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Ion Torrent PGM sequencing for genomic typing of Neisseria 1
meningitidis for rapid determination of multiple layers of typing 2
information 3
Running title: Whole genome typing of meningococci 4
5
Ulrich Vogel 1*, Rafael Szczepanowski 2 , Heike Claus1, Sebastian Jünemann2, Karola Prior2 and Dag 6
Harmsen2 7
8
1 Institute for Hygiene and Microbiology, University of Würzburg, Germany; 2Department for 9
Periodontology, University of Münster, Germany 10
11
12
Corresponding footnote 13
Ulrich Vogel, Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2 (E1), 14
97080 Würzburg, Germany, Email: [email protected], T ++49 931 31 46802, F ++49 931 15
31 46445 16
17
18
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00038-12 JCM Accepts, published online ahead of print on 29 March 2012
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Abstract 19
Neisseria meningitidis causes invasive meningococcal disease in infants, toddlers and adolescents 20
worldwide. DNA sequence based typing has become the standard for molecular epidemiology of the 21
organism including multilocus sequence typing, analysis of genetic determinants of antibiotic resistance, 22
and sequence typing of vaccine antigens. However, PCR of multiple targets and consecutive Sanger 23
sequencing provides logistic constraints to reference laboratories. Taking advantage of the recent 24
development of benchtop next generation sequencers (NGS) and of BIGSdb, a database accommodating 25
and analyzing genome sequence data, we therefore explored the feasibility and accuracy of Ion Torrent 26
Personal Genome Machine™ (PGM™) sequencing for genomic typing of meningococci. Three strains 27
from a previous meningococcus B community outbreak were selected to compare conventional typing 28
results with data generated by semiconductor chip based sequencing. In addition, sequencing of the 29
meningococcal type strain MC58 provided information about the general performance of the 30
technology. The PGM™ technology generated sequence information for almost all target genes 31
addressed. The results were 100% concordant with conventional typing results with no further editing 32
necessary. In addition, the amount of typing information, i.e. nucleotides and target genes analyzed, 33
could be substantially increased by the combined use of genome sequencing and BIGSdb compared to 34
conventional methods. In a near future, affordable and fast benchtop-NGS machines like the PGM™ 35
might enable reference laboratories to switch to genomic typing on a routine basis. This will reduce 36
workload and rapidly provide information for laboratory surveillance, outbreak investigation, assessment 37
of vaccine preventability and antibiotic resistance gene monitoring. 38
39
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Introduction 41
Neisseria meningitidis is a Gram-negative facultative pathogen which causes invasive disease mostly in 42
infants, toddlers and adolescents (34). Despite of a relatively low incidence in industrialized countries, 43
the disease is considered to be of highest priority (3), because of a substantial case fatality rate and the 44
risk of secondary cases, outbreaks, and even epidemics. Many countries therefore established reference 45
laboratories for laboratory surveillance of disease in addition to statutory notification. Typing of the 46
organism is a major task of reference laboratories (47). DNA sequence based typing has replaced earlier 47
immunotyping (46). Capsular serogroup determination in conjunction with antigen sequence typing of 48
PorA and FetA, immunodominant outer membrane proteins, is highly discriminatory (11,44). Multilocus 49
sequence typing (MLST) (28) provides information about the clonal descent of an isolate (27). Sequence 50
based typing has furthermore been developed to determine variations in antibiotic resistance genes 51
associated with reduced antimicrobial susceptibility (37,41). Sequence typing of subcapsular antigens in 52
addition has become a major aspect of molecular typing of meningococci with the increasing attention 53
drawn towards protein based vaccines against serogroup B meningococci (17,18,26). This includes 54
sequence typing of the genes which encode the factor H binding protein (fHbp) (15,33), the Neisserial 55
heparin binding antigen (36) and the Neisserial adhesin A (8). Other protein based vaccines in 56
development comprise PorA (45), ZnuD (38), or Opc (24). Therefore, simultaneous assessment of a large 57
variety of target genes by DNA sequencing is highly desirable. 58
The meningococcal field is especially well suited as a paradigm for typing by whole genome sequencing 59
because of the development of sequence databases for meningococcal typing, which probably represent 60
the most powerful resource in the bacterial world (20,22,23). As a latest development, the typing data 61
are now powered by BIGSdb, an open source software accommodating genome sequence data (23). The 62
new database engine flexibly integrates numerous genomic loci for genetic analysis and serves for 63
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phylogenetic analysis, bacterial typing and functional assessment. Most importantly for this project, 64
submission of discontinuous genome sequence data from whatever source can be used to query MLST 65
with seven, but also 20 loci, antigen sequences not only of variable regions, but also of the whole coding 66
gene, multiple vaccine and antibiotic resistance genes. 67
The introduction of affordable and fast benchtop NGS machines like the Ion Torrent Personal Genome 68
Machine™ (PGM™) or illumina® MiSeq, makes bacterial whole genome sequencing (WGS) feasible for 69
small and medium sized laboratories (35). As the price per base continues to drop these machines will be 70
soon applicable for routine surveillance WGS. In this proof of principal study we assessed the 71
performance of PGM™ for meningococcal typing with three strains from a serogroup B meningococcal 72
outbreak (12) and a genome sequenced type strain as examples. We took advantage of the Neisseria 73
PubMLST database (http://pubmlst.org/neisseria/) to rapidly analyze data. We were interested in the 74
accuracy of data, whether editing of raw data was necessary, how much information beyond the 75
standard typing scheme can be achieved, and what the time from DNA preparation to the result is. 76
77
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Materials and Methods 79
Bacterial strains 80
Serogroup B strains DE9622, DE9686, and DE9938 were isolated in 2003 and 2004 in the neighboring 81
German counties Düren, Aachen and Heinsberg as part of a long lasting community outbreak caused by 82
strains of sequence type (ST)-42 (ST-41/44 clonal complex [cc]) with PorA variable region 1 (VR1) being 7-83
2, and VR2 being 4, and FetA F1-5 (12). The German outbreak strains were highly similar to the New 84
Zealand outbreak strain and were identical upon analysis by Multiple Locus Variable Number of Tandem 85
Repeat Analysis (MLVA) (2,12). The strains used in this study were isolated from children aged two to 86
four years and were sent to the National reference laboratory in Würzburg, Germany, for typing in the 87
frame of National laboratory surveillance. Strain MC58 (serogroup B, ST-74, ST-32 cc) was chosen as a 88
reference strain because it was the first meningococcal isolate to be completely sequenced (43). The 89
strain was isolated from invasive meningococcal disease in the UK in the 1980ies and was kindly provided 90
by E.R. Moxon (Oxford). Minimal inhibitory concentrations towards penicillin and rifampicin were 91
determined according to the manufacturer´s instructions by Etest (bioMérieux, France). Bacteria were 92
grown on Mueller-Hinton agar supplemented with 5% sheep blood (BectonDickinson, Germany). 93
Breakpoints defined by CLSI (http://www.clsi.org/) and EUCAST (http://www.eucast.org/) were applied. 94
95
DNA isolation 96
Meningococci were incubated on sheep blood agar (bioMérieux, France) over night at 37 °C and 5% CO2. 97
DNA isolation was performed as described (32). The quality of the genomic DNA was checked by gel 98
electrophoresis, the purity was measured with a NanoDrop 1000 (NanoDrop Products, USA), and the 99
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quantity was estimated by a fluorescence based method using the Qubit® dsDNA BR Assay Kit and the 100
Qubit® Fluorometer (Life Technologies, Germany) according to the manufacturer’s instructions. 101
102
Sequencing of selected strains on the Personal Genome Machine™ system 103
The genome sequences of the selected strains were determined on the PGM™ (Life Technologies, 104
Germany). Libraries were generated using 1 µg of the genomic DNA and the Ion Xpress™ Plus Fragment 105
Library Kit comprising the Ion Shear™ chemistry according to the user guide. After a dilution of each 106
library to 2.66 x 107 molecules/µL, 4.5 x 108 molecules were used as templates for clonal amplification on 107
Ion Spheres™ particles during the emulsion PCR according to the Ion Xpress™ Template 200 Kit manual. 108
The quality of the amplification was estimated on the Guava easyCyte 5 system (Millipore, Germany), 109
loaded onto an Ion 316™ chip and subsequently sequenced using 105 sequencing cycles according to the 110
Ion Sequencing 200 Kit user guide. 105 sequencing cycles approximately result in an average reading 111
length of 200 nucleotides. Genome projects have been registered as NCBI Bioprojects PRJNA78229, 112
PRJNA78227, and PRJNA78225. 113
114
Bioinformatics 115
MIRA (v. 3.4.0) was used for de novo assembly of all four genomes (7). Consecutively, the draft genomes 116
were uploaded to the BIGSdb website (http://pubmlst.org/software/database/bigsdb/) and analyzed 117
(23). DNA sequences in FASTA format were submitted online to the Neisserial locus/sequence definitions 118
database at http://pubmlst.org/neisseria. The database was interrogated for each locus in succession. 119
“Exact match” was searched for and recorded. SNPs and indels of the MC58 genome sequence were 120
extracted with the newest version of the CLC Genomics Workbench software (CLCbio, Aarhus, Denmark). 121
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For the comparison of the syntenic conservation of the chromosomal location of multiple genes between 122
two genomes, synteny plots were generated using the MUMmer 3.0 software suite (25). 123
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Results 125
Performance data 126
The turnaround time from DNA isolation to sequence reads was about 32 hours. The MIRA de novo 127
assembly on an ordinary PC then took approximately 3h per strain and the following BIGSdb analysis 128
needed another 20 minutes per genome. The accuracy of sequencing protocol was first assessed by 129
comparison of the MC58 draft sequence with the published genome sequence of the strain retrieved 130
from GenBank accession number AE002098.2 (43). The draft genome had a 49-fold sequencing coverage 131
resulting in 181 contigs. The sequence length of the consensus was 2, 194,618 nucleotides, the length of 132
the largest contig was 132,117 nuleotides, the median length 25,538. Twelve single nucleotide 133
polymorphisms were determined; four nucleotides differed and at eight loci the chip technology 134
revealed ambiguous results. Re-sequencing of eight of the twelve loci by PCR and Sanger sequencing on 135
both strands revealed identity to the original genome sequence in four cases and to the semiconductor 136
technology derived sequence also in four cases. Furthermore, 1,538 indels were recorded. We selected 137
ten loci for control by Sanger sequencing. All indels identified by semiconductor technology turned out 138
to be false. However, this did not affect any of the typing loci described below. The accuracy of the 139
sequence assembly was further demonstrated by the syntenic dot plot comparing the published MC58 140
sequence (43) with the de novo assembled MC58 sequence generated herein (Fig.1). 92% of the 141
published open reading frames were identified in the MC58 draft genome. The syntenic dot plot in 142
addition demonstrates a satisfactory agreement of the assemblies with very few discontinuities. 143
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Typing data 145
Three meningococcus B strains from a previous community outbreak in Western Germany (12) and the 146
type strain MC58 (43) were analyzed by semiconductor based sequencing. De novo assembled genomes 147
were analyzed by querying sequences in batch against BIGSdb in order to compare the data with our 148
previous conventional typing results (12), and – in addition - to assess the multitude of typing 149
information retrievable by the novel sequencing approach. The accuracy of extended typing beyond the 150
data achieved with our previous conventional typing results (12) was assessed by strain comparison 151
assuming a very high strain identity. For this purpose, BIGSdb was employed (23), to which whole 152
genome data can be uploaded and which allows comparison to genes of various functional categories. 153
Table 1 summarizes the results. For all four strains (three outbreak strains with prefix DE and reference 154
strain MC58) conventional typing results available at the laboratory were confirmed without any further 155
editing of the sequences. In addition to MLST and antigen sequence typing data, the following typing 156
information were retrieved: 13 additional housekeeping gene loci (extended or eMLST (9)), complete 157
gene of porA, porin B (porB) partial and complete gene, vaccine antigen gene fhbp (6), antimicrobial 158
resistance genes penA (41) and rpoB (40). Twelve of the additional thirteen housekeeping loci of the 159
eMLST (9) were identical among the outbreak strains, suggesting that they were correctly assessed. One 160
locus differed in one strain by 14 nucleotide exchanges. This finding was highly suggestive of a 161
recombination event, which is not unlikely even in highly related and epidemiologically linked strains 162
(21,39). This recombination event was independently confirmed by Sanger sequencing on both strands 163
(Fig. 2). The complete porA and porB genes were also fully identical among the outbreak strains, as were 164
the sequences of the fhbp gene. Finally sequence analyses of the antimicrobial resistance genes penA 165
and rpoB revealed identical allelesThe phenotypic susceptibility determined as minimal inhibitory 166
concentration by Etest was in line with the molecular analyses. In the penA database at www.Neisseria 167
.org (41) penA allele 1 is associated with a susceptible phenotype, because it lacks the typical mutations 168
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associated with reduced susceptibility. The same held true for rifampicin, where allele 18 is in line with a 169
susceptible phenotype. 170
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Discussion 172
Meningococcal typing serves a variety of purposes (16). Whereas the so-called fine-type, which includes 173
serogroup, PorA and FetA type (11), and the sequence type provides a framework for strain 174
discrimination and phylogenetic assignment (19), prediction of antimicrobial resistance (37,41) and 175
vaccine strain coverage (18,26) assist clinical management and preventive measures. Especially vaccine 176
antigen typing needs to be flexible used due to various approaches to meningococcus B subcapsular 177
antigen vaccine development (42,48). 178
In Europe, many National reference laboratories for logistic and financial reasons seem to be 179
overburdened by the effort to fulfill the typing requirements of the European Centre for Disease Control 180
and Prevention (ECDC) which include serogroup, PorA and FetA type and the sequence type (16,47). 181
Running seven PCR reactions and 14 sequencing reactions for a complete MLST scheme despite of all 182
possibilities of automation is an obvious challenge if done on hundreds of isolates. Not surprisingly, with 183
the advent of deep sequencing technologies such as Illumina technology (5) and 454 technology (29), 184
interest in replacement of time consuming PCR combined with Sanger sequencing by genome data 185
acquisition has increased considerably, but until now was hampered by cost and demands for rapid data 186
procession. A further major advantage of whole genome sequencing would be to archive abundant strain 187
information for rapid retrospective re-analysis if necessary. 188
This report describes the first application of 200 base reads for the Ion PGM™ platform. Increased 189
reading length improves substantially the results of the de novo assembly. Longer reading length and 190
scalability are the most discriminating features in comparison to the just released illumina® MiSeq 191
platform. Because of the PGM´s pyro-sequencing procedure quite a high number of indel errors due to 192
homopolymers were observed. However, indels did not affect the results of this study and further 193
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editing was not necessary. Furthermore, this systematic error can in principal be well compensated when 194
employing a genome wide gene by gene analysis coupled with allele reference databases like BIGSdb. It 195
is important to note that substitution errors, which are not compensable by comparison to allele 196
reference databases, appeared at a very low rate. 197
198
The semiconductor technology and the related 454 technology are sensitive to homopolymeric tracts. 199
We therefore wondered to what extent homopolymeric tracts in the meningococcal genome will cause 200
difficulties for the typing approach. In fact, the meningococcal genome contains a variety of long intra- 201
and intergenic homopolymeric tracts whose erroneous replication causes phase variation (31). 202
Fortunately, none of the typing loci addressed in this study belonged to the category of contingency loci. 203
Most surprisingly, the de novo sequence was highly robust and no further editing of sequences was 204
required. Furthermore, the detection of numerous indels, which were incorrectly identified by the novel 205
sequencing technology, was without consequence for the typing of three strains, as no indels were 206
identified in the numerous loci addressed for typing. This is an important finding as manual editing 207
consecutive to genome sequence assembly would otherwise be detrimental to broad application of 208
genomic typing. 209
The availability of the Neisseria sequence typing home page powered by BIGSdb greatly facilitated the 210
approach (23). Typing data of thousands of strains have been compiled herein, and the query platform 211
allows the interrogation of multiple loci within a negligible amount of time. The concept behind BIGSdb 212
was a prerequisite for this study. It should be highlighted that comparable database structures are also 213
needed for the application of genomic typing to other organisms of public health importance. 214
Genomic typing of microorganisms such as Neisseria meningitidis does not alter the typing philosophies 215
per se, it simply facilitates data acquisition. Typing of meningococci by MLST with seven loci is sufficient 216
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to define the clonal framework of a strain. Extended MLST (eMLST) of 20 loci mostly serves refined 217
phylogenetic analyses (9). The combination of serogroup, PorA and FetA has been validated for its 218
discriminatory power to identify possible epidemiological links between cases (10). For 1616 strains 219
isolated over a period of 42 months the Simpson´s index was high with 0.963. Discriminatory power 220
needs not to be extended by inclusion of other targets for most purposes. However, genomic typing 221
greatly facilitates the portfolio for on the fly analysis of vaccine antigens and antimicrobial resistance 222
genes. It provides the unique possibility of data storage for retrospective analysis of strains with regards 223
to antigen encoding genes included in future generations of vaccines. Currently, retrospective analyses 224
of this kind are initiated regularly for investigational vaccines and require novel repetitive sequencing of 225
hundreds of strains (4). It will greatly facilitate the search for specific markers in the event of emergence 226
of a new, highly virulent clone, such as the so-called ET-15 clone (1), which is typed by a single nucleotide 227
polymorphism and an insertion element (13,49). Maintaining physical strain collections will continue to 228
be an indispensible requirement also for the future, because besides the antigenic variant protein 229
expression is another predictor of strain coverage by bactericidal antibodies elicited by vaccines. 230
Nevertheless, constant re-addressing of stored genome data will greatly speed-up analyses and facilitate 231
vaccine implementation. 232
Taken together, our first experience with the use of Ion Torrent PGM™ for genomic typing of 233
meningococci was very positive with respect to speed, accuracy and the lack of necessity of further data 234
editing. For broad use in many reference laboratories, cost for both hardware and consumables must be 235
within the range of average budgets for laboratories. Laboratory partnering is a possible model to offer 236
service to small countries. Alternatively, efficient networking as exemplified by PulseNet (14) might help 237
distributing technology at a large scale in the future. Furthermore, bioinformatics tools need to be 238
developed that enable non-specialists to perform data processing at all steps of the procedure. 239
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Acknowledgements 241
The authors are indebted to the many senders of strains supporting the laboratory surveillance of 242
meningococcal disease at the Reference Laboratory in Würzburg for. The Reference Laboratory is funded 243
by the Robert Koch Institute, Berlin. We thank Johannes Elias for helpful discussions. Craig A. Cummings 244
from Life Technologies is thanked for giving advice for bioinformatics analysis. Finally, the authors wish 245
to thank Anjali Shah from Life Technologies for inclusion and support for the PGM™ 200 nucleotide read 246
length early access program. 247
This publication made use of the Neisseria Multi Locus Sequence Typing website (http://pubmlst.org/ 248
neisseria/) developed by Keith Jolley and sited at the University of Oxford (23). The development of this 249
site has been funded by the Wellcome Trust and European Union. 250
251
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Reference List 252
253
1. Ashton, F. E., J. A. Ryan, A. Borczyk, D. A. Caugant, L. Mancino et al. 1991. Emergence of a 254 virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococcal group C 255 disease in Canada. J.Clin.Microbiol. 29:2489-2493. 256
2. Baker, M. G., D. R. Martin, C. E. Kieft, and D. Lennon. 2001. A 10-year serogroup B meningococcal 257 disease epidemic in New Zealand: descriptive epidemiology, 1991-2000. J.Paediatr.Child Health 258 37:S13-S19. 259
3. Balabanova, Y., A. Gilsdorf, S. Buda, R. Burger, T. Eckmanns et al. 2011. Communicable diseases 260 prioritized for surveillance and epidemiological research: results of a standardized prioritization 261 procedure in Germany, 2011. PLoS.ONE. 6:e25691. 262
4. Bambini, S., A. Muzzi, P. Olcen, R. Rappuoli, M. Pizza et al. 2009. Distribution and genetic 263 variability of three vaccine components in a panel of strains representative of the diversity of 264 serogroup B meningococcus. Vaccine 27:2794-2803. 265
5. Bentley, D. R., S. Balasubramanian, H. P. Swerdlow, G. P. Smith, J. Milton et al. 2008. Accurate 266 whole human genome sequencing using reversible terminator chemistry. Nature 456:53-59. 267
6. Brehony, C., D. J. Wilson, and M. C. Maiden. 2009. Variation of the factor H-binding protein of 268 Neisseria meningitidis. Microbiology 155:4155-4169. 269
7. Chevreux, B., T. Pfisterer, B. Drescher, A. J. Driesel, W. E. Muller et al. 2004. Using the miraEST 270 assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced 271 ESTs. Genome Res. 14:1147-1159. 272
8. Comanducci, M., S. Bambini, B. Brunelli, J. Adu du-Bobie, B. Arico et al. 2002. NadA, a novel 273 vaccine candidate of Neisseria meningitidis. J.Exp.Med. 195:1445-1454. 274
9. Didelot, X., R. Urwin, M. C. Maiden, and D. Falush. 2009. Genealogical typing of Neisseria 275 meningitidis. Microbiology 155:3176-3186. 276
10. Elias, J., H. Claus, M. Frosch, and U. Vogel. 2006. Evidence for Indirect Nosocomial Transmission of 277 Neisseria meningitidis Resulting in Two Cases of Invasive Meningococcal Disease. J.Clin.Microbiol. 278 44:4276-4278. 279
11. Elias, J., D. Harmsen, H. Claus, W. Hellenbrand, M. Frosch et al. 2006. Spatiotemporal analysis of 280 invasive meningococcal disease, Germany. Emerg.Infect.Dis. 12:1689-1695. 281
12. Elias, J., L. M. Schouls, I. van de Pol, W. C. Keijzers, D. R. Martin et al. 2010. Vaccine preventability 282 of meningococcal clone, Greater Aachen Region, Germany. Emerg.Infect.Dis. 16:465-472. 283
13. Elias, J. and U. Vogel. 2007. IS1301 fingerprint analysis of Neisseria meningitidis strains belonging 284 to the ET-15 clone. J.Clin.Microbiol. 45:159-167. 285
on August 19, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
16
14. Gerner-Smidt, P., K. Hise, J. Kincaid, S. Hunter, S. Rolando et al. 2006. PulseNet USA: a five-year 286 update. Foodborne.Pathog.Dis. 3:9-19. 287
15. Giuliani, M. M., J. Adu-Bobie, M. Comanducci, B. Arico, S. Savino et al. 2006. A universal vaccine 288 for serogroup B meningococcus. Proc.Natl.Acad.Sci.U.S.A 103:10834-10839. 289
16. Harrison, O. B., A. B. Brueggemann, D. A. Caugant, A. van Der Ende, M. Frosch et al. 2011. 290 Molecular typing methods for outbreak detection and surveillance of invasive disease caused by 291 Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review. 292 Microbiology 157:2181-2195. 293
17. Jacobsson, S., S. T. Hedberg, P. Molling, M. Unemo, M. Comanducci et al. 2009. Prevalence and 294 sequence variations of the genes encoding the five antigens included in the novel 5CVMB vaccine 295 covering group B meningococcal disease. Vaccine 27:1579-1584. 296
18. Jiang, H. Q., S. K. Hoiseth, S. L. Harris, L. K. McNeil, D. Zhu et al. 2010. Broad vaccine coverage 297 predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup 298 B meningococcal disease. Vaccine 28:6086-6093. 299
19. Jolley, K. A., C. Brehony, and M. C. Maiden. 2007. Molecular typing of meningococci: 300 recommendations for target choice and nomenclature. FEMS Microbiol.Rev. 31:89-96. 301
20. Jolley, K. A., M. S. Chan, and M. C. Maiden. 2004. mlstdbNet - distributed multi-locus sequence 302 typing (MLST) databases. BMC.Bioinformatics. 5:86. 303
21. Jolley, K. A., J. Kalmusova, E. J. Feil, S. Gupta, M. Musilek et al. 2000. Carried meningococci in the 304 Czech Republic: a diverse recombining population. J.Clin.Microbiol. 38:4492-4498. 305
22. Jolley, K. A. and M. C. Maiden. 2006. AgdbNet - antigen sequence database software for bacterial 306 typing. BMC.Bioinformatics. 7:314. 307
23. Jolley, K. A. and M. C. Maiden. 2010. BIGSdb: Scalable analysis of bacterial genome variation at 308 the population level. BMC.Bioinformatics. 11:595. 309
24. Keiser, P. B., S. Biggs-Cicatelli, E. E. Moran, D. H. Schmiel, V. B. Pinto et al. 2011. A phase 1 study 310 of a meningococcal native outer membrane vesicle vaccine made from a group B strain with 311 deleted lpxL1 and synX, over-expressed factor H binding protein, two PorAs and stabilized OpcA 312 expression. Vaccine 29:1413-1420. 313
25. Kurtz, S., A. Phillippy, A. L. Delcher, M. Smoot, M. Shumway et al. 2004. Versatile and open 314 software for comparing large genomes. Genome Biol. 5:R12. 315
26. Lucidarme, J., M. Comanducci, J. Findlow, S. J. Gray, E. B. Kaczmarski et al. 2009. Characterization 316 of fHbp, nhba (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in 317 group B meningococcal ST269 clonal complex isolates from England and Wales. J.Clin.Microbiol. 318 47:3577-3585. 319
27. Maiden, M. C. 2008. Population genomics: diversity and virulence in the Neisseria. 320 Curr.Opin.Microbiol. 11:467-471. 321
on August 19, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
17
28. Maiden, M. C., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell et al. 1998. Multilocus sequence 322 typing: a portable approach to the identification of clones within populations of pathogenic 323 microorganisms. Proc.Natl.Acad.Sci.U.S.A 95:3140-3145. 324
29. Margulies, M., M. Egholm, W. E. Altman, S. Attiya, J. S. Bader et al. 2005. Genome sequencing in 325 microfabricated high-density picolitre reactors. Nature 437:376-380. 326
30. Martin, D. R., N. Ruijne, L. McCallum, J. O'Hallahan, and P. Oster. 2006. The VR2 epitope on the 327 PorA P1.7-2,4 protein is the major target for the immune response elicited by the strain-specific 328 group B meningococcal vaccine MeNZB. Clin.Vaccine Immunol. 13:486-491. 329
31. Martin, P., V. van de Ven, N. Mouchel, A. C. Jeffries, D. W. Hood et al. 2003. Experimentally 330 revised repertoire of putative contingency loci in Neisseria meningitidis strain MC58: evidence for 331 a novel mechanism of phase variation. Mol.Microbiol. 50:245-257. 332
32. Mellmann, A., D. Harmsen, C. A. Cummings, E. B. Zentz, S. R. Leopold et al. 2011. Prospective 333 genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by 334 rapid next generation sequencing technology. PLoS.ONE. 6:e22751. 335
33. Murphy, E., L. Andrew, K. L. Lee, D. A. Dilts, L. Nunez et al. 2009. Sequence Diversity of the Factor 336 H Binding Protein Vaccine Candidate in Epidemiologically Relevant Strains of Serogroup B Neisseria 337 meningitidis. J.Infect.Dis. 200:279-289. 338
34. Rosenstein, N. E., B. A. Perkins, D. S. Stephens, T. Popovic, and J. M. Hughes. 2001. 339 Meningococcal disease. N.Engl.J.Med. 344:1378-1388. 340
35. Rothberg, J. M., W. Hinz, T. M. Rearick, J. Schultz, W. Mileski et al. 2011. An integrated 341 semiconductor device enabling non-optical genome sequencing. Nature 475:348-352. 342
36. Serruto, D., T. Spadafina, L. Ciucchi, L. A. Lewis, S. Ram et al. 2010. Neisseria meningitidis 343 GNA2132, a heparin-binding protein that induces protective immunity in humans. 344 Proc.Natl.Acad.Sci.U.S.A 107:3770-3775. 345
37. Skoczynska, A., C. Ruckly, E. Hong, and M. K. Taha. 2009. Molecular characterization of resistance 346 to rifampicin in clinical isolates of Neisseria meningitidis. Clin.Microbiol.Infect. 15(12):1178-81. 347
38. Stork, M., M. P. Bos, I. Jongerius, N. de Kok, I. Schilders et al. 2010. An outer membrane receptor 348 of Neisseria meningitidis involved in zinc acquisition with vaccine potential. PLoS.Pathog. 349 6:e1000969. 350
39. Swartley, J. S., A. A. Marfin, S. Edupuganti, L. J. Liu, P. Cieslak et al. 1997. Capsule switching of 351 Neisseria meningitidis. Proc.Natl.Acad.Sci.U.S.A. 94:271-276. 352
40. Taha, M. K., S. T. Hedberg, M. Szatanik, E. Hong, C. Ruckly et al. 2010. Multicenter study for 353 defining the breakpoint for rifampin resistance in Neisseria meningitidis by rpoB sequencing. 354 Antimicrob.Agents Chemother. 54:3651-3658. 355
41. Taha, M. K., J. A. Vazquez, E. Hong, D. E. Bennett, S. Bertrand et al. 2007. Target gene sequencing 356 to characterize the penicillin G susceptibility of Neisseria meningitidis. Antimicrob.Agents 357 Chemother. 51:2784-2792. 358
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.org/D
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42. Tan, L. K., G. M. Carlone, and R. Borrow. 2010. Advances in the development of vaccines against 359 Neisseria meningitidis. N.Engl.J.Med. 362:1511-1520. 360
43. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C. Jeffries, K. E. Nelson et al. 2000. Complete genome 361 sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809-1815. 362
44. Urwin, R., J. E. Russell, E. A. Thompson, E. C. Holmes, I. M. Feavers et al. 2004. Distribution of 363 surface protein variants among hyperinvasive meningococci: implications for vaccine design. 364 Infect.Immun. 72:5955-5962. 365
45. van den Dobbelsteen, G. P., H. H. van Dijken, S. Pillai, and L. van Alphen. 2007. Immunogenicity 366 of a combination vaccine containing pneumococcal conjugates and meningococcal PorA OMVs. 367 Vaccine 25:2491-2496. 368
46. Vogel, U. 2010. Molecular epidemiology of meningococci: Application of DNA sequence typing. 369 Int.J.Med.Microbiol. 300:415-420. 370
47. Vogel, U. 2011. European efforts to harmonize typing of meningococci. Int.J.Med.Microbiol. 371 301:659-662. 372
48. Vogel, U. and H. Claus. 2010. Vaccine development against Neisseria meningitidis. 373 Microb.Biotechnol. 4:20-31. 374
49. Vogel, U., H. Claus, M. Frosch, and D. A. Caugant. 2000. Molecular basis for distinction of the ET-375 15 clone within the ET- 37 complex of Neisseria meningitidis [letter]. J.Clin.Microbiol. 38:941-942. 376
377 378
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Figure legends 380
381
Figure 1: 382
Syntenic dot plot of MC58 GenBank accession number AE002098.2 (x-axis) and the de novo assembled 383
MC58 sequence (y-axis). 384
385
Figure 2: 386
Depiction of the recombination event in the carB gene of strain DE9622, which harbors carB allele 32 in 387
contrast to the ST-41/44 cc isolates DE9686 and DE9938 with carB-8. A total of 2,233 bp were inserted 388
en bloc importing 81 single nucleotide changes. The figure represents a contig of 3,617 bp. CarB spans 389
the positions 501 to 3617. The polymorphic sites are numbered according to the sequenced contig. The 390
position numbers are presented vertically. 391
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Table 1
Category Locus DE9622 DE9938 DE9686 MC58 (outgroup)
MLST (ref. 28) abcZ 10 10 10 4 adk 6 6 6 10 aroE 9 9 9 5 fumC 5 5 5 4 gdh 9 9 9 5 pdhC 6 6 6 3 pgm 9 9 9 2 ST 42 42 42 74 cc ST-41/44cc ST-41/44cc ST-41/44cc ST-32cc eMLST (ref. 9) aspA 8 8 8 1 carB 32 9 9 3 dhpS 11 11 11 5 glnA 3 3 3 8 gpm 7 7 7 4 mtgA 7 7 7 5 pilA 5 5 5 3 pip 4 4 4 2 ppk 3 3 3 9 pykA 9 9 9 6 rpiA 1 1 1 5 serC 4 4 4 3 talA 7 7 7 3 AST (ref. 22, 44) PorA VR1/VR2 7-2,4 7-2,4 7-2,4 7,16-2 porA partial 39 39 39 12
porA full length 26 26 26 2
FetA 1-5 1-5 1-5 1-5 porB partial 3-1 3-1 3-1 3-24
porB full length 42 42 42 10
4CMenB (ref. 6) fhbp 14 14 14 1 FHBP 14 14 14 1 AR (ref. 40, 41) penA 1 1 1 3 rpoB 18 18 18 2
NOTE: MLST, multilocus sequence typing; ST, sequence type; cc, clonal complex; eMLST, extended MLST; AST, antigen sequence typing; VR, variable region; 4CMenB, Novartis investigational MenB vaccine; AR, antimicrobial resistance
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Table 2
Strain MIC a penicillin (Etest, bioMérieux)
penA allele b MIC rifampicin (Etest, bioMérieux)
rpoB allele c
DE9622 0,047 µg/ml (S)d 1 0,004 µg/ml (S) 18 DE9686 0,047 µg/ml (S) 1 0,006 µg/ml (S) 18 DE9938 0,047 µg/ml (S) 1 0,008 µg/ml (S) 18
NOTE: a MIC, minimal inhibitory concentration; b,c penA allele 1 and rpoB allele 18 are considered as predictive for a sensitive phenotype (http://pubmlst.org/neisseria/). d (S), sensitive as determined by Etest and breakpoints defined by CLSI (http://www.clsi.org/) and EUCAST (http://www.eucast.org/).
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