LegioType AS-1: Rapid microarray-based genotyping of Legionella pneumophila Sg1 isolates M. Petzold1, S. Jarraud2,3, R. Ehricht4, N. Jacotin2, T. Meyer4, P. Slickers4, A. Ziegler4, S. Monecke1,4 and C. Lück1 1
Faculty of Medicine ‚Carl Gustav Carus‘, Institute of Medical Microbiology and Hygiene/Institute of Virology, University of Technology Dresden, Germany 2 Centre National de Référence des légionelles, Centre de Biologie Est, Bron, France 3 Centre International de Recherche en Infectiologie (CIRI), Université Lyon 1, Faculté de médecine Lyon Est - Claude Bernard, Lyon, France 4 Alere Technologies GmbH, Jena, Germany
Objectives Reliable typing of clinical and environmental L. pneumophila isolates is necessary in routine testing of as well in outbreak situations. Sequence based typing (SBT) and monoclonal antibody (mAb)-based subgrouping reach more and more its limits as gold standard typing methods. There is an unmet clinical need for new typing methods is ongoing – with strong focus on whole genome sequencing (WGS). Although WGS became more affordable during the last decade, this technique is not yet fully evaluated and it is not yet applicable for routine diagnostic laboratories. We focused therefore on a method with similar properties regarding handling, speed and analysis as SBT and mAb-subgrouping but with increased discriminatory power. Using the well established ArrayMate platform (Alere Technologies, Jena, Germany) allowing the simultaneous processing of multiple arrays, we designed a DNAmicroarray to analyse L. pneumophila serogroup 1 isolates. The LegioType-AS1 represents the perfect link between the current gold-standard methods (SBT/mAbsubgrouping) and the oncoming WGS-area that is, so far, not yet affordable for all routine laboratories and not yet fully evaluated.
Application and Conclusion
100
99
98
97
96
95
94
93
92
91
90
89
Chip 4
88
87
Chip 4 ()
L CPL A A 1C A P1L 2 W 1LLC HP6r L 1 W A6W PxW x _t _o Co H 6 P 1 yW 2 3 4 5 6 1yc7 W2 1A o2 19ble1 b8n 2 3 4 5 6r6 7 8 1 2 3 4 5 1P 2 3 1in4 2 3 4 5f5 H C 1rK 2 3 4 5 f6L6 1 2 3 A4 31o10 4 5 7 8 9 0 6 1 2h2 3 4 K11 1 2 3 4 5 e_M1o8 11 0660 _ 6 1 W C 1 16 0 6PC 86C 0 1 _7 __G 66 1_ n2 1 2 _3 i-S_A __1 BB _t_L A BgB C P1 C DS IG rL YbBcluPS 3E o airlgrap P4b e 3 idria n B arM sIT n L lh m vtrp
Outbreak investigation A
99.5
99.2
Epidemic strain 98.5
99.5 98.1 99.2
97.8
97.4
97.3
97.0 98.3 99.5
97.7
99.0
99.5 99.2
97.8 98.2 99.5
99.3
98.1
99.5 86.0 99.3
96.9
99.5
99.1 99.5 99.3 98.6 96.7 99.5 98.1 98.8
99.5
96.0
99.3
98.8
99.0
97.3
Stamm
ST
SG-Mab
source
Key Modified Level In fla MALDI pil asd mip momp pro neu Classif epid. Orig-Nr/ Isolationsdatum IQ KbE/ml MLVA gsp CC dot ANR SBT PF Orig-name Not Chip_CC VNTR Sfi Arbeit CC related CCPat/Alter date
W13-886-1
345
1 Knox
env - D
.
W13-885-1
345
1 Knox
env - D
.
W13-884-2
345
1 Knox
env - D
.
W13-883-1
345
1 Knox
env - D
.
W13-882-2
345
1 Knox
env - D
.
L13-439
345
1 Knox
clin
.
W13-845-4
345
1 Knox
env - A
.
W13-873-1
345
1 Knox
env - A
.
W13-959-4
345
1 Knox
env - B
.
W13-954-3
345
1 Knox
env - B
.
W13-888-1
345
1 Knox
env - D
.
W13-957-2
345
1 Knox
env - B
.
W13-953-4
345
1 Knox
env - B
.
W13-953-3
345
1 Knox
env - B
.
W13-952-4
345
1 Knox
env - B
.
W13-881-1
345
1 Knox
env - D
.
W13-845-4_A
345
1 Knox
L13-435
345
1 Knox
clin
.
W13-1089-1
345
1 Knox
env - A
.
W13-967
345
1 Knox
env - B
.
W13-957-3
345
1 Knox
env - B
.
L13-473
345
1 Knox
clin
.
L13-477
345
1 Knox
clin
.
L13-446
345
1 Knox
clin
.
L13-445
345
1 Knox
clin - dupl
.
L13-444
345
1 Knox
clin
.
W13-871-2
345
1 Knox
env - B
.
W13-887-1
345
1 Knox
env - D
.
W13-875-15
345
1 Knox
env - D
.
W13-876-13
345
1 Knox
env - C
.
W13-875-17
345
1 Knox
env - D
.
W13-875-14
345
1 Knox
env - D
.
W13-874-15
345
1 Knox
env - A
.
W13-1096-2
345
1 Knox
env - B
.
W13-878-1
345
1 Knox
env - D
.
W13-871-1
345
1 Knox
env - B
.
W13-959-3
345
1 Knox
env - C
.
L13-438
345
1 Knox
clin
.
W13-877-2
345
1 Knox
env - D
.
W13-879-1
345
1 Knox
env - D
.
W13-1093
345
1 Knox
W13-1090-1
600
1 Knox
W13-845-31
600
1 Knox
env - A
.
W13-872-2
600
1 Knox
env - D
.
W13-1087-1
600
1 Knox
W13-845-23
600
1 Knox
env - A
.
W13-845-9
600
1 Knox
env - A
.
W13-876-16
600
1 Knox
env - C
.
W13-874-16
600
1 Knox
env - A
.
W13-876-15
600
1 Knox
env - C
.
W13-875-13
600
1 Knox
env - D
.
W13-875-16
600
1 Knox
env - D
.
W13-874-13
600
1 Knox
env - A
.
W13-1089-2
600
1 Knox
W13-845-28
600
1 Knox
env - A
.
W13-880-1
600
1 Knox
env - D
.
W13-845-22
600
1 Knox
env - A
.
W13-958-4
600
1 Knox
env - B
.
W13-845-15
600
1 Knox
env - A
.
W13-845-14
600
1 Knox
env - A
.
W13-1090-2
600
1 Knox
W13-845-25
600
1 Knox
env - A
.
W13-845-20
600
1 Knox
env - A
.
W13-845-21
600
1 Knox
env - A
.
W13-845-11
600
1 Knox
env - A
.
W13-1094-2
600
1 Knox
W13-845-27
600
1 Knox
env - A
.
W13-845-8
600
1 Knox
env - A
.
W13-845-34
600
1 Knox
env - A
.
W13-845-26
600
1 Knox
env - A
.
W13-845-12
600
1 Knox
env - A
.
W13-845-5
600
1 Knox
env - A
.
W13-845-18
600
1 Knox
env - A
.
W13-876-18
600
1 Knox
env - C
.
W13-876-17
600
1 Knox
env - C
.
W13-876-14
600
1 Knox
env - C
.
W13-875-18
600
1 Knox
env - D
.
W13-874-18
600
1 Knox
env - A
.
W13-874-17
600
1 Knox
env - A
.
W13-874-14
600
1 Knox
env - A
.
W13-845-19
600
1 Knox
env - A
.
•
The LegioType-AS1 is a rapid and reliable tool to investigate outbreak situations (Petzold et al., 2016).
•
Especially, when isolate numbers are high (e.g. in an outbreak situation), the multiplexed assay shows its advantages by combining rapidity (isolates can be analysed within 5 hours) and a high discriminatory power (0.965).
•
An essential point of the array for isolate analysis is reflected by six probes of the lag-1 gene that allows the subdivision into mAb 3/1+-isolates (predominantly found in patient samples) and mAb 3/1- -isolates.
•
The hybridisation profiles allow a higher resolution of complex ST’s with several mAb-subgroups (eg. ST1).
•
An user-friendly software was implemented into the ArrayMate reading device that assigns the HPsimilarity score and the corresponding CC or sub-complex to the tested isolate.
.
. .
.
Methods and results Biotin
.
.
.
An optimized protocol was established based on the HybridizationPlus Kit (Alere) to hybridise single stranded and biotin labeled amplicons to 97 probes. Probes were spotted in quadruplicate on DNA microarrays that were mounted onto ArrayStrips (Alere).
0
2
4
6
8
10
12
14
16
18
20
22
24
Chip 4
26
28
30
32
L CPL A A 1C A P1L 2 W 1LLC HP6r L 1 W A6W PxW x _t _o Co H 6 P 1 yW 2 3 4 5 6 1yc7 W2 1A o2 19ble1 b8n 2 3 4 5 6r6 7 8 1 2 3 4 5 1P 2 3 1in4 2 3 4 5f5 H C 1rK 2 3 4 5 f6L6 1 2 3 A4 31o10 4 5 7 8 9 0 6 1 1h2 2 3 K14 1 2 3 4 5 e_M1o8 11 0660 _ 6 1 W C 6 11 0 6PC 86C 0 1 _7 __G _6 16 n2 1 2 _3 i-S_A __1 BB _t_L A BgB C P1 C D IG L rS luPS YbBc 3E o airlgrap P4b e 3 idria n B arM IT s n L lh m trp v
A: Outbreak in Warstein (2013) Clear distinction between epideminic strain (Sg1, ST345, Knoxville) and predominant environmental strains of same mAb-subgroup Knoxville (Sg1, ST600). (Petzold et al., 2015) Chip 4 ()
B
Stamm
SG-Mab
Key Modified Level In fla MALDI pil asd mip momp pro neu Classif epid. Orig-Nr/ Isolationsdatum IQ KbE/ml ANR MLVA gsp CC dot source Arbeit related CCPat/Alter date
WL16-211-57
ST
1 Knox
.
WL16-204-1
1 Knox
.
WL16-219-43
1 Phil
.
WL16-192-2
1 Knox
.
WL16-204-83
1 Knox
.
WL16-211-27
600
1 Beni
.
WL16-202-10
345
1 Knox
.
WL16-204-40
474
1 Knox
.
WL16-204-16
2208
1 Beni
.
WL16-213-54
Clinical isolate
SBT CC PF Orig-name Not Chip_CC VNTR Sfi
Raw data signal intensities were normalised, quality checked and converted into a 97-ternary hybridization profile that categorises signals as positive (POS), negative (NEG) or ambiguous (AMB).
.
WL16-204-9
1 Olda-Oxford
.
WL16-204-15
1 Knox
.
WL16-407-3
2195 Chip?
1 Beni
.
WL16-204-47
2195
1 Beni
.
WL16-219-28
110
1 Beni
.
WL16-192-1
2209
1 Beni
.
WL16-211-38
2209
1 Beni
.
L16-142
2151
1 Beni
.
L16-086-1
2151
1 Beni
.
WL16-202-9
new?
1 Knox
.
WL16-405-1
.
WL16-219-42
1 Phil
.
WL16-173-29
1 Heys
.
WL16-213-32
1 Knox
.
CC-A ST51
0
2
4
6
8
10
12
14
16
18
20
22
24
Chip 4
26
28
30
32
34
L CPL A A 1C A P1L 2 W 1LLC HP6r L 1 W A6W PxW x _t _o Co H 6 P 1 yW 2 3 4 5 6 1yc7 W2 1A o2 19ble1 b8n 2 3 4 5 6r6 7 8 1 2 3 4 5 1P 2 3 1in4 2 3 4 5f5 H C 1rK 2 3 4 5 f6L6 1 2 3 A4 31o10 4 5 7 8 9 0 6 1 1h2 2 3 K14 1 2 3 4 5 e_M1o8 11 0660 _ 6 1 W C 6 11 0 6PC 86C 0 1 _7 __G _6 16 n2 1 2 _3 i-S_A __1 BB _t_L A BgB C P1 C D IG L rS luPS YbBc 3E o airlgrap P4b e 3 idria n B arM IT s n L lh m trp v
B: Outbreak in Bremen (2015/2016) The epidemic strain (Sg1, ST2151, Benidorm) could not be found in environmental samples. Chip 4 ()
C Epidemic strain
DNA-extraction of L. pneumophila colonies. Linear amplification of 25 target genes (incl. LPS-biosynthesis genes, variable elements and the genes pilE and neuA by using 62 primers and simultaneous biotin labeling of amplicons.
CC-A ST20 CC-A ST77 Stamm
ST
SG-Mab
Orig-Nr/ IQ Key Modified Level In MALDI fla pil asd mip momp pro neu Classif epid. Isolationsdatum KbE/ml dot ANR gsp CC MLVA source Arbeit related CCPat/Alter date
L10-069
62
1 Knox
.
L10-071
62
1 Knox
.
L10-070
62
1 Knox
.
L10-072
62
1 Knox
.
L09-451
62
1 Knox
.
L10-073
62
1 Knox
.
W10-080
62
1 Knox
. env
W10-078
62
1 Knox
. env
W10-081
62
1 Knox
. env
L10-040
62
1 Knox
. clin
L10-023
62
1 Knox
. clin
W10-079
62
1 Knox
. env
L10-034
62
1 Knox
. clin
W10-079a
62
1 Knox
.
W10-201
1
1 OLDA
.
W10-069
1
1 OLDA
.
W10-039
1
1 OLDA
.
EUL 119
1
1 OLDA
.
W10-241
1
1 OLDA
.
W10-053
1
1 OLDA
.
W10-030
608
1 OLDA
.
W10-029
608
1 OLDA
.
W10-046
595
1 OLDA
.
W10-044
595
1 OLDA
.
L10-232
1
1 OLDA
.
W10-056
334
1 Bell
.
W10-154
334
1 Bell
.
W10-052
104
1 OLDA
.
W10-054
196
1 Bell
.
Not CC PF Orig-name SBT Sfi VNTR Chip_CC
CC-A ST62 CC-A ST35/60
.
CC-A ST23
CC-A ST47
CC-001
.
C: Outbreak in Ulm (2010) Epidemic strain (Sg1, ST62, Knoxville) is clearly distinguishable from other environamtal isolates.
CC-012
CC-B ST45
Reference and acknowledgements Petzold et al., 2016 Rapid genotyping of Legionella pneumophila serogroup 1 strains by a novel DNA microarraybased assay during the outbreak investigation in Warstein, Germany 2013, IJHEH, online 22nd Feb. 2016 We gratefully thank all colleagues that helped us to investigate the outbreak scenarios. We thank Kerstin Lück for technical assistance. The study was partially supported by the RKI/German Federal Ministry of Health grant 1369-351.
Contact:
[email protected] Fetscherstraße 74, 01307 Dresden, Germany Phone: +49 351 458-6575
CC-B ST39/93
CC-B ST36
Approximately 700 isolates were screened. More than 100 well described strains including strains of the EUL-strain collection were tested at least twice in independent experiments. Furthermore, sets of related strains were analyzed in order to define a threshold that could assign related isolates to each other based on a HP-similarity score. Hybridisation profiles that had similarities > 94 % and that showed identity in key markers were clustered to a specific hybridisation pattern (HP). Patterns were combined to clonal complexes (CC) when the criteria of an HPsimilarity > 85 % and the same ST were met. We identified 165 HP’s and were able to cluster four mayor CC’s and 13 minor CC’s.
CC-B ST42
CC-B ST15
Contact:
[email protected] Download: http://alere-technologies.com/en/science-technologies/publications.html
