculosis complex is of importance The NTM are rarely clinically significant In the cases in which NTM are clinically significant their identification at species level is of little use for the clinician

11. NTM, frequently raised criticisms. Replay 1 NTN differentiation is unnecessary, only their distinction from M. tuberculosis complex is of importance –The identification of “new” NTM is essential to enlarge the current poor knowledge of such organisms and, first of all, of their clinical relevance

12. The NTM are rarely clinically significant –Among NTM isolated from human samples, for 5 species only the involvement in disease has never been proved NTM, frequently raised criticisms. Replay 2

13. In the cases in which NTM are clinically significant their identification at species level is of little use for the clinician –Several NTM differ for virulence –Some NTM are preferentially involved in specific pathologies –Various NTM are characterized by different susceptibility pattern NTM, frequently raised criticisms. Replay 3

14. Phenotypic identification methods Conventional approach –Biochemical tests –Cultural tests –Selective inhibition tests Lipid approach –Thin layer chromatography –Gas-chromatography –High performance liquid chromatography

15. Phenotypic identification methods Conventional approach –Biochemical tests –Cultural tests –Selective inhibition tests Lipid approach –Thin layer chromatography –Gas-chromatography –High performance liquid chromatography

16. Phenotypic identification methods Conventional approach –Biochemical tests –Cultural tests –Selective inhibition tests Lipid approach –Thin layer chromatography –Gas-chromatography –High performance liquid chromatography

17. Nucleic acid probes PCR restriction analysis Genetic sequencing of conserved regions Genotypic identification methods 10

18. Conventional approach worked apparently well until the number of species remained limited It run into problems with the expansion of the number of species –Need of larger panels of tests –Still insufficient information available for many species Previously disregarded problems emerged –Low reproducibility of several tests –Biological variability of the strains Misidentification of strains belonging to new species as variants of well established ones –Time consumption Limits of conventional methods

19. Culture-dependent TLC and GLC –Large clusters of species exist sharing the same pattern HPLC –Emergence, among new mycobacteria, of species with similar profiles (rapid growers) Limitations of lipid methods

20. DNA probes –Highly reliable but available for common species only PRA –Unidentified strains with patterns overlapping to the ones of well defined species –Species with multiple patterns (up to 8) Genetic sequencing (16S rDNA) –Species with overlapping sequences –Species with multiple sequevars –Poor quality control of major databases Limitations of genetic methods

21. Identification for diagnostic purpose DNA probe 16S rDNA sequence + identification new sequence

22. Major biochemical and cultural features Lipid structure –TLC –GLC –HPLC Full 16S rDNA sequence Description of new species, minimal standard

23. Conclusions Genetic sequencing of 16S rDNA is at present the only method that, even when used alone, provides reliable identifications Identifications emerging from PRA of 65kD hsp are satisfactory but many are the species whose pattern has been not yet determined Among lipid investigations the HPLC is the one with higher discriminatory power, species do exist whose identification cannot be determined With conventional tests a correct identification is achievable for “classical” mycobacteria only; selected tests may help in resolving ambiguities due to overlapping of genetic characters