Genotyping The NARSA Collection

1
Genotyping The NARSA Collection

• Barry N. Kreiswirth
• PHRI

2
- 8
- 56
- 16
- 16
- 8
- 26
- 84
- 28
- 0
3
Clinical Relevant Strains
4
(No Transcript)
5
Genotyping Methods

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

6
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

7
(No Transcript)
8
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
9
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
10
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
11
An MLST Clonal Complex (CC) BURST Analysis
Robinson Enright - 2003, AAC473926
12
Cooper and Feil - 2006, Microbiology1521297
13
Cooper and Feil - 2006, Microbiology1521297
14
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

15
ST45 - Australian
ST36 - EMRSA16
ST22 - EMRSA15
ST8 Archaic/USA300
ST1 - MW2
ST5 - North American
MRSA Major Lineages
16
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
17
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

18
Protein A Gene Organization
19
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

20
(No Transcript)
21
MLST and spa Typing

• Consensus between two typing methods
• Database give comparable groups
• spa typing - faster molecular clock

22
Fowler et al - 2007, JID196738
23
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)

24
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
Group 2
Spa916 ZFGU2DMGGU2 Spa867 ZGM Spa910 ZGU2 Spa2
TJMBMDMGMK
ST 25
25
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
26
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  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 
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
27
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
28
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
CC25 ST25 spa 868 Reynolds
CC5 ST5 spa 230 502A
Cooper and Feil - 2006, Microbiology1521297
29
Neighbor-Joining Tree of Concatenated MLST
Allelic Sequences from the NARSA Collection
CC45
CC30
CC1
CC5
CC8 ?
CC8
CC1 ?
30
Neighbor-Joining Tree of Concatenated MLST
Allelic Sequences from the NARSA Collection
31
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
32
NRS-001  NRS-002  NRS-003  NRS-004  NRS-011 
NRS-012  NRS-013  NRS-014  NRS-017  NRS-018 
NRS-019  NRS-023  NRS-024  NRS-049  NRS-051 
NRS-052  NRS-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-403  NRS-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 
59
NRS-408
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
105
NRS-177  NRS-189  NRS-191  NRS-247  NRS-264
372
231
NRS-054  NRS-056
239
34
NRS-079 
109
NRS-256
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-036  NRS-037  NRS-039  NRS-118  NRS-209 
NRS-218  NRS-249  NRS-272
NRS-198  NRS-201  NRS-202  NRS-274  NRS-275
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
33
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

34
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

35
(No Transcript)
36
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