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Mycobacteria Identification Challenges in a Reference Laboratory

This study at the European Society for Mycobacteriology Meeting in 2001 delves into biochemical and sequencing analyses for identifying unreported mycobacteria species, highlighting the burden of unidentifiable MOTTs. The research presents disagreements between 16 rRNA sequencing and biochemical analyses, covering phenotypic classifications of various slow and rapid grower MOTTs found in a sample of 1,035. Notably, the study identifies rare and common HPLC motifs, including hypervariable regions in the 16S rRNA loci, with a focus on Mycobacterium tuberculosis sequences.

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Mycobacteria Identification Challenges in a Reference Laboratory

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  1. The burden of unidentifiable mycobacteria in a reference laboratory presented at the European Society for Mycobacteriology Meeting, Berlin July 1-4, 2001

  2. biochemical and HPLC analyses agreement species identification disagreement 16 rRNA sequencing species identification reported sequence unreported sequence 72 unidentified MOTTs Study plan 1,035 MOTTs

  3. Phenotypic Runyon’s classification • Photochromogenic slow growers 2 • Scotochromogenic slow growers 27 • Nonchromogenic slow growers 10 • Rapid growers 33

  4. FI-2494 & FI-2595 FI-22098 FI-2389 & FI-30796 FI-15495 & FI-8298 Most frequent HPLC motifs

  5. FI-7297 a rare HPLC profile

  6. Hypervariable region B of 16S rRNA M. tuberculosis 5’ GGGTTGTAAACC-TCTTTCACCAT-CGACGAAGGTCCGGG--TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACT FI-24800 5’ ............-.......GT..-..G.........A..AT..T...T..G-...........AC.................... FI-2897 5’ ............-.......GT..-..G.......TXC..GT..TCTCGCAGG...........AC.................... FI-24198 5’ ............-.......GT..-..G.......TGC..-T..TCTCGCAGG...........AC.................... FI-22000 5’ ............-.......GT..-..G.........C..CT..TGT.-..GG.........-.AC......-............. FI-4696 5’ ..........G.-.......GT..-..G.........A..AT.CT...T..GG...........AC.................... FI-14797 5’ ............-.......GT..-..G..........T.GT..TCTG...GG...........AC.................... FI-19295 5’ ............-.......GT..-..G...........AGT..TCT.T..GG...........AC.................... FI-5198 & 5 others 5’ ............-.......GT..-..G..........T.GT..TCTG...GG...........AC.................... FI-12796 5’ ............-...........-..........T....--..T......................................... FI-2398 & six others 5’ ............-...........-...............--..T......................................... FI-2400 5’ ............-...........-...............--..T......................................... FI-23297 5’ ............-...........-..........T....--..T...........................-............. FI-4898 5’ ............-...........-...............--..T......................................... FI-17499 5’ ............-...........-...............--..T......................................... FI-9197 5’ ............-...........-..........T....--..T......................................... FI-14199 5’ ............-...........-........T..--..--..T.TAT..................................... FI-1500 5’ ............-...........-.........CT.-----ACT..GT.AG.................................. FI-7499 5’ ............-.......G.C.A..........G....--..T.....TGG..........G.C.................... FI-7297 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-9994 & FI-22995 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-23395 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-22895 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-12100 & FI-25197 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-24599 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-2389 & 2 others 5’ ............-.......G..C-A........G.--------------A.G........T...C........G........... FI-1191 5’ ............-.......G..C-A........G.--------------A.G........T..KC........G........... FI-27599 5’ ............-.......G..C-A........G.--------------A.G........T...C........G....-...... FI-7494 5’ ............-.......G..C-A........G.--------------A.G........T...C........G........... FI-11097 5’ ............-.......G..C-A........G.--------------..G........T...C........G........... FI-17697 5’ ............-.......GT.C-.........G.--------------A.G...........AC........G........... FI-16194 5’ ............-.......AT.G-G........G.--------------A.G........CC.AT.................... FI-100 5’ ............-.......AT.G-G........G.--------------A.G........CC.AT.................... FI-23299 5’ ............-...C...AT.G-G........G.--------------A.G........CC.AT.................... FI-29396 5’ ............-......G...G-G........G.--------------A.G........CC...........G........... FI-26200 5’ ............-......G...G-G........G.--------------A.G........CC...........G........... FI-19499 5’ ............-..........G-G........G.--------------A.G........CC....................... FI-5196 5’ ............-..........G-G........GT--------------A.G........CC....................... FI-19696 5’ ............-.......G.CA-T........G.--------------A.G........ATG.C.................... FI-4896 5’ ............-.......G..S-R........G.--------------A.G........C...C.................... FI-1994 5’ ............-.......G..G-G........G.--------------A.G........CC..T........G........... FI-18799 5’ ............-..........G-G-.......G.--------------T.G........CC..-.................... FI-2494 & FI-2595 5’ ............-.......GT.G-G........GT--------------A.G........CC.AT.................... FI-8196 & FI-7796 5’ ...........Y-.......AT.G-G........-G.------------AAG-........CC.AT........G........... FI-32498 5’ ............-.......AT.G-G........-G.------------AAG-........CC.AT........G........... FI-15495 & FI-8298 5’ ............-C......GT..-.........CGT------------.AGG...........AC.................... FI-5896 & FI-11096 5’ ............-C.....G...G-G.......A-GA------------.AT-........CC...........G........... FI-17496 5’ ............-.......G...-.........-G.------------AAG-.........A..C.................... FI-28696 5’ ............-.......G...-.........-G.------------AAG-.........A..C.................... FI-19000 5’ ............-......-G.TCA.........-GT------------AAG-.........TGA.........-........... FI-26099 5’ ............-.......G.TCA.........-GT------------AAG-.........TGA..................... FI-8499 5’ ............-......-G.TCA.........-G.------------AAC-.........TGA..................... FI-27897 5’ ............-.......GT.C-.........-G.------------AAG-...........AC.................... FI-22098 5’ ............-.......GTGC-.........-GG------------AAG-..........CAC........G...........

  7. Major phylogenetic clusters

  8. M. terrae-related mycobacteria M. tuberculosis 5’ GGGTTGTAAACC-TCTTTCACCAT-CGACGAAGGTCCGGG--TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACT FI-24800 5’ ............-.......GT..-..G.........A..AT..T...T..G-...........AC.................... FI-2897 5’ ............-.......GT..-..G.......TXC..GT..TCTCGCAGG...........AC.................... FI-24198 5’ ............-.......GT..-..G.......TGC..-T..TCTCGCAGG...........AC.................... FI-22000 5’ ............-.......GT..-..G.........C..CT..TGT.-..GG.........-.AC......-............. FI-4696 5’ ..........G.-.......GT..-..G.........A..AT.CT...T..GG...........AC.................... FI-14797 5’ ............-.......GT..-..G..........T.GT..TCTG...GG...........AC.................... FI-19295 5’ ............-.......GT..-..G...........AGT..TCT.T..GG...........AC.................... FI-5198 & 5 others 5’ ............-.......GT..-..G..........T.GT..TCTG...GG...........AC....................

  9. Thermotolerant rapid growersregion B M. tuberculosis 5’ GGGTTGTAAACC-TCTTTCACCAT-CGACGAAGGTCCGGG--TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACT FI-8196 & FI-7796 5’ ...........Y-.......AT.G-G........-G.------------AAG-........CC.AT........G........... FI-32498 5’ ............-.......AT.G-G........-G.------------AAG-........CC.AT........G........... FI-15495 & FI-8298 5’ ............-C......GT..-.........CGT------------.AGG...........AC.................... FI-5896 & FI-11096 5’ ............-C.....G...G-G.......A-GA------------.AT-........CC...........G........... FI-17496 5’ ............-.......G...-.........-G.------------AAG-.........A..C.................... FI-28696 5’ ............-.......G...-.........-G.------------AAG-.........A..C.................... FI-19000 5’ ............-......-G.TCA.........-GT------------AAG-.........TGA.........-........... FI-26099 5’ ............-.......G.TCA.........-GT------------AAG-.........TGA..................... FI-8499 5’ ............-......-G.TCA.........-G.------------AAC-.........TGA..................... FI-27897 5’ ............-.......GT.C-.........-G.------------AAG-...........AC.................... FI-22098 5’ ............-.......GTGC-.........-GG------------AAG-..........CAC........G...........

  10. Thermotolerant rapid growersregion A M. tuberculosis 5’ TGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGACCA--CGGGATGCATGTCTTG-TGGTGGA FI-8196 & FI-7796 5’ .................T................................A...TTT..T-TTT..TC.....G.-..GGA.G... FI-32498 5’ .................T................................A...TTT..T-TTT..TC.....G.-..GGA.G... FI-15495 & FI-8298 5’ .................T................................A...CAC..T-G.T..TC.....G.C..G...G... FI-5896 & FI-11096 5’ .................T................................A...CAC..T-G.T..TC.....G.C..G...G... FI-17496 5’ .................T................................A...CAC.TC-ATT..TC.....G.C..G...G... FI-28696 5’ .................T................................A...CAC.TT-GYT..TC.....G.C..G...G..- FI-19000 5’ .................T................................A...CAC..T-TTT..TC.....G.C-.T.A.G... FI-26099 5’ .................T................................A...CAC..T-TTT..TC.....G.C-.T.A.G... FI-8499 5’ .................T................................A...CAC..T-TTT..TC.....G.C..G.A.G... FI-27897 5’ .................T................................A...CAC.TT-G.T..TC.....G.C..G...G... FI-22098 5’ .................T................................A...CAC..T-G.T..TC.....G.C..G...G...

  11. Slow growers M. tuberculosis 5’ GGGTTGTAAACC-TCTTTCACCAT-CGACGAAGGTCCGGG--TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACT FI-12796 5’ ............-...........-..........T....--..T......................................... FI-2398 & six others 5’ ............-...........-...............--..T......................................... FI-2400 5’ ............-...........-...............--..T......................................... FI-23297 5’ ............-...........-..........T....--..T...........................-............. FI-4898 5’ ............-...........-...............--..T......................................... FI-17499 5’ ............-...........-...............--..T......................................... FI-9197 5’ ............-...........-..........T....--..T......................................... FI-14199 5’ ............-...........-........T..--..--..T.TAT..................................... FI-1500 5’ ............-...........-.........CT.-----ACT..GT.AG.................................. FI-7499 5’ ............-.......G.C.A..........G....--..T.....TGG..........G.C....................

  12. M. simiae-related mycobacteria M. tuberculosis 5’ GGGTTGTAAACC-TCTTTCACCAT-CGACGAAGGTCCGGG--TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACT FI-7297 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-9994 & FI-22995 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-23395 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-22895 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-12100 & FI-25197 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C.................... FI-24599 5’ ............-.......G..G-G.......-------------.G.AAG.-.......CC..C....................

  13. FI-9994 & FI-22995 FI-7297 FI-23395 M. simiae M. simiae-related HPLC profiles

  14. Rapid growers M. tuberculosis 5’ GGGTTGTAAACC-TCTTTCACCAT-CGACGAAGGTCCGGG--TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACT FI-2389 & 2 others 5’ ............-.......G..C-A........G.--------------A.G........T...C........G........... FI-1191 5’ ............-.......G..C-A........G.--------------A.G........T..KC........G........... FI-27599 5’ ............-.......G..C-A........G.--------------A.G........T...C........G....-...... FI-7494 5’ ............-.......G..C-A........G.--------------A.G........T...C........G........... FI-11097 5’ ............-.......G..C-A........G.--------------..G........T...C........G........... FI-17697 5’ ............-.......GT.C-.........G.--------------A.G...........AC........G........... FI-16194 5’ ............-.......AT.G-G........G.--------------A.G........CC.AT.................... FI-100 5’ ............-.......AT.G-G........G.--------------A.G........CC.AT.................... FI-23299 5’ ............-...C...AT.G-G........G.--------------A.G........CC.AT.................... FI-29396 5’ ............-......G...G-G........G.--------------A.G........CC...........G........... FI-26200 5’ ............-......G...G-G........G.--------------A.G........CC...........G........... FI-19499 5’ ............-..........G-G........G.--------------A.G........CC....................... FI-5196 5’ ............-..........G-G........GT--------------A.G........CC....................... FI-19696 5’ ............-.......G.CA-T........G.--------------A.G........ATG.C.................... FI-4896 5’ ............-.......G..S-R........G.--------------A.G........C...C.................... FI-1994 5’ ............-.......G..G-G........G.--------------A.G........CC..T........G........... FI-18799 5’ ............-..........G-G-.......G.--------------T.G........CC..-.................... FI-2494 & FI-2595 5’ ............-.......GT.G-G........GT--------------A.G........CC.AT....................

  15. Strains’ source • Human clinical samples 47 • Environmental samples 14 • Animal clinical samples 3 • Unknown 8

  16. Strains of human origin • Respiratory tract (33 out of 47) • Origin of potentially significant isolates • pus • pleural biopsy • AIDS patient blood culture • central venous catheter tip

  17. Unidentified strains clustering 7 M. bohemicum-related strains 6 M. hiberniae-related strains 3 mycobacterium SRB1151-113 strains 6 couples of identical strains • 40 single-isolate strains • Strains sharing previously reported sequences were also present: MCRO7, MCRO17, MCRO33, “M. ratisbonense”

  18. Conclusion • Many pages of mycobacterial taxonomy still wait to be written. We hope new reports, maybe stimulated by our study too, will further elucidate, in adjunct to taxonomy, epidemiology and potential pathogenicity for humans and animals of still unknown mycobacteria. Fine della presentazione, fare clic per uscire

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