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Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence… Adamiak, Paul; Vanderkooi, Otto G.; Kellner, James D.; Schryvers, Anthony B.; Bettinger, Julie A.; Alcantara, Joenel 2014

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METHODOLOGY ARTICLEEffectiveness of the standm,3,Streptococcus pneumoniae, Bacterial typingAdamiak et al. BMC Microbiology 2014, 14:143http://www.biomedcentral.com/1471-2180/14/143gitidis are readily able to incorporate environmentalHospital Dr. NW, Calgary, AB T2N 4 N1, CanadaFull list of author information is available at the end of the articleBackgroundAccurate, reproducible isolate characterization data helpsepidemiologists, scientists, physicians, public healthofficials, and many other professions, better monitorand manage endemic and epidemic infectious diseasetrends [1]. Historically, bacterial typing schemes havebeen based on immunological and electrophoretic ap-proaches [2]. Immunological based schemes classifystrains on the specificity of antibodies raised againstantigenic bacterial components. This approach hasbeen widely applied in the form of capsular serotyping,whereby the antigenic specificity of different intra-speciescapsule types are used to classify the bacteria [3,4].However, many globally significant bacterial pathogenssuch as Streptococcus pneumoniae and Neisseria menin-* Correspondence: jalcanta@ucalgary.ca1Department of Microbiology, Immunology and Infectious Diseases, 3330Keywords: Multi-locus sequence typing, MLST, Invasive pand Joenel Alcantara1*AbstractBackground: Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterialisolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardizedmeans of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genessuch that each unique combination of sequences defines an isolate’s sequence type. In order to reliably determinethe sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. Thisstudy assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers tocompletely sequence, in both directions, the required typing alleles.Results: The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles,the standard primers were unable to obtain the complete forward and reverse sequences. This is due to thestandard primers annealing too closely to the target regions, and current sequencing technology failing tosequence the bases that are too close to the primer. The alternative primer set described here, which includes acombination of primers proposed by the CDC and several designed as part of this study, addresses this limitationby annealing to highly conserved segments further from the target region. This primer set was subsequentlyemployed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization MonitoringProgram ACTive (IMPACT) over a period of 18 years.Conclusions: The inability of several of the standard S. pneumoniae MLST primers to fully sequence the requiredregion was consistently observed and is the result of a shift in sequencing technology occurring after the originalprimers were designed. The results presented here introduce clear documentation describing this phenomenoninto the literature, and provide additional guidance, through the introduction of a widely validated set of alternativeprimers, to research groups seeking to undertake S. pneumoniae MLST based studies.neumococcal disease, Molecular epidemiology,set of Streptococcus pneusequence typing primersPaul Adamiak1, Otto G Vanderkooi1,3,4, James D Kellner1,2© 2014 Adamiak et al.; licensee BioMed CentrCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.Open Accessard and an alternativeoniae multi locusAnthony B Schryvers1, Julie A Bettinger5al Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Adamiak et al. BMC Microbiology 2014, 14:143 Page 2 of 8http://www.biomedcentral.com/1471-2180/14/143genetic material into their genomes allowing for rapidgenetic variation and interchange of immunogenic com-ponents; including those on which serotyping is based[5]. This phenomenon has been observed recently withS. pneumoniae capsular typing following the introduc-tion of the seven-valent pneumococcal conjugate vaccine(PCV7) [6]. As a result of the component specificity ofimmunological based typing methods, it has becomewell recognized that strains possessing the same serotypeare not necessarily clonally related, nor expected topossess the same repertoire of virulence factors. Im-munogenic approaches are now used in more focusedways to explore specific factors, particularly those rele-vant to guiding vaccine evaluation and development, aswas demonstrated with a recent serotype B meningococ-cal vaccine investigation [7].Multi-locus enzyme electrophoresis (MLEE) is anothertyping method, and is based on the relative electrophor-etic mobility of a set of ubiquitously present bacterialenzymes [8]. This approach is not dependent on a singleimmunogenic component and as such is less influencedby horizontal exchange or positive selection events.However, it is complicated to perform and it is difficultto compare the resulting electrophoretic types betweendifferent groups [2]. Similar to the MLEE, pulse field gelelectrophoresis (PFGE) classifies individual strains basedon the gel electrophoretic mobility of bacterial compo-nents: in this case the relative mobility of DNA frag-ments which have been obtained through restrictionenzyme digestion [9]. PFGE has been widely used fortyping and has been considered a gold standard for someepidemiological studies, however, there have been chal-lenges in standardizing protocols between different researchgroups [10].Multi-locus sequence typing (MLST) is a classificationscheme whereby isolates are typed based on the nucleotidesequences from a set of housekeeping genes that are neces-sary for the maintenance of basic cellular functions. Thenucleotide sequences for each of these housekeeping genesare used to define a unique bacterial sequence type [1].Each gene is sequenced from individual strains and thencompared against existing sequences in a publically access-ible, globally maintained database. Those submitted se-quences matching ones already in the database are assignedthe gene type number of the sequence in the database; if anovel sequence is submitted, the curator of the database as-sesses the sequencing results and assigns an appropriategene number. While this approach does address several ofthe limitations encountered by other typing methods, thecost of sequencing can be a barrier to large scale typingprojects. Particularly, because of the potential for error insequencing reads the standard for determining a gene typerequires matching forward and reverse sequences. The S.pneumoniae typing system is based on the partial sequenceof seven genes coding for the housekeeping proteins:Shikimate dehyrogenase (aroE), glucose-6-phosphate de-hydrogenase (gdh), glucose kinase (gki), transketolase (recP),signal peptidase I (spi), xanthine phosphoribosyltransferase(xpt), and D-alanine-D-alanine ligase (ddl) [11].Some preliminary results, and information provided bythe curator of the S. pneumoniae MLST database indi-cated that several of the provided MLST sequencingprimers were unable to obtain the full sequence requiredin each direction. As a result, in cases where a novelgene type is identified based on sequences from thestandard primers (Table 1), the investigators are requiredto design new primers and re-sequence the particulargene (Cynthia Bishop, personal communication, May,2012). In these circumstances, investigators are requiredto expend additional time and resources developing newprimers, as well as purchasing additional sequencing andvalidating results. While several investigators in thefield are aware of this issue, and all sequences in theMLST database have been correctly verified throughsubsequent primer redesign and re-sequencing, thislimitation has not been specifically addressed in theliterature [12,13] (Cynthia Bishop, personal communi-cation, May 2012).The lack of description of this limitation in the litera-ture is evidenced by the prevalence of recent studiesonly referencing the original primers, and not providingany discussion pertaining to the sequencing challenge[6,14-18]. The purpose of this study is to systematicallyidentify the primers unable to obtain the correct se-quence, describe an alternative set of primers, and intro-duce documentation to the literature offering additionalguidance to groups undertaking S. pneumoniae MLSTstudies. In this investigation, the effectiveness of thestandard MLST sequencing primers, and an alternate setof primers were evaluated for their ability to completelysequence, in both directions, the appropriate typing re-gions of each gene.ResultsThis analysis consistently observed that the forward andreverse sequences obtained with the standard MLSTprimers only completely covered the typing region fortwo of the seven genes: gki and gdh. The reverse primerfor the aroE, and recP genes failed to sequence thelast 21 and 10 bases of their respective typing regions(Figure 1A, and B). The forward spi and xpt MLSTprimers do not sequence the first 6 and 17 bases of theirrespective typing regions (Figure 1C and D). In the caseof ddl, the forward primer was unable to sequence thefirst 8 bases (Figure 1E) and the reverse did not se-quence the last 26 bases (Figure 1F). These observationswere consistent across all of the different isolates, bothsequencing services, and each replicate. In each of theAdamiak et al. BMC Microbiology 2014, 14:143 Page 3 of 8http://www.biomedcentral.com/1471-2180/14/143Table 1 Standard S. pneumoniae amplification andsequencing primers proposed by Enright and Spratt [11]Typing gene Primer sequences Annealingtemperature °CaroE shikimatedehydrogenaseF: 5′-GCCTTTGAGGCGACAGC55R: 5′-TGCAGTTCA(G/A)AAACAT(A/T)TTCTAAgdh glucose-6-phosphatedehydrogenaseF: 5′-ATGGACAAACCAGC(G/A/T/C)AG(C/T)TT55R: 5′-GCTTGAGGTCCCAT(G/A)CT(G/A/T/C)CCgki glucose kinase F: 5′-GGCATTGGAATGGGATCACC60R: 5′-TCTCCCGCAGCTGACACrecP transketolase F: 5′-GCCAACTCAGGTCATCCAGG65R: 5′-TGCAACCGTAGCATTGTAACspi signal peptidase I F: 5′-TTATTCCTCCTGATT 50cases that the full sequence was not obtained, thealignment of the primers with publically available gen-omic sequences for S. pneumoniae identified that thoseprimers annealed less than 30 base pairs from the re-quired typing region (Figure 1).A partial set of modified MLST primers for S. pneu-moniae were designed and introduced by the US Centersfor Disease Control (CDC) [12]. The CDC primers foraroE, the reverse primer for recP, and the forward primerof ddl each annealed within the coding sequence for thegene possessing the typing region, and were able to com-pletely cover the required sequence. However, the CDCforward primer for recP, and both sets of spi and xptprimers annealed to regions of genomic DNA outside oftheir target gene. While these primers successfully se-quenced the correct region, the highly plastic nature ofpneumococcal genome suggests these genes may not al-ways be in the same region, and in this case primers thatbind outside of the gene may not always be specific tothe target region of the genome [19]. To address sequen-cing errors potentially resulting from this phenomenon,the recP CDC forward primer was replaced with thepillary separation is employed, the base pairs immedi-CTGTCR: 5′-GTGATTGGCCAGAAGCGGAAxpt xanthinephosphoribosyltransferaseF: 5′-TTATTAGAAGAGCGCATCCT55R: 5′-AGATCTGCCTCCTTAAATACddl D-alanine-D-alanine ligaseF: 5′-TGC(C/T)CAAGTTCCTTATGTGG65R: 5′-CACTGGGT(G/A)AAACC(A/T)GGCATately after the sequencing primer will not be clearlysequenced [20]. This is a characteristic of the size separat-ing technology used by chain termination sequencing.When the terminated segments are separated based on size,there is poor resolution between the smaller fragments atthe start of the sequence. This results in unclear and am-biguous sequencing results for approximately the first 20 –50 base pairs of the sequence.Next generation sequencing approaches such as 454,Illumina, and ABI function by determining the sequencefor overlapping segments of 35 to 200 base pairs, de-pending on the specific method, and then assemblingthese segments into the complete sequence [21]. Thesenext generation techniques have recently been applied toMLST with some success, however, the assembly processcan be hindered by highly repetitive sequence in the over-lapping sections of the sequence reads. This can poten-tially result in inaccurate assemblies within sequencetyping regions. Additionally, the infrastructure and expert-ise required to employ next generation sequencing tech-nologies still limits their accessibility to many researchgroups [21,22]. Given these limitations, and noting thestandard MLST recP forward primer, as this primerannealed within the recP gene and can correctly se-quence the typing region. Novel primers that annealedwithin the gene were also designed to replace the spiand xpt CDC primers. Lastly, the CDC reverse primerfor ddl bound only 19 base pairs away from the typingregion, and a modified primer binding 57 base pairsfrom the typing region was designed as a replacement. Ana-lysis of the alternate primer sets (Table 2) using the samefive test isolates revealed that, each primer set that was suffi-ciently down/upstream from the typing region was able tocorrectly amplify and sequence the appropriate DNAfragment (Figure 2). The effectiveness of the alternativeprimer set was subsequently validated through sequencetyping of 105 diverse isolates collected by the CanadianImmunization Monitoring Program ACTive (IMPACT)surveillance network (Additional file 1: Table S1). In allcases investigated in this study, the modified primers wereable to obtain the complete typing sequence, in both direc-tions, for the gene/primer combinations not obtained bythe standard primers.DiscussionThese results demonstrate that the current inability ofthe standard sequencing primers to effectively sequencethe S. pneumoniae MLST typing regions is a result ofhow close the primers anneal to the typing region of thegene. When sequencing by Sanger chain termination ca-number of recent studies still making unaltered referenceto the standard primers, it remains valuable for researchersFigure 1 (See legend on next page.)Adamiak et al. BMC Microbiology 2014, 14:143 Page 4 of 8http://www.biomedcentral.com/1471-2180/14/143in this field to be more aware of the limitations presentedby the standard MLST sequencing primers.ConclusionThe alternative primer set described here addressesthe limitation of the standard S. pneumoniae MLSTprimers by annealing sufficiently far from the target re-gion such that the sequence for the correct segment isconsistently obtained. Clear documentation defining(See figure on previous page.)Figure 1 S. pneumoniae MLST typing regions for each of the segmentsection of the corresponding genomic DNA. Panels (A) through (F) idencomplete typing region is not obtained. The black arrows depict the bindinDNA. The line marked boxes identify the segment that is consistently not oand top sequence identify either the 5’ or 3’ end of the typing region depeTable 2 Alternate primers used for amplifying andsequencing each of the seven genes for multi locussequencing typing S. pneumoniaeTyping gene Primer sequences Annealingtemperature °C1aroE shikimatedehydrogenaseF: 5′-TCCTATTAAGCATTCTATTTCTCCCTTC55R: 5′-ACAGGAGAGGATTGGCCATCCATGCCCACACTG2gdh glucose-6-phosphatedehydrogenaseF: 5′-ATGGACAAACCAGC(G/A/T/C)AG(C/T)TT55R: 5′-GCTTGAGGTCCCAT(G/A)CT(G/A/T/C)CC2gki glucose kinase F: 5′-GGCATTGGAATGGGATCACC60R: 5′-TCTCCCGCAGCTGACAC1,2GATACGGGTGATTGG1Adamiak et al. BMC Microbiology 2014, 14:143 Page 5 of 8http://www.biomedcentral.com/1471-2180/14/143xpt xanthinephosphoribosyltransferaseF: 5′-TTAACTTTTAGACTTTAGGAGGTCTTATG55R: 5′-CGGCTGCTTGCGAGrecP transketolase F: 5′-GCCAACTCAGGTCATCCAGG65R: 5′-TGCTGTTTCGATAGCAGCATGGATGGCTTCC3spi signal peptidase I F: 5′-GAATTCATTTAAAAATTTCTTAAAAGAGTGG50R: 5′-TTAAAATGTTCCTGTTTTTCTTGAG1,3ddl D-alanine-D-alanine ligaseF: 5′-TAAAATCACGACTAAGCGTGTTCTGG65R: 5′-CGCTCGATTAGTTTTGGGTAGCTGATCCC1The aroE primers, the recP reverse primer, the xpt primers and the ddl forwardprimer were designed by the US Centers for Disease Control [12].2The recP forward primer, and the gdh and gki primers are the originalsdeveloped by Spratt and Enright [11].3The spi primers and the ddl reverse primer were designed as part ofthis study.the limitations of the standard S. pneumoniae MLSTprimers and describing an effective set of alternativeprimers is of particular importance as automated Sangercapillary sequencing remains a highly optimized, and stillwidely employed method for S. pneumoniae MLST basedstudies.MethodsStreptococcus pneumoniae strains and genomicpreparationEvaluation of the standard and alternative MLST primerswas carried out on five randomly selected isolates fromstrains collected provided by the Canadian ImmunizationMonitoring Program ACTive (IMPACT) [23-26]. Isolateswere obtained from patients aged 0 – 16 presentingwith invasive pneumococcal infection at tertiary carecentres across Canada; diagnosis was confirmed bypositive S. pneumoniae culture from normally sterilebody fluid (blood/cerebrospinal fluid). The IMPACTsurveillance study has research ethics board approvalat each participating centre to obtain demographic,clinical and microbiologic information on all caseswithout the requirement for written informed consent.S. pneumoniae strains were verified and serotyped aspart of IMPACT’s routine surveillance protocol. Theinvestigation described here was undertaken usingIMPACT's 19A invasive strains, collected with ethicalapproval between 1991 and 2009. Strains were grownovernight at 5% CO2 on Columbia Blood Agar (pre-pared according to manufacturer’s instructions, BectonDickinson and Company, Difco™, Sparks, Maryland,USA) plates with Optochin Disk (used according tomanufacturer’s instructions, Sigma-Aldrich, Oakville,Ontario, Canada) susceptibility and the presence alphahemolysis used for species verification. Genomic DNAwas then isolated with the QIAamp DNA Mini Kit(used according to manufacturer instructions, Qiagen,Toronto, Ontario, Canada).s not fully sequenced by the standard primers aligned with atify each individual gene and direction combination, for which theg sites of the standard primers to the up or downstream genomicbtained by sequencing with the standard primers. The angle bracketnding on the specific MLST gene.Sequencing methodologyEach of the seven typing alleles was evaluated with boththe standard (Table 1) and alternative (Table 2) MLSTprimers. PCR solutions were prepared for each primerset consisting of: 11 μl sterile distilled water, 2.5 μl of10× reaction buffer (5 ml 1 M KCL, 5 ml 1 M (NH4)2SO4,5 ml 2 M Tris–HCl pH 8.8, 5 ml 200 mM MgSO4, 5 ml10% Triton X-100, water to 50 ml), 2.5 μl of 2 mM dNTPs,Adamiak et al. BMC Microbiology 2014, 14:143 Page 6 of 8http://www.biomedcentral.com/1471-2180/14/1432.5 μl of each primer at 5 μM, 1 unit pfu enzyme (ThermoScientific, Ottawa, Ontario, Canada) and 2 μl of genomicDNA template at 50 – 300 ng/μl. All PCRs were performedin a BioRad (Mississauga, Ontario, Canada) Thermocyclerwith annealing temperatures specific to each primer setFigure 2 5’ or 3’ end of the S. pneumoniae MLST typing region that isaligned with the sequencing results from both the forward and reverresults of the alternative primers in relation to their corresponding typing rthe typing region, the middle sequence is the result from sequencing withfrom sequencing with the reverse alternative primer.(Table 1 and 2). Amplification was verified by visual-izing gene products with gel electrophoresis on a 1% eth-idium bromide agarose gel with a voltage of 110 V for25 minutes. Verified PCR products were purified with theE.Z.N.A Cycle Pure Kit (used according to manufacturer’snot obtained by both the forward and reverse standard primersse alternative primers. Panels (A) through (F) depict the sequencingegion. The angle bracket and top sequence identify the 5’ or 3’ end ofthe forward alternative primer, and the bottom sequence is the resultquality with the open source program 4Peaks, and se-isolate, along with 5 known typing regions from theAdamiak et al. BMC Microbiology 2014, 14:143 Page 7 of 8http://www.biomedcentral.com/1471-2180/14/143Authors’ contributionsPA contributed to the study’s conception, conducted the experiments,drafted the manuscript, and approved the final submission. Dr. OV is theIMPACT site co-investigator in Calgary Alberta, and was involved with theconception and design of the study, as well as the acquisition of the data.He also revised and approved the submitted manuscript. Dr. JK was involvedin the conception and design of the study, and assisted in data acquisition.Dr. K also revised and approved the submitted manuscript. Dr. AS participated inthe development of the project, provided technical support, and assisted in theacquisition of data and analysis of results. He revised and approved the submittedmanuscript. Dr. JB is the IMPACT epidemiologist; she was involved in theconception and design of the study, provided the data and supervisedthe data analysis. She revised and approved the submitted manuscript.Dr. JA contributed substantially to the conception, implementation, andMLST database. The annealing site of each primer wasidentified by BLASTing the primer’s sequence againstpublically accessible S. pneumoniae genomic sequencesavailable through the National Center for BiotechnologyInformation [28,29]. These results identified where eachprimer annealed relative to the typing region, and whetherthe sequencing resulting from the primer was able to con-sistently cover the required region. This full process wasreplicated twice for each primer set and each test isolate toconfirm the reproducibility of the observations.Additional fileAdditional file 1: Table S1. S. pneumoniae strains sequence typed withalternative MLST primers.Competing interestsThe authors declare no competing financial or personal interests withrespect to the presentation of these results.quence coverage was inspected by using the Multiple Se-quence Alignment by Fast Fourier Transform (MAFFT)program, available through the European BioinformaticsServer [27]. MAFFT was used to align the forward andreverse sequence reads from each test primer set, andAssessing sequence coverageThe sequencing results were manually inspected forinstructions OMEGA Biotek, Norcross, Georgia, USA).Purified products were subsequently verified via spectro-photometry (used according to manufacturer’s instructionsNanoDrop 1000 Spectrophotometer, Thermo Scientific,Ottawa, Ontario, Canada). Purified samples with a con-centration of greater than 3 ng/μl, and 260 nm/280 nmabsorbance values between 1.0 and 2.0 were accepted tosend for sequencing. Sequencing was carried out at bothMacrogen Corporation, Rockville USA, and the Universityof Calgary, Calgary Canada, DNA Core Services facility.interpretation of the results presented in this study. Dr. JA, also revisedand approved the submitted manuscript. All authors read and approvedthe final manuscript.AcknowledgementsThe authors would like to acknowledge the Canadian ImmunizationMonitoring Program Active Investigators for collecting the S. pneumoniaeisolates that made this project possible. The Canadian ImmunizationMonitoring Program Active is a national surveillance initiative managed bythe Canadian Pediatric Society (CPS) and conducted by the IMPACTinvestigators on behalf of the Public Health Agency of Canada’s (PHAC)Centre for Immunization and Respiratory Infectious Diseases. The authorswould also like to acknowledge Cynthia Bishop for providing her guidanceduring this investigation and her permission to reference the personalcommunications between herself and the author’s research team.FundingFunding for collection of the pneumococcal isolates used in this studywas provided by an unrestricted grant to CPS from Wyeth Pharmaceuticals(1991–2005), and the PHAC (2005–2009). Funding to support the laboratoryanalysis was provided by Pfizer Canada through an investigator-initiatedresearch grant in aid to Dr. James D. Kellner.Author details1Department of Microbiology, Immunology and Infectious Diseases, 3330Hospital Dr. NW, Calgary, AB T2N 4 N1, Canada. 2Alberta Children’s HospitalResearch Institute for Child and Maternal Health, 3330 Hospital Dr. NW, T2N4N1 Calgary, AB, Canada. 3Department of Pediatrics University of Calgary,Alberta Children’s Hospital, 2888 Shaganappi Trail NW, T3B 6A8 Calgary, AB,Canada. 4Department of Pathology and Laboratory Medicine CalgaryLaboratory Services, 3535 Research Rd NW, T2L 2 K8 Calgary, AB, Canada.5Vaccine Evaluation Center BC Children’s Hospital, University of BritishColumbia, 4480 Oak St, V6H 3 V4 Vancouver, British Colombia, Canada.Received: 1 November 2013 Accepted: 14 May 2014Published: 3 June 2014References1. 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