research article genomic analysis of a serotype 5 streptococcus...

Research ArticleGenomic Analysis of a Serotype 5 Streptococcus pneumoniaeOutbreak in British Columbia Canada 2005ndash2009

Ruth R Miller1 Morgan G I Langille23 Vincent Montoya4 Anamaria Crisan4

Aleksandra Stefanovic5 Irene Martin6 Linda Hoang57 David M Patrick1

Marc Romney58 Gregory Tyrrell910 Steven J M Jones31112

Fiona S L Brinkman3 and Patrick Tang13

1 School of Population and Public Health University of British Columbia Vancouver BC Canada2 Department of Pharmacology Dalhousie University Halifax NS Canada3 Department of Molecular Biology and Biochemistry Simon Fraser University Burnaby BC Canada4 British Columbia Centre for Disease Control Vancouver BC Canada5 Department of Pathology and Laboratory Medicine University of British Columbia Vancouver BC Canada6 National Microbiology Laboratory Public Health Agency of Canada Winnipeg MB Canada7 British Columbia Public Health Laboratory Vancouver BC Canada8 Department of Pathology and Laboratory Medicine St Paulrsquos Hospital Providence Health Care Vancouver BC Canada9 Department of Laboratory Medicine and Pathology University of Alberta Edmonton AB Canada10The Provincial Laboratory for Public Health (Microbiology) Edmonton AB Canada11Department of Medical Genetics University of British Columbia Vancouver BC Canada12Genome Sciences Centre BC Cancer Agency Vancouver BC Canada13Department of Pathology Sidra Medical and Research Center Doha Qatar

Correspondence should be addressed to Patrick Tang ptangsidraorg

Received 2 April 2015 Accepted 4 October 2015

Copyright copy 2016 Ruth R Miller et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Streptococcus pneumoniae can cause a wide spectrum of disease including invasive pneumococcal disease (IPD) From2005 to 2009 an outbreak of IPD occurred in Western Canada caused by a S pneumoniae strain with multilocus sequence type(MLST) 289 and serotype 5 We sought to investigate the incidence of IPD due to this S pneumoniae strain and to characterize theoutbreak in British Columbia using whole-genome sequencing Methods IPD was defined according to Public Health Agency ofCanada guidelines Two isolates representing the beginning and end of the outbreak were whole-genome sequencedThe sequenceswere analyzed for single nucleotide variants (SNVs) and putative genomic islands Results The peak of the outbreak in BritishColumbia was in 2006 when 57 of invasive S pneumoniae isolates were serotype 5 Comparison of two whole-genome sequencedstrains showed only 10 SNVs between themA 155 kb genomic islandwas identified in outbreak strains allowing the design of a PCRassay to track the spread of the outbreak strain Discussion We show that the serotype 5 MLST 289 strain contains a distinguishinggenomic island which remained genetically consistent over time Whole-genome sequencing holds great promise for real-timecharacterization of outbreaks in the future and may allow responses tailored to characteristics identified in the genome

1 Introduction

Streptococcus pneumoniae is a ubiquitous bacterium that cancause a wide spectrum of disease as well as being found inthe normal flora of the respiratory tract in healthy individ-uals Streptococcal disease ranges from ear and respiratorytract infections to invasive pneumococcal disease (IPD)

which includes bacteremia and its complications such asmeningitis arthritis and endocarditis In many countriesincluding Canada IPD is a reportable disease to publichealth authorities Prior to the introduction of pneumococ-cal conjugate vaccines the incidence of IPD was highestamongst infants However since then IPD rates from vaccineserotypes in children and adults have dropped significantly

Hindawi Publishing CorporationCanadian Journal of Infectious Diseases and Medical MicrobiologyVolume 2016 Article ID 5381871 7 pageshttpdxdoiorg10115520165381871

2 Canadian Journal of Infectious Diseases and Medical Microbiology

Table 1 Glossary of genomics and bioinformatics terms

Term Definition

Read Short fragment of DNA sequence output by genome sequencer Commonly 50ndash250 bp in lengthRaw read refers to reads taken directly from the genome sequencer and in no way filtered

Paired-end Pairs of reads that are two ends of the same region of DNA a standard distance apartDepth Number of reads mapped to a given position in the referenceReference based assembly Construction of reads by mappingaligning to a known reference sequenceDe novo assembly Assembly of reads without a referenceAdapter Short nucleotide sequences found at the end of reads which are part of the sequencing reactionInsertion Short regions of DNA present in our samples but not the reference sequenceContig Contiguous regions of DNA generated by joining together raw sequence reads

Genomic island Regions of the genome with suspected horizontal origins in that they were likely acquired fromother bacteria of the same or similar species

Sequence composition Proportion of each nucleotide base present

Codon usage bias Regions with different sequence composition or amino acid composition compared to the rest of thegenome

11989950 Length for which all the contigs of that length or longer contain at least half of the total of the lengthsof the contigs

Nonsynonymous Nucleotide substitution that alters the amino acid sequenceOpen reading frame (ORF) The part of a gene that has the potential to code for a proteinbp base pairs

with concomitant shifts in the ages of those affected andthe circulating serotypes of S pneumoniae [1] Pneumococcalserotype is determined by the polysaccharide capsule whichitself is also a virulence factor [2]

In Vancouver Canada a large cluster of IPD associatedwith S pneumoniae serotype 5 (multilocus sequence type orMLST 289) was identified in 2006 [3] following its initialemergence in Alberta in 2005 [4] This increase in IPDresulted in a large outbreak of pneumococcal disease thatspread throughout Western Canada (namely the provincesof British Columbia Alberta Saskatchewan and Manitoba)from 2005 to 2009 [3 4] In addition to being associated witha particular serotype-MLST profile the outbreak was largelyassociated with certain demographic groups affectingmostlyyoungmale adults (aged 16ndash65) ofAboriginal heritage Peopleaffected were alsomore likely to be homeless and intravenousdrug users (IDU) [3 4]

Here we provide an update on the incidence of IPDassociatedwith S pneumoniae serotype 5 since 2009 inBritishColumbia (BC) Furthermore we used a combination ofwhole-genome sequencing and PCR to genetically character-ize the outbreak strain This included the identification of agenomic island which enabled us to track the extent to whichthe outbreak strain was causing IPD in BC

2 Methods

21 Identification of S pneumoniae Isolates Associated withIPD All S pneumoniae isolates throughout Canada fittingthe definition of S pneumoniae IPD are required to bereported to regional public health authorities In BritishColumbia invasive isolates of S pneumoniae are forwardedto the BC Public Health Microbiology and Reference Lab-oratory for confirmation and initial serotyping followed by

additional serotyping at the National Centre for Streptococ-cus [4] For the purpose of this study all IPD cases from BCwith S pneumoniae recovered in culture were included

A confirmed case of IPD was defined as clinical illnesssuch as pneumonia with bacteremia bacteremia without aknown site of infection and meningitis Laboratoryconfirmation of S pneumoniae was either through culture ordetection of DNA from a normally sterile site (excluding themiddle ear) (httpwwwphac-aspcgccapublicatccdr-rmtc09vol3535s2Pneumoco-engphp) All isolates confirmed asS pneumoniae are stored at minus80∘C

22 Whole-Genome Sequencing Two isolates were selectedfor whole-genome sequencing BCSP1 and BCSP2They wereisolated in August 2006 and February 2010 respectivelyboth from patients admitted to a tertiary care hospital inVancouver These isolates were chosen since BCSP1 wasthought to be the index case inBCandBCSP2was a randomlyselected strain from the tail end of the outbreak in BC

The bacteria were cultured on sheep blood agar platesand approximately 4mm3 of colonial growth was used forDNA extraction using the Qiagen DNA Mini Kit as per themanufacturerrsquos instructions Library preparation for BCSP1was performed as described previously [5] and for BCSP2wasperformed using the Nextera kit (Illumina San Diego USA)as per the manufacturerrsquos instructions BCSP1 was sequencedon the Illumina GAIIx (50 bp paired-end reads) while BCSP2was sequenced on the Illumina MiSeq (250 bp paired-endreads) Sequence reads were deposited to the EuropeanNucleotide Archive under accession number PRJEB7803(httpwwwebiacukenadataviewPRJEB7803) A glos-sary of genomics and bioinformatics terms is listed inTable 1

Canadian Journal of Infectious Diseases and Medical Microbiology 3

23 Bioinformatics Methods For single nucleotide variant(SNV) detection we chose to perform reference based assem-bly whereby the raw reads are mapped or aligned to a chosenreference sequence This involves matching each individualread to its best place in the reference genome and resultsin a nucleotide-by-nucleotide comparison of every sequencecompared to every position in the reference To performreference based assembly the software bowtie2 v0128 wasused to align raw reads compared to the Streptococcuspneumoniae 70585 reference (NCBI accession CP000918)using software input parameters ndashphred33 ndashlocal ndashdovetailndashmaxins 850 to maximize the quality of the assembly Itwas then necessary to convert the alignment results intoSNVs compared to the reference using the software SAMtoolsv0119 [6] with the parameter indel detection turned offThe SNVs generated using SAMtools were then filtered toensure high confidence The parameters for filtering were (i)minimum read depth of five with at least one read in each ofthe forward and reverse direction (ii) maximum read depthnot greater than the highest 25 of the depth distributionfor every nucleotide in the sample (iii) minimum root-mean-square read mapping quality (statistic generated bySAMtools) of 20 (iv) minimum of 75 of reads supportingthe consensus call of the nucleotide present at each positionand (v) calls required to be homozygous under the diploidmodel assumed by SAMtools (as determined from an outputstatistic generated by SAMtools)

Additionally we performed de novo assembly in orderto identify any regions present in the outbreak strains butnot the reference sequence Prior to assembly the softwarecutadapt [7] was used to remove Illumina adapters usingthe parameter ndasha for sample BCSP2 The software programVelvet v1207 [8] was then used for de novo assemblyin conjunction with the VelvetOptimiser v220 (httpbioinformaticsnetausoftwarevelvetoptimisershtml)

After de novo assembly we identified insertions notpresent in the 70585 reference using the software programsprogressiveMauve v231 [9] and NUCmer [10] Insertionswere investigated using all contigsgt500 bp generated from denovo assembly of the reads unmapped after reference basedassembly Regions were considered potentially unique to onestrain if they did not have a match according to the backboneoutput file generated byMauve or if the output of the softwareNUCmer indicated that lt5 of one contig was not coveredor gt5 of a contig had double or more coverage from contigsfrom the other sample Regions were verified as unique bychecking that they did not match to any regions generatedfrom the de novo assemblies from the entire read set usingthe software BLAST and only regions gt500 bp which didnot match according to BLAST were considered uniqueDeletions with respect to the 70585 reference were identifiedusing a window-based approach whereby any region of 1 kbwith gt500 bp missing was considered a deletion

24 Genomic Island Identification Genomic islands areregions of the genome with suspected horizontal origins inthat they were likely acquired from other bacteria of the sameof similar species To identify putative genomic islands in

our samples we used the software IslandPick [11] IslandPickis a comparative genomics approach that uses both genomealignments and BLAST to identify genomic regions that areunique to a genome when compared to a set of closely relatedspecies IslandPick was used with a minimum island sizeof 4000 base pairs and with the genomes of StreptococcuspneumoniaeD39 S pneumoniaeTIGR4 S pneumoniaeR6 SpneumoniaeG54 S pneumoniaeCGSP14 and S pneumoniaeHungary Abnormal dinucleotide and codon usage bias wastested using the software IslandViewer [12] while insertionsequences were identified using the software ISfinder [13]

25 MLST Seventeen isolates were available for multilocussequence typing (MLST) MLST was performed usingprimers and conditions described at httppubmlstorgspneumoniae Resulting sequences were edited and assem-bled using DNAStar Inc and submitted to the MLSTdatabase

Six strains sequenced by Chewapreecha et al [14] werealso included in a comparative analysis of the genomic islandFive of these six strainswere investigated because they had thesame serotype and sequence type (ST) as outbreak strains inthis study and the sixth was investigated because a BLASTnsearch of the genomic island to the NCBI wgs databaseidentified a match to a contig from this strain The six strainswere downloaded from the NCBI short read archive in sraformat and converted to fastq using the software programSRA toolkit fastq-dump [15]Theywere then assembled usingidentical methods to those used for the two outbreak strainsNeighbor joining trees to compare the genomic island in thestrains sequenced by Chewapreecha et al to the outbreakstrains were constructed using the software MEGA 6 [16]

26 PCR Detection of the Genomic Island Two regions of thegenomic islandwere targeted for PCR amplification A 923 bpproduct at the 51015840 end of the island was amplified using BF1 51015840-ATCTTTTCCGCGAATCACTG-31015840 (Venter 70585 2049624-2049643) and BR1 51015840-TTGTATTGGAGGACCAAGC-31015840(2050519-2050537) primers Also a 1323 bp productwas amplified targeting the 31015840 end with EF1 51015840-GCGCTGTAATATAGGCAAAGC-31015840 (2054079-2054099)and ER1 51015840-ATAAGCGTGTCCGCTATCGT-31015840 (2055382-2055401) primers Bacterial colonies were inoculated intoeach PCR reaction consisting of 125 120583L of HotStart Taqmastermix (Qiagen) 125 120583L of each 10 120583M primer and10 120583L of ddH

2O Reactions were incubated at 95∘C for 15

minutes followed by 30 cycles each of 94∘C for 30 s x∘C for30 s and 72∘C for 60 s and finally 72∘C for 10min where 119909equals 525∘C for BF1BR1 and 540∘C for EF1ER1 primersAdditional reactions were also carried out with Ready-To-GoPCR beads (GE Healthcare) with 05 120583L of each 10 120583Mprimer and 24120583L of ddH

2O Reactions were carried out

with the same conditions for each respective primer pairAny samples with discrepant or negative PCR results wererepeated


0

50

100

150

200

250To

tal n

umbe

r of S

pne

umon

iae i

sola

tes

Serotype 5

Non-serotype 5Not typed

2013

q42013

q32013

q22013

q12012

q42012

q32012

q22012

q12011

q42011

q32011

q22011

q12010

q42010

q3

2010

q12009

q42009

q32009

q22009

q12008

q42008

q32008

q22008

q12007

q42007

q32007

q22007

q12006

q42006

q32006

q22006

q12005

q42005

q32005

q22005

q12004

q42004

q32004

q22004

q12003

q42003

q32003

q22003

q1

lowast2010

q2

00

83

56

164

74

35

69

26

41

00

00

00

00

63

235

572

425

289

353

286

133

168

157

82

31

42

43

55

24

00

00

00

00

00

00

00

08

34

00

72

08

00

00

00

00

Figure 1 Quarterly prevalence of serotype 5 pneumococcal IPD in British Columbia Numbers above bars are serotype 5 isolates perquarter lowast is introduction of PCV13 q quarterly (ie three-month period)

3 Results

31 Outbreak Epidemiology Investigation of S pneumoniaeserotypes recorded from IPD samples in BC shows thatthe outbreak occurred from the third quarter of 2006 to2009 with only sporadic detections thereafter (Figure 1)During the peak occurring in the fourth quarter of 200657 of all invasive S pneumoniae isolates were serotype 5After this the number of S pneumoniae isolates that wereserotype 5 continued to decrease until June 2010 when anew multivalent pneumococcal conjugate vaccine (PCV13)was introduced in Canada which included serotype 5

32 Genomic Island Genomic islands contiguous regions ofgenomic sequence that have probable horizontal origins wereinitially identified using IslandPick This approach identifieda 155 kb genomic island which is found to be present instrains isolated from the outbreak This island is widelypresent in Streptococcus suis however in S pneumoniaeit has only been identified in six serotype 5 isolates thereference strain 70585 (NCBI accession CP000918) and fiveisolates from Maela Thailand in 2010 [14] (ENA acces-sion ERR054237 ERR066355 ERR064008 ERR067846 andERR084189) Additionally the genomic island has been

identified in a single serotype 19B ST5095 strain also fromMaela isolated in 2008 (ENA ERR052634)

Notably the sequence composition (ie proportion ofeach nucleotide base present) did not differ between thegenomic island regions reported using IslandViewer and therest of the genome This suggests that the region was trans-ferred froma genomewith similar sequence composition or isan ancient transfer that has allowed amelioration of the regionto the host genome

33 PCR Based Characterization of the Genomic Island in OurSamples From 23rd of August 2006 to 24th of July 2007150 serotype 5 S pneumoniae isolates plus 10 isolates withother serotypes all fromBC were randomly selected for PCRtesting for the outbreak-specific genomic islandAdditionallyto characterize more recent isolates one isolate was selectedfrom the most prevalent serotypes causing invasive diseasein 2013 (119873 = 38) including serotype 5 (119873 = 1) Nearlyall of the serotype 5 samples were PCR positive for thegenomic island (146149 980) However 4149 serotype5 samples were negative for the genomic island of which214 were invasive It should be noted that MLST and repeatserotyping were not performed on these four serotype 5 PCR-negative isolates so we are unable to confirm that they areST289 All other non-serotype 5 isolates were PCR-negative


Table 2 SNVs between BCSP1 and BCSP2

Position70585

referencebase

BCSP1base

BCSP2base Qualifier Gene product Synonymous

Aminoacid

change384518 T A T SP70585 0437 Hypothetical protein No Krarr lowast425383 C C T SP70585 0473 Helicase RecDTraA family No R rarr H457503 C C T ⟨Noncoding⟩ Not applicable mdash mdash568204 C C T ⟨Noncoding⟩ Not applicable mdash mdash923459 T T G SP70585 1004 Dihydroorotate dehydrogenase 1B No Srarr A936681 A A T SP70585 101 Competence protein No Krarr N1389846 C A mdash SP70585 1509 Pyridoxal biosynthesis lyase PdxS No Erarr lowast1675901 G G A SP70585 1804 Preprotein translocase subunit SecY No Trarr I2044455 T G T SP70585 2234 beta-N-Acetylglucosaminidasebeta-glucosidase (3-

beta-N-acetyl-D-glucosaminidasebeta-D-glucosidase)(Nag3)

No Erarr A

2104679 T T G SP70585 2289 alpha-L-Fucosidase No Srarr RSNV is located in the genomic island

(Supplementary Table 1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520165381871)

34 Whole-Genome Sequencing (WGS) 976 of 12160592reads and 973 of 941010 reads for samples BCSP1 andBCSP2 respectively were mapped to the 70585 referenceresulting in 956 of the reference being covered by sequencereads for both of the samples After filtering to ensure highconfidence in calls made 905 and 887 of positionsin the 70585 reference were called in BCSP1 and BCSP2respectively De novo assembly of unmapped reads produced11 contigsgt500 bp for BCSP1with 11989950 1521median depth 176and total length 13337 For BCSP2 eight contigs gt500 bp wereproduced with 11989950 1204 median depth 81 and a total lengthof 9475 bp

Comparison of the two reference based assembliesrevealed that they were highly similar with only 10 variantpositions between the two samples nine of which werenonsynonymous (Tables 1 and 2) Assembly of the unmappedreads identified one region 1476 bases in length present insample BCSP1 but not in BCSP2 which encoded a probablephage integrase

Comparison of the two outbreak strains to the 70585reference revealed 1429 SNVs where both BCSP1 and BCSP2differ from the reference seven genetic regions present inour samples but not present in the reference and 13 regionswhere the strains have deletions compared to the reference

Identification of 10 SNVs between the two isolates sep-arated in time by 35 years is suggestive of an evolutionaryrate of sim131 times 10minus6 substitutions per site per year which isslightly lower than the S pneumoniae evolutionary rate of 157times 10minus6 substitutions per site per year (95CI 134ndash179times 10minus6)and 145ndash481 times 10minus6 estimated by Chewapreecha et al andCroucher et al respectively [14 17] The difference of 1429SNVs between the two outbreak samples sequenced for this

study and the reference strain suggest a much more distantcommon ancestor

35 Genomic Comparison of the Genomic Island betweenSamples The outbreak-specific genomic island was detectedin both samples from the velvet contigs generated It differedby one variant position between the two samples in the geneproducing beta-N-acetylglucosaminidase verified from thereference based and de novo assembly

Comparison of the genomic islands identified in thisstudy to reference strain 70585 identified two nonsynony-mous substitutions (Figure 2)The genomic islands identifiedfrom Maela were more distantly related to those in theoutbreak strains The first four serotype 5 samples fromMaela isolated from February to April 2010 were identical toone another and differed by four and three SNVs fromBCSP1and BCSP2 respectively The genomic island from the fifthserotype 5 strain from Maela isolated in July 2010 differedfrom BCSP1 and BCSP2 by five and four SNVs respectivelyThese SNVs were completely different from those in theother serotype 5 Maela strains Finally the serotype 19Bstrain containing the genetic island was isolated considerablyearlier in July 2008 and was more divergent from the otherislands with eight and nine SNVs from the two outbreakstrains (Figure 2)

In comparison to the homologous genomic island presentin S suis strain TL13 [18] the S pneumoniae genomic islanddiffers by nearly 2000 variant positions of which sim350 arenonsynonymous Furthermore the S suis island containsthree large deletions compared to the S pneumoniae homologof 19 27 and 105 nucleotides respectively as well as foursmaller deletions of 1 (119873 = 4) or 2 (119873 = 1) bp Of these the105 bp deletion spans the region between the end of the openreading frame (ORF) 2244 and the start of the subsequentORF 2245 encoding components of the endA gene andrepresents the insertion of the RUP unit in the pneumococcalversion of the genomic island Finally the S suis genomic


ERR052634

S pneumoniae 70585

ERR084189

ERR067846

ERR064008

ERR054237

BCSP2

BCSP1

ERR066355

2

2

5

3

11

A A A AA

A A A AA

A A A AA

A A A

A AA A

A A

A

A

A A A

A

A A

A A

A A A

A

A

A

A

A

A

C

C

C

C

C

C

C

C

C

C

CC

C CC C

CC

CC

CC

CC

CC

CC

CC GGGG

G

G

G

G

GG

G

G

G G

G

GG

GG

GG

GG

GG

GG

GG

G

G

G

G

TT

TT

TT

TT

TT

TT

T

T

T

T T T

TT

2041246

2041378

2041771

2043727

2044455

2044708

2045304

2045914

2045915

2049998

2050506

2051060

2052056

2055330

Figure 2Neighbor joining tree of single nucleotide variants (SNVs)within the genomic island showing the evolutionary relationship betweenBCSP1 and BCSP2 (sequenced as part of this study) the 70585 reference and six strains sequenced by Chewapreecha et al [16] (ERR067846ERR066355 ERR064008 ERR054237 ERR084189 and ERR052634) The table to the right shows the individual SNVs that separate theisolates with their position in the 70585 reference at the top

island also contains six insertions of 1 (119873 = 4) 2 (119873 = 1)and 9 (119873 = 1) bp respectively of which the 9 bp insertionalso falls in the 2245 endA gene

4 Discussion

Wehave reported an update on the continuation of the ST289serotype 5 pneumococcal outbreak in Western Canada from2009 to the present date including genetic characterizationof the strain Whole-genome sequencing of two strainsrepresenting the beginning and the end of the outbreak inBC identified a distinct 155 kb genetic island present in theoutbreak strain which has remained genetically consistentover time From this sequence a PCR assay was designedwhich was able to detect the genomic island in almostall of serotype 5 strains tested from the outbreak perioddemonstrating its utility as a marker for the outbreak It ispossible that the genomic island could provide an explanationfor the increased severity of IPD seen during the outbreakhowever further functional experiments would be requiredto confirm this

The most closely related sequence to the two strainsinvestigated for this study is an uncharacterized strain ofS pneumoniae (serotype 5 and ST289) also containing thegenetic island However five other ST289 serotype 5 strainsisolated in Thailand four years after the peak of the outbreakin BC have also been demonstrated to contain the genomicisland The fact that this genomic island is present in strainsfrom such a wide geographical range suggests worldwidespread of this S pneumoniae strain Alternatively it could be

possible that the genomic island has been present in serotype5 pneumococci for quite some time as suggested by the lackof sequence compositional bias between the genomic islandand the rest of the genome and the variation present withinthe island sequence Interestingly the genomic island wasalso identified in a serotype 19B ST5059 isolate in Maelasuggesting recombination between this and a serotype 5strain or similar phage origins for the island region

Comparison of BCSP1 and BCSP2 sequenced for thisstudy showed that throughout the approximately four-yearoutbreak the responsible strain remained genetically similarand evolved only 10 SNVs between the isolates examinedan evolutionary rate marginally slower than previous esti-mates However this rate may have been slightly greater ifless stringent filtering criteria were used to identify SNVsDespite this the 70585 reference strain had more than 1000SNVs compared to the two outbreak strains when the sameanalysis parameters were used The conserved nature of theoutbreak strain together with its success over the outbreakperiod suggests that the strain is highly adaptive to its cur-rent environment however further functional experimentswould be required to determine which genetic features causethis One limitation of the comparison performed betweenthe two isolates is that they were each sequenced using adifferent platform (the Illumina GAIIx andMiseq) Howeverit has previously been demonstrated that different Illuminatechnologies have no impact on the conclusions drawn [19]

Genomic sequencing used in conjunction with clinicaland epidemiological analysis that is genomic epidemiol-ogy holds much promise in allowing rapid identification


of outbreaks and notable new features associated withthem [5 20] Furthermore use of genomic epidemiologyis likely to become increasingly routine as the cost ofwhole-genome sequencing decreases and technologies forsequencing improve As well as finding genomic islandsgenomic epidemiology may identify virulence factors orantimicrobial resistance genes warranting further study [2122] The identification of such features can then directlyimpact public health decisions for example it may benecessary to implement a wider vaccine campaign if a newstrain harbors a genetic island with potential increasedvirulence and transmissibility Furthermore such featurescan also be used to design specific diagnostic assays to rapidlydetect and track the spread of the outbreak strain such asthe PCR designed to identify the genomic island in thisoutbreak investigation As we obtainmore genome sequencesand more functional genomics data our ability to performsuch analyses will only improve and become an increasinglyvaluable tool in infectious disease outbreak analysis andcontrol

Competing Interests

The authors declare that they have no competing interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by grants from the BritishColumbiaCentre for Disease Control Foundation for Population andPublic Health andGenomeCanadaWhole genome sequenc-ing was performed at the Michael Smith Genome SciencesCentre and the McGill University and Genome QuebecInnovation Centre

References

[1] X Deng D Church O G Vanderkooi D E Low and DR Pillai ldquoStreptococcus pneumoniae infection a Canadianperspectiverdquo Expert Review of Anti-InfectiveTherapy vol 11 no8 pp 781ndash791 2013

[2] S D Bentley DM Aanensen AMavroidi et al ldquoGenetic anal-ysis of the capsular biosynthetic locus fromall 90 pneumococcalserotypesrdquo PLoS Genetics vol 2 no 3 p e31 2006

[3] M G Romney M W Hull R Gustafson et al ldquoLargecommunity outbreak of Streptococcus pneumoniae serotype5 invasive infection in an impoverished urban populationrdquoClinical Infectious Diseases vol 47 no 6 pp 768ndash774 2008

[4] G J TyrrellM LovgrenQ Ibrahim et al ldquoEpidemic of invasivepneumococcal disease western Canada 2005ndash2009rdquo EmergingInfectious Diseases vol 18 no 5 pp 733ndash740 2012

[5] J L Gardy J C Johnston S J Sui et al ldquoWhole-genomesequencing and social-network analysis of a tuberculosis out-breakrdquoTheNew England Journal of Medicine vol 364 no 8 pp730ndash739 2011

[6] H Li B Handsaker A Wysoker et al ldquoThe sequence align-mentmap format and SAMtoolsrdquoBioinformatics vol 25 no 16pp 2078ndash2079 2009

[7] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet vol 17 no 1 pp 10ndash122011

[8] D R Zerbino and E Birney ldquoVelvet Algorithms for de novoshort read assembly using de Bruijn graphsrdquo Genome Researchvol 18 no 5 pp 821ndash829 2008

[9] A E Darling B Mau N T Perna and J E Stajich ldquoprogres-siveMauve multiple genome alignment with gene gain loss andrearrangementrdquo PLoS ONE vol 5 no 6 Article ID e11147 2010

[10] S Kurtz A Phillippy A L Delcher et al ldquoVersatile and opensoftware for comparing large genomesrdquo Genome Biology vol 5no 2 article R12 2004

[11] M G Langille W W Hsiao and F S Brinkman ldquoEvaluationof genomic island predictors using a comparative genomicsapproachrdquo BMC Bioinformatics vol 9 article 329 2008

[12] M G Langille and F S Brinkman ldquoIslandViewer an integratedinterface for computational identification and visualization ofgenomic islandsrdquo Bioinformatics vol 25 no 5 pp 664ndash6652009

[13] P Siguier J Perochon L Lestrade J Mahillon and MChandler ldquoISfinder the reference centre for bacterial insertionsequencesrdquo Nucleic Acids Research vol 34 pp D32ndashD36 2006

[14] C Chewapreecha S R Harris N J Croucher et al ldquoDensegenomic sampling identifies highways of pneumococcal recom-binationrdquo Nature Genetics vol 46 no 3 pp 305ndash309 2014

[15] R Leinonen H Sugawara and M Shumway ldquoThe sequenceread archiverdquo Nucleic Acids Research vol 39 supplement 1 ppD19ndashD21 2011

[16] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[17] N J Croucher S R Harris C Fraser et al ldquoRapid pneumococ-cal evolution in response to clinical interventionsrdquo Science vol331 no 6016 pp 430ndash434 2011

[18] K Wang H Yao C Lu and J Chen ldquoComplete genomesequence of Streptococcus suis serotype 16 strain TL13rdquo GenomeAnnouncements vol 1 no 3 Article ID e00394-13 2013

[19] J G Caporaso C L Lauber W A Walters et al ldquoUltra-high-throughputmicrobial community analysis on the IlluminaHiSeq andMiSeq platformsrdquoThe ISME Journal vol 6 no 8 pp1621ndash1624 2012

[20] RMMiller J R Price EM Batty et al ldquoHealthcare-associatedoutbreak of meticillin-resistant Staphylococcus aureus bacter-aemia role of a cryptic variant of an epidemic clonerdquo TheJournal of Hospital Infection vol 86 no 2 pp 83ndash89 2014

[21] AMellmann D Harmsen C A Cummings et al ldquoProspectivegenomic characterization of the German enterohemorrhagicEscherichia coli O104H4 outbreak by rapid next generationsequencing technologyrdquo PLoS ONE vol 6 no 7 Article IDe22751 2011

[22] C U Koser M T Holden M J Ellington et al ldquoRapid whole-genome sequencing for investigation of a neonatal MRSAoutbreakrdquo The New England Journal of Medicine vol 366 no24 pp 2267ndash2275 2012

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Table 1 Glossary of genomics and bioinformatics terms

Term Definition

Read Short fragment of DNA sequence output by genome sequencer Commonly 50ndash250 bp in lengthRaw read refers to reads taken directly from the genome sequencer and in no way filtered

Paired-end Pairs of reads that are two ends of the same region of DNA a standard distance apartDepth Number of reads mapped to a given position in the referenceReference based assembly Construction of reads by mappingaligning to a known reference sequenceDe novo assembly Assembly of reads without a referenceAdapter Short nucleotide sequences found at the end of reads which are part of the sequencing reactionInsertion Short regions of DNA present in our samples but not the reference sequenceContig Contiguous regions of DNA generated by joining together raw sequence reads

Genomic island Regions of the genome with suspected horizontal origins in that they were likely acquired fromother bacteria of the same or similar species

Sequence composition Proportion of each nucleotide base present

Codon usage bias Regions with different sequence composition or amino acid composition compared to the rest of thegenome

11989950 Length for which all the contigs of that length or longer contain at least half of the total of the lengthsof the contigs

Nonsynonymous Nucleotide substitution that alters the amino acid sequenceOpen reading frame (ORF) The part of a gene that has the potential to code for a proteinbp base pairs

with concomitant shifts in the ages of those affected andthe circulating serotypes of S pneumoniae [1] Pneumococcalserotype is determined by the polysaccharide capsule whichitself is also a virulence factor [2]

In Vancouver Canada a large cluster of IPD associatedwith S pneumoniae serotype 5 (multilocus sequence type orMLST 289) was identified in 2006 [3] following its initialemergence in Alberta in 2005 [4] This increase in IPDresulted in a large outbreak of pneumococcal disease thatspread throughout Western Canada (namely the provincesof British Columbia Alberta Saskatchewan and Manitoba)from 2005 to 2009 [3 4] In addition to being associated witha particular serotype-MLST profile the outbreak was largelyassociated with certain demographic groups affectingmostlyyoungmale adults (aged 16ndash65) ofAboriginal heritage Peopleaffected were alsomore likely to be homeless and intravenousdrug users (IDU) [3 4]

Here we provide an update on the incidence of IPDassociatedwith S pneumoniae serotype 5 since 2009 inBritishColumbia (BC) Furthermore we used a combination ofwhole-genome sequencing and PCR to genetically character-ize the outbreak strain This included the identification of agenomic island which enabled us to track the extent to whichthe outbreak strain was causing IPD in BC

2 Methods

21 Identification of S pneumoniae Isolates Associated withIPD All S pneumoniae isolates throughout Canada fittingthe definition of S pneumoniae IPD are required to bereported to regional public health authorities In BritishColumbia invasive isolates of S pneumoniae are forwardedto the BC Public Health Microbiology and Reference Lab-oratory for confirmation and initial serotyping followed by

additional serotyping at the National Centre for Streptococ-cus [4] For the purpose of this study all IPD cases from BCwith S pneumoniae recovered in culture were included

A confirmed case of IPD was defined as clinical illnesssuch as pneumonia with bacteremia bacteremia without aknown site of infection and meningitis Laboratoryconfirmation of S pneumoniae was either through culture ordetection of DNA from a normally sterile site (excluding themiddle ear) (httpwwwphac-aspcgccapublicatccdr-rmtc09vol3535s2Pneumoco-engphp) All isolates confirmed asS pneumoniae are stored at minus80∘C

22 Whole-Genome Sequencing Two isolates were selectedfor whole-genome sequencing BCSP1 and BCSP2They wereisolated in August 2006 and February 2010 respectivelyboth from patients admitted to a tertiary care hospital inVancouver These isolates were chosen since BCSP1 wasthought to be the index case inBCandBCSP2was a randomlyselected strain from the tail end of the outbreak in BC

The bacteria were cultured on sheep blood agar platesand approximately 4mm3 of colonial growth was used forDNA extraction using the Qiagen DNA Mini Kit as per themanufacturerrsquos instructions Library preparation for BCSP1was performed as described previously [5] and for BCSP2wasperformed using the Nextera kit (Illumina San Diego USA)as per the manufacturerrsquos instructions BCSP1 was sequencedon the Illumina GAIIx (50 bp paired-end reads) while BCSP2was sequenced on the Illumina MiSeq (250 bp paired-endreads) Sequence reads were deposited to the EuropeanNucleotide Archive under accession number PRJEB7803(httpwwwebiacukenadataviewPRJEB7803) A glos-sary of genomics and bioinformatics terms is listed inTable 1















0

50

100

150

200

250To

tal n

umbe

r of S

pne

umon

iae i

sola

tes

Serotype 5


2013

q42013

q32013

q22013

q12012

q42012

q32012

q22012

q12011

q42011

q32011

q22011

q12010

q42010

q3

2010

q12009

q42009

q32009

q22009

q12008

q42008

q32008

q22008

q12007

q42007

q32007

q22007

q12006

q42006

q32006

q22006

q12005

q42005

q32005

q22005

q12004

q42004

q32004

q22004

q12003

q42003

q32003

q22003

q1

lowast2010

q2

00

83

56

164

74

35

69

26

41

00

00

00

00

63

235

572

425

289

353

286

133

168

157

82

31

42

43

55

24

00

00

00

00

00

00

00

08

34

00

72

08

00

00

00

00


3 Results








Position70585

referencebase

BCSP1base


Aminoacid



No Erarr A












ERR052634

S pneumoniae 70585

ERR084189

ERR067846

ERR064008

ERR054237

BCSP2

BCSP1

ERR066355

2

2

5

3

11

A A A AA

A A A AA

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C

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CC

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G

G

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GG

GG

GG

GG

GG

GG

GG

G

G

G

G

TT

TT

TT

TT

TT

TT

T

T

T

T T T

TT

2041246

2041378

2041771

2043727

2044455

2044708

2045304

2045914

2045915

2049998

2050506

2051060

2052056

2055330



4 Discussion








Competing Interests


Acknowledgments


References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























0

50

100

150

200

250To

tal n

umbe

r of S

pne

umon

iae i

sola

tes

Serotype 5


2013

q42013

q32013

q22013

q12012

q42012

q32012

q22012

q12011

q42011

q32011

q22011

q12010

q42010

q3

2010

q12009

q42009

q32009

q22009

q12008

q42008

q32008

q22008

q12007

q42007

q32007

q22007

q12006

q42006

q32006

q22006

q12005

q42005

q32005

q22005

q12004

q42004

q32004

q22004

q12003

q42003

q32003

q22003

q1

lowast2010

q2

00

83

56

164

74

35

69

26

41

00

00

00

00

63

235

572

425

289

353

286

133

168

157

82

31

42

43

55

24

00

00

00

00

00

00

00

08

34

00

72

08

00

00

00

00


3 Results








Position70585

referencebase

BCSP1base


Aminoacid



No Erarr A












ERR052634

S pneumoniae 70585

ERR084189

ERR067846

ERR064008

ERR054237

BCSP2

BCSP1

ERR066355

2

2

5

3

11

A A A AA

A A A AA

A A A AA

A A A

A AA A

A A

A

A

A A A

A

A A

A A

A A A

A

A

A

A

A

A

C

C

C

C

C

C

C

C

C

C

CC

C CC C

CC

CC

CC

CC

CC

CC

CC GGGG

G

G

G

G

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G

G

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G

GG

GG

GG

GG

GG

GG

GG

G

G

G

G

TT

TT

TT

TT

TT

TT

T

T

T

T T T

TT

2041246

2041378

2041771

2043727

2044455

2044708

2045304

2045914

2045915

2049998

2050506

2051060

2052056

2055330



4 Discussion








Competing Interests


Acknowledgments


References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Position70585

referencebase

BCSP1base


Aminoacid



No Erarr A












ERR052634

S pneumoniae 70585

ERR084189

ERR067846

ERR064008

ERR054237

BCSP2

BCSP1

ERR066355

2

2

5

3

11

A A A AA

A A A AA

A A A AA

A A A

A AA A

A A

A

A

A A A

A

A A

A A

A A A

A

A

A

A

A

A

C

C

C

C

C

C

C

C

C

C

CC

C CC C

CC

CC

CC

CC

CC

CC

CC GGGG

G

G

G

G

GG

G

G

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G

GG

GG

GG

GG

GG

GG

GG

G

G

G

G

TT

TT

TT

TT

TT

TT

T

T

T

T T T

TT

2041246

2041378

2041771

2043727

2044455

2044708

2045304

2045914

2045915

2049998

2050506

2051060

2052056

2055330



4 Discussion








Competing Interests


Acknowledgments


References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ERR052634

S pneumoniae 70585

ERR084189

ERR067846

ERR064008

ERR054237

BCSP2

BCSP1

ERR066355

2

2

5

3

11

A A A AA

A A A AA

A A A AA

A A A

A AA A

A A

A

A

A A A

A

A A

A A

A A A

A

A

A

A

A

A

C

C

C

C

C

C

C

C

C

C

CC

C CC C

CC

CC

CC

CC

CC

CC

CC GGGG

G

G

G

G

GG

G

G

G G

G

GG

GG

GG

GG

GG

GG

GG

G

G

G

G

TT

TT

TT

TT

TT

TT

T

T

T

T T T

TT

2041246

2041378

2041771

2043727

2044455

2044708

2045304

2045914

2045915

2049998

2050506

2051060

2052056

2055330



4 Discussion








Competing Interests


Acknowledgments


References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests


Acknowledgments


References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article genomic analysis of a serotype 5 streptococcus...

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