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Genotyping The NARSA Collection

Genotyping The NARSA Collection. Barry N. Kreiswirth PHRI . - 8. - 56. - 16. - 16. - 8. - 26. - 84. - 28. - 0. Clinical Relevant Strains. Genotyping Methods. Pulsed-Field Gel Electrophoresis (PFGE) Multilocus Sequence Typing (MLST)

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Genotyping The NARSA Collection

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  1. Genotyping The NARSA Collection Barry N. Kreiswirth PHRI

  2. - 8 - 56 - 16 - 16 - 8 - 26 - 84 - 28 - 0

  3. Clinical Relevant Strains

  4. Genotyping Methods • Pulsed-Field Gel Electrophoresis (PFGE) • Multilocus Sequence Typing (MLST) • Sequence protein A repeats (spa Typing)

  5. MLST - Housekeeping Genes • Carbamate kinase (arcC) • Shikimate dehyrogenase (aroE) • Glycerol kinase (glpF) • Gunylate kinase (gmk) • Triosephosphate isomerase (tpi) • Phosphate acetyltransferase (pta) • Acetyl coenzyme A acetyltransferase (yqiL) Note: genes not scattered around the genome

  6. MLST analysis of COL • Carbamate kinase Allele 3 • Shikimate dehyrogenase Allele 3 • Glycerol kinase Allele 1 • Guanylate kinase Allele 1 • Phosphate acetyltransferase Allele 4 • Triosephosphate isomerase Allele 4 • Acetyl coenzyme A acetyltransferase Allele 16 MLST = 3 3 1 1 4 4 16 = ST250

  7. MLST analysis of USA300 • Carbamate kinase Allele 3 • Shikimate dehyrogenase Allele 3 • Glycerol kinase Allele 1 • Guanylate kinase Allele 1 • Phosphate acetyltransferase Allele 4 • Triosephosphate isomerase Allele 4 • Acetyl coenzyme A acetyltransferase Allele 3 MLST = 3 3 1 1 4 4 3 = ST8

  8. MLST – Single Locus Variant (SLV) Strain: COL ST250 = 3 3 1 1 4 4 16 Strain: USA300 ST8 = 3 3 1 1 4 4 3 BOTH MEMBERS OF CLONAL COMPLEX 8

  9. An MLST Clonal Complex (CC) – BURST Analysis Robinson & Enright - 2003, AAC;47:3926

  10. Cooper and Feil - 2006, Microbiology;152:1297

  11. Cooper and Feil - 2006, Microbiology;152:1297

  12. MRSA – Major Lineages Defined by Multilocus Sequence Typing (MLST) Clonal Complex – 8 Archaic, USA300 Clonal Complex – 22 EMRSA 15 Clonal Complex - 45 Australian Clonal Complex – 5 North American Clonal Complex – 30 EMRSA 16

  13. ST45 - Australian ST36 - EMRSA16 ST22 - EMRSA15 ST8 – Archaic/USA300 ST1 - MW2 ST5 - North American MRSA Major Lineages

  14. ST45; USA600 ST36: USA200 ST22 – USA900 ST239: USA1100 ST72: USA700 ST59: USA1000 ST8: USA300, USA500 ST1: USA400 ST15: USA1100 ST5: USA100, USA800 MRSA - USA TYPES

  15. spa Typing • Protein A gene (spa) • Present in all S. aureus strains • Sequence analysis of the variable number tandem repeats in the 3’ region of the S. aureus protein A gene

  16. Protein A Gene Organization

  17. spa Typing • Sequence PCR amplicons of the repeat region • Amplicons range in size: ~200-600 bp • Web-based programs to analyze repeats • Ridom (Germany) - eGenomics (US) • Identify and name each repeat cassette • The repeat profile equals the spa type

  18. MLST and spa Typing • Consensus between two typing methods • Database give comparable groups • spa typing - faster molecular clock

  19. Fowler et al - 2007, JID;196:738

  20. NARSA COLLECTION • Typed 208 / 242 isolates • 72 MRSA • MLST and spa typing • 49 Sequence Types (STs) • 79 spa Types • 101 different genotypes (ST and spa Types)

  21. Spa16 WGKAKAOMQQQ Spa205 WGKAKAOMQQQQ Spa10 A2AKEEEMBKB Spa15 A2AKEEMBKB Spa278 XKAKB Spa73 XKAKBEMBKB Spa433 XKAX2BMB ST 36 ST 45 ST 30 Spa915 WFGKAKAO Spa158 WFGKAKAOMQQ Spa43 WGKAKAOMQ Spa33 WGKAKAOMQQ Spa16 WGKAKAOMQQQ Spa887 WGKAOMOMQQ Spa251 WGKKAKAOMQ Spa909 WGMKAKAOMQQ Spa468 XKAKAOMQQ Spa99 XKKAKAOMQQ Group 1a Spa295 ZZ2PNGKBKGOLB Spa469 ZZ2PGKBKGOLB ST 207 ST 10 Spa683 I2Z2EGJH2M Spa913 I2Z2EGMM Spa312 I2Z2EGMMJH2M Spa287 I2Z2EMMMJH2M Spa490 I2Z2GMJH2M Spa454 XMJH2M ST 121 Spa885 TJEJCMQOMOKR Spa113 TJEJNCMOMOKR Spa912 TJEJOCMOMOKR Spa382 TJJEJNF2MNF2MOMOKR ST 22 ST 182 Spa888 ZDMMMNNQMMMMO ST 49 Spa3 WGKAOMQ Spa1 YHGFMBQBLO Spa7 YHGCMBQBLO Spa59 YHGGFMBQBLO Spa68 YGCMBQBLO Spa363 YGFMBQBLO Spa882 YHGAMBQBLO  ST 240 ST 239 ST 59 ST 50 Spa 17 ZDMDMNKB ST 8 ST 17 ST 20 Spa35 UJFKBPE Spa194 UJFKKPFKPE Spa131 UJJFKBPE Spa227 UJJJJFE Spa8 UKJFKBPE ST 55 Group 1b ST 1 ST 97 Spa 92 UJGFMBBPB ST 9 ST 15 Spa21 UJGBBGGJAGJ Spa90 UBGGJAGJ Spa232 TJMBBMDMGMK Spa29 TJMBMDMGGMK Spa2 TJMBMDMGMK Spa25 TJMDMGMK Spa24 TJMEMDMGMK Spa12 TJMGMK Spa230 TMBMDMGMK ST 5 ST 7 ST 101 ST 13 Spa916 ZFGU2DMGGU2 Spa867 ZGM Spa910 ZGU2 Spa2 TJMBMDMGMK Group 2 ST 25

  22. NRS022  NRS027  NRS168  NRS169  NRS236  NRS029  NRS071  NRS283  NRS383  ST 36 ST 45 ST 30 NRS104  NRS112  NRS131  NRS162 NRS174  NRS176  NRS180  NRS181  NRS196  NRS204  NRS207  NRS210  NRS212  NRS213  NRS216  NRS219  NRS222  NRS223  NRS231 NRS246 NRS252  NRS261  NRS267 Group 1a ST 207 NRS113  NRS170  ST 10 NRS148  NRS153  NRS165 NRS166  NRS185  NRS186  NRS215  NRS217  NRS225 NRS228  NRS237  NRS239  NRS242  NRS258  NRS266 NRS230  NRS259  NRS152  NRS157  NRS161  NRS184  NRS232  NRS235  NRS271  NRS154  ST 121 ST 22 ST 182 NRS021  NRS026  NRS028  NRS073  NRS077  NRS106  NRS107  NRS108  NRS114  NRS128  NRS129  NRS130  NRS133  NRS134  NRS135  NRS136  NRS137  NRS139  NRS140  NRS141  NRS142  NRS144  NRS145  NRS146  NRS147  NRS150  NRS151  NRS156  NRS158  NRS171  NRS172  NRS211  NRS214  NRS251  NRS254  NRS257  NRS384  NRS385  ST 49 NRS178 ST 240 NRS054  NRS056  ST 239 ST 59 NRS241 NRS243 ST 50 ST 8 ST 17 ST 20 NRS072  NRS123 NRS192  NRS193 NRS194  NRS226 NRS229  NRS248 ST 55 Group 1b ST 1 NRS105  NRS220  NRS224 ST 97 ST 9 ST 15 NRS164  NRS183  NRS234  NRS250  ST 5 NRS001 NRS002  NRS003  NRS004  NRS011  NRS012  NRS013  NRS014  NRS017  NRS018  NRS019  NRS023  NRS024  NRS049  NRS051  NRS052  NRS068  NRS070  NRS076  NRS110  NRS126  NRS127  NRS149  NRS155  NRS173  NRS179  NRS238  NRS240  NRS244  NRS269  NRS382  NRS387  NRS402  NRS403  NRS404 ST 7 ST 101 ST 13 Group 2 ST 25 NRS102  NRS187  NRS227  NRS260

  23. NRS022  NRS027 NRS168  NRS169  NRS236  VISA STRAINS NRS029  NRS071  NRS283  NRS383  ST 36 ST 45 ST 30 NRS104  NRS112  NRS131  NRS162 NRS174  NRS176  NRS180  NRS181  NRS196  NRS204  NRS207  NRS210  NRS212  NRS213  NRS216  NRS219  NRS222  NRS223  NRS231 NRS246 NRS252  NRS261  NRS267 Group 1a ST 207 NRS113  NRS170 ST 10 NRS148  NRS153  NRS165 NRS166  NRS185  NRS186  NRS215NRS217  NRS225 NRS228  NRS237  NRS239  NRS242  NRS258  NRS266 NRS230  NRS259  NRS152  NRS157  NRS161  NRS184 NRS232  NRS235  NRS271  NRS154  ST 121 ST 22 ST 182 NRS021  NRS026  NRS028  NRS073  NRS077  NRS106  NRS107  NRS108  NRS114  NRS128  NRS129  NRS130  NRS133  NRS134  NRS135  NRS136  NRS137  NRS139  NRS140  NRS141  NRS142  NRS144  NRS145  NRS146  NRS147  NRS150  NRS151  NRS156  NRS158  NRS171  NRS172  NRS211 NRS214  NRS251  NRS254  NRS257  NRS384  NRS385  ST 49 NRS178 ST 240 NRS054  NRS056  ST 239 ST 59 NRS241 NRS243 ST 50 ST 8 ST 17 ST 20 NRS072  NRS123 NRS192  NRS193 NRS194  NRS226 NRS229  NRS248 ST 55 Group 1b ST 1 NRS105  NRS220  NRS224 ST 97 ST 9 ST 15 NRS164  NRS183  NRS234  NRS250  NRS001 NRS002  NRS003  NRS004 NRS011  NRS012  NRS013  NRS014  NRS017  NRS018  NRS019  NRS023 NRS024  NRS049  NRS051  NRS052  NRS068 NRS070  NRS076  NRS110  NRS126  NRS127  NRS149  NRS155  NRS173 NRS179  NRS238  NRS240  NRS244  NRS269  NRS382  NRS387  NRS402 NRS403  NRS404 ST 5 ST 7 ST 101 ST 13 Group 2 ST 25 NRS102  NRS187 NRS227  NRS260

  24. VRSA STRAINS ST 36 ST 45 ST 30 ST 207 ST 10 Group 1a Group 1b ST 121 ST 22 ST 182 ST 49 ST 240 ST 239 ST 59 ST 50 ST 8 ST 17 ST 20 ST 55 ST 1 Group 2 ST 97 ST 9 ST 15 VRSA 1 – spa 12: TJMGMK VRSA 2 – spa 1: TJMBMDMGMK VRSA 3 – spa 1: TJMBMDMGMK VRSA 4 – spa 1: TJMBMDMGMK VRSA 5 – spa 1: TJMBMDMGMK VRSA 6 – spa 1: TJMBMDMGMK VRSA 7 - spa 12: TJMGMK ST 5 ST 7 ST 101 ST5: USA100, USA800 ST 13 ST 25

  25. RESEARCH STRAINS CC45: ST508: spa 715 - Becker CC30: ST36: spa 16 – MRSA252 (EMRSA16) CC30: ST30: spa 99 – Cowan CC30: ST30: spa 43 – PS80 CC30: ST30: spa 33 – MN8 CC121, ST121; spa 312 - Smith Diffuse CC22: ST22: spa type 113 – EMRSA15 CC8: ST8: spa 59 – 8325, 4220 CC8: ST8: spa 1 – Newman, USA300 CC1: ST1: spa 131 – MW2, 476 CC97; ST97: spa 92 – Wood 46 CC5: ST5: spa 2 – N315, MU50 CC5: ST5: spa 230 – 502A CC25: ST25: spa 868 – Reynolds Cooper and Feil - 2006, Microbiology;152:1297

  26. Neighbor-Joining Tree of Concatenated MLST Allelic Sequences from the NARSA Collection CC45 CC30 CC1 CC5 CC8 ? CC8 CC1 ?

  27. Neighbor-Joining Tree of Concatenated MLST Allelic Sequences from the NARSA Collection

  28. 582 CC 45 15 97 508 395 45 47 25 CC 1 CC 5 188 59 371 1 5 105 372 231 239 34 109 CC 8 CC 30 121 30 8 36 507 250 572 254 247 72 398 22 890 10 12 49 846 80 707 88 STs are indicated by single numbers eBURST of MLST data

  29. NRS-001NRS-002NRS-003 NRS-004NRS-011NRS-012 NRS-013NRS-014NRS-017 NRS-018NRS-019NRS-023 NRS-024NRS-049NRS-051 NRS-052NRS-068  NRS-070  NRS-076  NRS-110  NRS-126 NRS-127  NRS-149  NRS-155  NRS-173  NRS-179  NRS-238  NRS-240  NRS-244  NRS-269  NRS-382  NRS-387  NRS-402 NRS-403NRS-404 NRS-022 NRS-027 NRS-168  NRS-169  NRS-236  582 NRS-105  NRS-220  NRS-224 NRS-233 CC 45 15 97 NRS-102  NRS-187  NRS-227  NRS-260 NRS-103 508 395 NRS-111 45 NRS-164  NRS-183  NRS-234  NRS-250  47 NRS-109 25 CC 1 CC 5 NRS-072  NRS-123  NRS-192  NRS-193  NRS-194  NRS-226  NRS-229  NRS-248 NRS-262  NRS-263  188 NRS-104  NRS-112  NRS-131  NRS-162 NRS-174  NRS-176 NRS-180  NRS-181 NRS-196  NRS-204 NRS-207  NRS-210 NRS-212  NRS-213 NRS-216  NRS-219 NRS-222  NRS-223 NRS-231 NRS-246  NRS-252  NRS-261  NRS-267 NRS-241  NRS-243  NRS-408 59 371 1 NRS-063 NRS-064 NRS-065 5 NRS-074 NRS-021 NRS-026 NRS-028 NRS-073 NRS-077  NRS-106  NRS-107  NRS-108 NRS-128 NRS-133 NRS-177  NRS-189  NRS-191  NRS-247  NRS-264 105 372 231 NRS-054 NRS-056 239 34 NRS-079 NRS-256 109 CC 8 CC 30 121 NRS-114    NRS-129 NRS-130  NRS-134 NRS-135  30 NRS-148  NRS-153  NRS-165  NRS-166  NRS-185  NRS-186  NRS-215  NRS-217  NRS-225  NRS-228  NRS-237  NRS-239  NRS-242  NRS-258  NRS-266  NRS-230  NRS-259  8 NRS-119  NRS-120  NRS-121 NRS-136  NRS-137  NRS-139  NRS-140  NRS-141  NRS-142  NRS-144  NRS-145  NRS-146 NRS-147 NRS-150 NRS-151  36 507 250 NRS-029 NRS-071  NRS-283 NRS-383  572 NRS-156  NRS-158  NRS-171  NRS-172  NRS-211  NRS-214    NRS-100  NRS-281  NRS-035 254 247 72 NRS-251  NRS-254  NRS-257  NRS-384  NRS-385  NRS-175  NRS-386  NRS-198  NRS-201  NRS-202  NRS-274  NRS-275 NRS-036NRS-037 NRS-039NRS-118 NRS-209  NRS-218  NRS-249  NRS-272 398 22 NRS-253 890 10 NRS-199 12 NRS152  NRS157  NRS161  NRS184  NRS232  NRS235  NRS271  NRS154  NRS167  49 846 NRS-182  NRS-190  80 707 NRS-113  NRS-170  NRS-143 NRS-205 NRS-255 NRS-178 NRS-203 88 eBURST of MLST database showing locations of strains in NARSA S. aureus collection NRS-265 STs are indicated by single numbers VISA/GISA strains are underlined dotted lines indicate recombination events

  30. IN SUMMARY • NARSA Collection is representative of the species, but not comprehensive • MLST and spa provide an objective approach to distinguish the NARSA collection • How should the data be presented

  31. GOING FORWARD • Should all strains be catalogued with both MLST and spa? • Should we extend the genotype/phenotype? • Focus only on research strains? • How to update the NARSA strain information

  32. ST45; USA600 ST36: USA200 ST22 – USA900 ST239: USA1100 ST72: USA700 ST59: USA1000 ST8: USA300, USA500 ST1: USA400 ST15: USA1100 ST5: USA100, USA800 MRSA - USA TYPES

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