godschalk bio med central 1625157249777404 article

8/4/2019 Godschalk Bio Med Central 1625157249777404 Article

1/37

-1-

IdentificationofDNAsequencevariationinCampylobacterjejunistrainsassociatedwiththeGuillain-Barrsyndromebyhigh-throughputAFLP

analysis

PeggyCRGodschalk1,MathijsPBergman

1,RaymondFJGorkink

2,GuusSimons

2,3,

NicolevandenBraak1,AlbertJLastovica

4,HubertPEndtz

1,HenriAVerbrugh

1,

AlexvanBelkum1

1DepartmentofMedicalMicrobiology&InfectiousDiseases,ErasmusMC-

UniversityMedicalCenterRotterdam,Dr.Molewaterplein40,3015GDRotterdam,

TheNetherlands2DepartmentofMicrobialGenomics,KeygeneN.V.,AgroBusinesspark90,6708PW

Wageningen,TheNetherlands3PathofinderB.V.,CanisiusWilhelminaHospital,WegdoorJonkerbos100,6532SZ,

Nijmegen,TheNetherlands4DepartmentofClinicalLaboratorySciences,DivisionofMicrobiology,andIIDMM,

UniversityofCapeTown,AnzioRoad,Observatory7925,CapeTown,SouthAfrica

Correspondingauthor

Emailaddresses:

PCRG:[email protected]

MPB: [email protected]

RFJG:[email protected]: [email protected]

NVDB:[email protected]

AJL: [email protected]

HPE: [email protected]

HAV: [email protected]

AVB: [email protected]


2/37

-2-

Abstract

Background

Campylobacter jejuni is the predominant cause of antecedent infection in post-

infectious neuropathies such as the Guillain-Barr (GBS) and Miller Fisher

syndromes (MFS). GBS and MFS are probably induced by molecular mimicry

between human gangliosides andbacterial lipo-oligosaccharides (LOS). This study

describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for

detection and identification of DNA polymorphism, in general, and of putative

GBS/MFS-markers,inparticular.

Results

Wecompared6differentisolatesofthegenomestrainNCTC11168obtainedfrom

different laboratories.HtAFLP analysis generated approximately3000 markers per

stain, 19 of which were polymorphic. The DNA polymorphisms could not be

confirmed by PCR RFLP analysis, suggesting a baseline level of 0.6% AFLP

artefacts. Comparison ofNCTC 11168with 4 GBS-associated strains revealed 23

potentiallyGBS-specificmarkers,17ofwhichwereidentifiedbyDNAsequencing.A

collection of27GBS/MFS-associatedand17 enteritiscontrol strainswasanalyzed

withPCRRFLPtestsbasedon11ofthesemarkers.Weidentified3markers,located

in the LOS biosynthesis genes cj1136, cj1138 and cj1139c, thatwere significantly

associatedwithGBS (P=0.024, P=0.047andP


3/37

-3-

Conclusions

ThisstudyshowsthatbacterialGBSmarkersarelimitedinnumberandlocatedinthe

LOSbiosynthesisgenes,whichcorroboratesthecurrentconsensusthatLOSmimicry

maybetheprimeetiologicdeterminantofGBS.Furthermore,ourresultsdemonstrate

thathtAFLP,withitshighreproducibilityandresolution,isaneffectivetechniquefor

thedetectionandsubsequentidentificationofputativebacterialdiseasemarkers.


4/37

-4-

Background

The Guillain-Barr syndrome (GBS) is themost frequent form of acute immune-

mediated neuropathy. The Miller Fisher syndrome (MFS) is a variant of GBS,

affecting mainly the eye muscles [1]. A respiratory or gastro-intestinal infection

precedingtheneurologicalsymptomsisreportedbynearlytwo-thirdsofallpatients

[2].Themostfrequentlyidentifiedinfectiousagentis Campylobacterjejuni,whichis

alsothepredominantcauseofbacterialdiarrhoeaworldwide[3,4].Theneuropathyis

probably induced bymolecular mimicry between gangliosides in nerve tissue and

lipo-oligosaccharides (LOS)on theCampylobacter cell surface [5]. This structural

resemblanceleadstoacross-reactiveimmuneresponsecausingneurologicaldamage.

BiochemicalandserologicalstudieshaverevealedthatmanyC.jejunistrainsexpress

ganglioside-like structures in their LOS [6]. However, not all strains expressing

gangliosidemimics induceGBS. It isestimated that only 1 inevery 1000-3000C.

jejuniinfectionsisfollowedbyGBS[7,8],whichsuggeststhatadditionalbacterial

determinantsand/orhost-relatedfactorsareimportantaswell.

Many researchershavestudiedcollectionsofGBS-associatedandcontrolenteritis-

onlystrainsinsearchofGBS-specificmicrobialfeatures.Severalreportsdescribean

overrepresentationofspecificPenner (HS)serotypesamongGBS-associatedstrains

fromcertaingeographicalareas.TheHS:19andHS:41serotypesarethepredominant

serotypes precedingGBS in Japan andSouthAfrica, respectively [9, 10].Because

HS:19 and HS:41 strains represent a clonal population [11, 12], the observed

overrepresentation of these serotypes does not imply that the determinant of the

Penner serotyping system, the capsular polysaccharide, is involved in the


5/37

-5-

pathogenesisofGBS[13].Inaddition,thisphenomenonisnotseeninotherregions,

whereGBS-associatedstrainsaregeneticallyheterogeneous[14].Variousmolecular

typingtechniqueshavebeenusedinsearchofGBS-specificfeaturesinC.jejuni,such

asflaA PCR RFLP, pulsed field gel electrophoresis (PFGE), randomly amplified

polymorphic DNA (RAPD) analysis, ribotyping, amplified fragment length

polymorphism(AFLP)analysisandmultilocussequencetyping(MLST),butnoneof

thesehaveidentifiedGBS-specificmarkers[14-17].Veryrecently,Leonardetal.also

failed todetectGBS-specific featuresby the use ofanopen reading frame(ORF)-

specific C. jejuni DNA microarray [18]. However, this array was based on the

genomesequenceofstrainNCTC11168andORFsthatarenotpresentin thisstrain

will not be detected. In addition,possibleGBS-factorsother than those relating to

presenceorabsenceofcertaingeneswillnotbedetectedusingthisapproach.Based

on the molecular mimicry hypothesis, others focused on genes involved in LOS

biosynthesis and found significant associations withGBS [19-21].However, these

associations are not absoluteand the question remainswhether otherGBS-specific

microbialfactors,eitherLOS-relatedornot,mayexist.

Comparativegenomicstechnologyfacilitatesgeneticmarkeridentificationbutnotall

methods may be equally suited for high-throughput marker searches. Molecular

typing techniques for Campylobacter strains differ in sensitivity and the overall

coverageofgenomeregionsscreened.Forthedetectionofspecificdiseasemarkersit

isdesirabletouseatechniquethatscreensdiversityintheoverallgenomewithavery

high resolution.MLST andflaA PCRRFLPanalyzerestrictedpartsof thegenome

and are not suitablefor the detection ofadditionalGBS-markers [22,23].PFGEis

basedondigestionofgenomicDNAwithararecuttingrestrictionenzymeandonly


6/37

-6-

largeinsertionsordeletionsandmutationsintherestrictionsiteswillbedetected[14].

AFLPanalysis isconsiderablymore sensitive thanPFGE. In aconventionalAFLP

analysis, theuseoftworestriction enzymesanda primerpairwith1 or2 selective

nucleotides leads to a DNA fingerprint pattern consisting of approximately 50-80

fragments per strain. This approach physically covers in the order of 600-1000

nucleotidesofthetotalgenomeforsequencepolymorphism[24].Asindicatedabove,

DNA microarrays cover the full genome but will only detect differences in the

presence of known genes. Recently, we described a new high throughput AFLP

(htAFLP) approachfor the identificationofDNApolymorphisminMycobacterium

tuberculosis [25]. This method has the capacity to detect mutations in more than

30,000 nucleotides scattered throughout the genome, depending on the number of

restrictionenzymesandprimerpairsused.ThisenhancedresolutionmakeshtAFLP

an excellent candidate technique for the detection of potential disease-associated

markers.

InthepresentstudywedevelopedhtAFLPforC.jejuni.Weanalyzedsixisolatesof

strainNCTC11168,thegenomestrain,obtainedfromdifferentlaboratories,with

theaimtodetectbase-levelpolymorphismintroducedbysub-culturingorstorage.In

search of potential GBS-specific markers, we compared the NCTC 11168 AFLP

patterns with those of four GBS-associated strains. In addition, we analyzed a

collection of highly clonal GBS-associated and control strains from South Africa.

PotentialGBS-specifichtAFLPmarkerswerefurtheridentifiedbyDNAsequencing

andPCRRFLPtestsweredeveloped.ThesePCRRFLPtestswereusedtoscreena

larger collection of GBS-associated and control strains for confirmation of the

potentialGBSmarkers.


7/37

-7-

Results

Detectionand identificationofDNA sequencepolymorphism inNCTC11168

strainsofdiverseorigin

HtAFLPanalysisofC.jejuniwasperformedwithoneenzymecombinationandthe

64 possible combinations of +1/+2 selective primer pairs, which resulted in the

generation of approximately 3000 fragments per strain. The average fragment size

was243basepairs(bp),rangingfrom46to613bp.ComparativehtAFLPanalysisof

thesixNCTC11168isolatesrevealed19polymorphicfragments.Afterexcisionfrom

the gel, thesefragmentswere amplifiedand theDNA sequenceswere determined.

BLAST analysis of the DNA sequences resulted in the identification of 13/19

polymorphisms,whichwerespreadthroughoutthegenome(Table2).Fortheother

polymorphic fragments, repeated amplification and sequencing failed to generate

DNAsequencesofsufficientqualityforBLASTanalysis.

ValidationofNCTC11168polymorphismwithaPCRRFLPapproach

ToverifywhetherthehtAFLP-polymorphicfragmentsrepresenttrueDNAsequence

polymorphism,weanalyzedthesixNCTC11168isolatesbyPCRRFLPanalysis.An

AFLPpolymorphismthatisbasedonmutationsintherestrictionsitewillresultina

polymorphicRFLPpattern,whereasinsertionsordeletionsintheAFLPfragmentwill

result in size differences between the PCR products. Based on the BLAST hit

sequences (Table 2), PCR tests for amplifying twelve marker fragments and their

flankingregionsweredeveloped. Fragments ofcorrectsizewere producedwithall

primer sets and forall isolates (resultsnot shown).Next,PCR productsof the six

NCTC11168strainsweredigestedin separate reactionswith theAFLP restriction

enzymes (results not shown). None of the digests showed polymorphic RFLP


8/37

-8-

patterns. Thus, restriction site polymorphism or insertions/deletions could not be

confirmedascauseoftheobservedAFLPpolymorphisms.Nineoutoftwelve(75%)

digests with DdeI and six of twelve (50%) digests with MboI resulted in RFLP

patterns as expected based on the NCTC 11168 DNA sequence. An AFLP

polymorphismcanalsobetheresultofamutationin thenucleotidescomplementary

totheselectiveprimernucleotides.Becauseall64possiblecombinationsofthe+1/+2

primerpairswereusedinthishtAFLP,suchamutationwouldbeexpectedtoresultin

anadditionalpolymorphism,withthesamefragmentlengthandlocalizationonthe

genome,intheAFLPpatterngeneratedwithadifferentprimerpair.Wedidnotdetect

suchcomplementarypolymorphismsintheNCTC11168comparison.Inconclusion,

thepolymorphicAFLPbandsobservedintheNCTC11168comparison,representing

approximately0.6%ofallbands,probablyrepresentthelowbackgroundnoiseof

thehtAFLPtechnique.

ComparisonofNCTC11168withGBS-associatedstrainsforthedetectionand

identificationofpotentialmarkersforGBS

ForthedetectionofputativemarkersfortheGuillain-Barrsyndrome,wecompared

theNCTC11168isolateswithstrainGB11,whichwasisolatedfromaGBSpatient.

StrainNCTC11168wasoriginallyisolatedfromapatientwithgastroenteritiswithout

neurologicalsymptoms.IthadpreviouslybeenshownthatGB11andNCTC11168

aregeneticallycloselyrelated[14,15,26].Becauseofthisrelatedness,thesestrains

are very suitable for the detection of potential GBS-specific markers. HtAFLP

analysisofNCTC11168andGB11generated 241 putativeGBSmarkers.Overall,

156 of 241 markers could be successfully identified with DNA sequencing and

BLASTanalysis.Aproportionofthemarkerfragmentsthatwereexcisedfromthegel

could not be reliably reamplified and were excluded from the analysis. Although


9/37

-9-

BLASTsearcheswereconductedagainstallDNAsequencesinthePubmeddatabase,

themostsignificanthomologyforallAFLPDNAsequenceswaswithC.jejuniDNA

sequences. To further reduce this excessive number of putativeGBS markers,we

analyzedthreeadditionalGBS-associatedstrains,notrelatedtotheNCTC11168and

GB11 strains (Cura7, Cura276 and 260.94; Table 1). This reduced the number of

successfullysequencedputativeGBSmarkersto17(Table3).Threeofthesemarkers

were located in the LOS biosynthesis gene locus. Othergenes encoded a putative

periplasmic protein and proteins involved in signal transduction, metabolism,

transport, binding, amino acid biosynthesis, fatty acid biosynthesis and DNA

replication. Three genes were of unknown function. Markers 5 and 6 displayed

distinct restriction site polymorphism concordant with the AFLP polymorphism.

These markers contained largely overlapping DNA sequences and showed a

complementarypattern ofpresence andabsence in theGBS-associated andcontrol

strains(Table3).ComparisonoftheDNAsequencesrevealedthatthesemarkerswere

basedononeDNApolymorphism:thepresenceofanadditionalrestrictionsiteinthe

GBSstrainsduetoapointmutation(Figure1).

ScreeningofalargestraincollectionforpotentialGBSmarkersbyPCRRFLP

analysis

After htAFLP analysis of a limited number of strains to identify potential GBS

markers,wedeveloped PCRRFLP tests toscreen a largecollection ofGBS/MFS-

associatedandcontrolenteritisstrainsforthepresenceofthesemarkers(forasurvey

of these strains see Table 1). The strains used in the htAFLP analysis were also

included inthePCRRFLPanalysis.OnlyoneofthesixNCTC11168 isolateswas

included. Basedon the BLAST hit sequences (Table 3), PCR tests foramplifying

11/17markerfragmentsandtheirflankingregionsweredeveloped(Table4).Because


10/37

-10-

markers5and6representedthesameDNApolymorphism,theywereincludedinone

PCRtest.Fragmentsofcorrect,expectedsizewereproducedwithallprimersets.For

5/11markersaPCRproductwasabsentinavariableproportionofstrains(Table5),

probablyduetoprimersitesequenceheterogeneity.Forexample,wehaveobserved

previouslythatgenecj1136,partoftheLOSbiosynthesisgenelocusandcontaining

marker7,showsalargedegreeofDNAsequenceheterogeneitybetweenstrains(P.

Godschalk, unpublished results). This leads to primermismatches and absence of

PCR products inaproportionofstrains. In2/10 PCR tests (markers7 and8), the

htAFLP GBS-associated strains could be distinguished from strain NCTC 11168

through the pattern of presence and absence of PCR products. PCR products for

marker7wereabsentintheGBS-associatedstrainsusedinthehtAFLPandpresentin

NCTC11168,whichseemedtobeincontrastwiththeobservationthattheoriginal

AFLP fragment of marker 7was present in theGBS strains and absent in NCTC

11168.However, this apparent inconsistency can beexplained by the fact that the

primer sequencesofthePCR testwerebasedontheNCTC11168DNAsequence.

For marker 7, a PCR product was seen significantly more frequently in control

enteritisstrains(5/27(18.5%)GBS/MFSstrainsversus9/17(52.9%)controlenteritis

strains,P =0.024).Next,we subjected the PCR products toa combined digestion

withtheAFLPrestrictionenzymes(Table5).In4/10PCRtests(markers3,5/6,9and

13),theRFLPtypeswereconcordantwiththeAFLPanalysisi.e.thehtAFLPGBS-

associated strains shared the same RFLP type whereas NCTC 11168 displayed a

differentRFLPtype. In3/10PCR tests(markers11,12and14),thehtAFLPGBS-

associatedstrainsdidnothaveidenticalRFLPtypes(andformarker14therewasno

PCRproductintwoGBSstrains),buttheseRFLPtypeswerealsodifferentfromthat

ofNCTC11168.ThisisnotnecessarilyincontrastwiththehtAFLPresults,because


11/37

-11-

different RFLP types among the htAFLP GBS-associated strains may due to

heterogeneityintheflankingregionsoftheAFLPfragment.Formarker2,theRFLP

typesofthehtAFLPstrainswerenotconcordantwiththehtAFLPpolymorphism:the

NCTC11168andGB11RFLPtypeswerethesame.RFLPtypes3and4ofmarker9,

locatedingene cj1139c,were only detected inGBS/MFS-associated strains (RFLP

type3or4presentin15/27(55.6%)GBS/MFS-associatedstrainsversus0/17(0%)

enteritisstrains,P


12/37

-12-

DiscussionThis study describes a high-throughput AFLP approach for the detection and

identification of DNA polymorphism and putative GBS-markers in C. jejuni.

Previously,weshowedthathtAFLPisanexcellenttoolforassessingthepopulation

structureandtheexpansion ofpathogenic clones inStaphylococcus aureusandfor

identificationofgeneticpolymorphismintheclonalmicroorganismMycobacterium

tuberculosis[25,27].Theoptimalenzymeandselectiveprimerpaircombinationsare

determined by insilico calculationsusingthewholegenomeDNAsequenceofthe

target microorganism. The optimal number of AFLP fragments to be generated

depends on the aim of the study. For the detection and identification of potential

diseasemarkers,suchasGBS-specificmarkersinC.jejuniinthecurrentstudy,itis

desirabletoscreenthegenomewithhighresolution.Forthis,thegenerationofalarge

numberofAFLPfragmentsperstrainisneeded.ThishighresolutionAFLPapproach

limitsthenumberofstrainsthatcanbeanalyzed,butthiscanbeovercomebythe

subsequentanalysisofa largenumberofstrainsbyPCRRFLPtests,translatedfrom

thepotentialmarkersasdetectedbytheprecedinghtAFLPanalysis.Itis,ofcourse,

possible that a disease-specific marker is not detected by htAFLP because this

approachdoesnotresultin100%genomecoverage,whichcanonlybereachedwith

wholegenomesequencing.For C.jejuni,oneenzymecombination(MboIandDdeI)

and 64 different +1/+2 selective primer pair combinations physically covered

approximately30.000nucleotidesperstrain,whichapproximately2%ofthegenome.

TofindoutwhethersubculturingorstorageofC.jejunistrainsleadstotheemergence

ofDNApolymorphism,wecompared6isolatesofthegenomestrainNCTC11168

obtained from different laboratories worldwide by htAFLP analysis. The observed


13/37

-13-

polymorphisms, approximately 0.6% of the fragments per strain, could not be

confirmed by PCR RFLP analysis, which indicates that these polymorphisms

probablyrepresentthelowbackgroundnoiseofthehtAFLPtechnique.Thisequalsa

reproducibility of approximately 99.6%, still very high when compared to other

genotypingtechniques[28].

Our findings do not completely exclude the existence of DNA polymorphism in

differentNCTC11168isolates.Onlyarandomproportionofthegenomeisscreened

by htAFLP. Recently, two groups described differences in virulence properties

betweendifferentNCTC11168isolatesthatwerenotincludedinthecurrentstudy.

Theseobservations indicate thatselectiveDNAsequence polymorphism leading to

functional changes has occurred in some isolates [29, 30].Whereasmultiple high-

resolution genotyping methods had failed to detect any differences, transcriptional

analyses revealed expression differences for several gene families. Gaynor et al.

demonstrated sequence variation in the threemajor regulatory molecules for gene

expression in C. jejuni, the sigma factors. Whether these single-nucleotide

polymorphisms(SNPs)werealsoresponsiblefortheobservedfunctionaldifferences

betweenthetwoNCTC11168isolateswasnotdetermined.HtAFLPanalysisofthe

two NCTC 11168 isolates that were studied by Carrillo et al. failed to identify

polymorphismsresponsible for thedifference invirulence properties (P.Godschalk

andC.Szymanski,unpublisheddata).

InsearchofGBS-specificmarkers,wefirstcomparedtheNCTC11168patternswith

theAFLPpatternsofthegeneticallyrelatedGBS-associatedstrainGB11.However,

although NCTC 11168 was originally isolated from the faeces of a patient with


14/37

-14-

gastroenteritis,wecannotexcludethatNCTC11168caninduceGBSifapatientwith

the right host susceptibility factors becomes infected with this strain. The only

substantialbutprobablyveryimportantdifferencebetweenNCTC11168andGB11

thathasbeenfoundsofar,isthattheLOSbiosynthesisgenelocusstronglydiverges

between these strains, probably as result of a horizontal exchange event [31].

Comparison of NCTC 11168 with GB11 led to the detection of more than two

hundred possibleGBS markers, whichwas substantially higher than the expected

background noise of 0.6%, underscoring the phylogenetic relevance of the

polymorphisms.ThenumberofpossibleGBSmarkerswasreducedto23afteradding

threeadditionalGBS-associatedstrains.For17markers,thelocationonthegenome

could be identified after DNA sequence analysis. A relatively large proportion of

potentialGBSmarkers(3/17;18%)waslocatedintheLOSbiosynthesisgenelocus,

whereasthislocusonlycomprises1%ofthe C.jejunigenome(1.64Mbp).Although

this may represent a true pathogenic associationwith GBS, it is alsopossible that

htAFLPpreferentiallypickeduptheLOS locusbecause it isahighly polymorphic

region. However, analysis of a larger C. jejuni strain collection by PCR RFLP

analysis showed that the three LOS-specific markers were indeed associated with

GBS (marker 7, P = 0.024; marker 8,P = 0.047; marker 9, P


15/37

-15-

in the severity and spectrumof clinical symptoms inGBS patients [32].Different

ganglioside mimicking structures and anti-ganglioside antibody specificities have

been associatedwith certain clinical presentations [33-35], and therefore,C. jejuni

markersmaybeassociatedwithasubsetofvariousdiseaseentities.Becauseofthis

heterogeneityandthepresumedimportanceofhost-related factors, theexistenceof

featuresinC.jejunithatarespecificforGBSmaybequestionable.Second,acertain

combinationofmultipleC.jejunigenesmayberequiredfortheinductionofGBS.

Detection of such combinations of markers (polygenic markers) is extremely

labour-intensive and cannot be achieved with the current approach. Finally, it is

possiblethathtAFLPfailedtodetectGBS-specificmarkersbecausehtAFLPdoesnot

accomplish100%genomecoverage.

One of the three additional GBS-associated strains mentioned above was from a

collection of South-African HS:41 strains. In SouthAfrica, the HS:41 serotype is

over-representedamongGBS-associatedstrains[10].Acertainfeatureofthesestrains

mayberesponsiblefortheircapacitytotriggerGBS.HS:41strainswerefoundtobe

indistinguishablebypreviousgenotypingstudies,indicatingthatHS:41strainsforma

genetically stable clone [12]. It is important to note that the enteritis-only HS:41

strains may have the same GBS-inducing capacity as the GBS-associated strains,

becausehost-relatedfactorsarealsocrucialfordevelopingGBS.Weanalyzedboth

GBS-associated and control enteritis-only HS:41 strains, as well as an MFS-

associated isolatebyhtAFLP.Althoughwedidnot detectGBS-specificbands,we

foundthattheMFS-associatedisolateandhalfof theenteritis-onlystrainscontained

several additional fragments that appeared to be linked. DNA sequences of these

fragments showed homologies with plasmidal DNA sequences, indicating that a


16/37

-16-

subsetoftheHS:41strainscontainedaplasmid.Toourknowledge,theSouthAfrican

HS:41strainswe used in this study haveneverbeenanalyzedfor the presenceof

plasmids.Whether the presence ofa plasmid isof importance for the virulenceor

neuropathogenic potential ofHS:41 strains currently remains unknown, but seems

unlikelybasedonthedistributionofplasmidalDNAinthetestedstrains.

Conclusions

PrevioussearchesforC.jejunimarkersforGBS-invokingpotentialwereunsuccessful

whenperformedwithgeneralgenotyping techniques.Somestudiesthatfocussed at

specificlociorsometimesevenspecificgenesfoundpotential,thoughnotabsolute,

GBSmarkerswithin the LOSbiosynthesisgenes [19-21].Wehave usedamethod,

htAFLP,thatdetectssequencepolymorphismsinawide,non-genedependentscale.

Theoretically approximately 2% of the total genome is covered by this approach.

However,westillconclude thatbacterialGBSmarkersare not absolute, limited in

numberandlocatedintheLOSbiosynthesisgenelocus.Thiscorroboratesthecurrent

consensus that LOSmimicry with humangangliosidesmay be the primeetiologic

determinantofGBS.Inadditiontobacterialfactors,host-relatedfactorsprobablyplay

animportantroleinthepathogenesisofGBSaswell.

Furthermore,ourresultsdemonstratethathtAFLP,withitshighreproducibilityand

resolution,isanadequatetechniqueforthedetectionandsubsequentidentificationof

putativediseaseandepidemiologicalmarkers.Analysisofalimitednumberofstrains

ingreat detail byhtAFLP and subsequent screeningofa large collectionofstrains

withsimplePCRRFLPtestscombineshighsensitivitywiththepossibilitytoscreen

large groups of strains. This allows for the identification of regions of genomic


17/37

-17-

instability or variability. Finally, htAFLP does not require complete genome

sequences and it isnot influencedby the presence ofsequences not present in the

genome strain(s). As such, htAFLP is the second best option, after complete

sequencingofthegenomeofmultiplestrains,fortheunbiaseddetectionofgenome

polymorphismsassociatedwithpathogenicity or other featuresof bacterial isolates

fromdiverseclinicalandenvironmentalorigin.


18/37

-18-

Methods

Bacterialstrains,cultureconditionsandDNAisolation

TheC.jejunistrainsusedforhtAFLPanalysisaredescribedinTable1.Wecollected

6isolatesofthegenomestrainNCTC11168strainsfromdifferentlabsworldwide

[36].ForthedetectionofpotentialGBSmarkers,weincludedfour C.jejunistrains

isolatedfromthediarrhoealstoolsofGBSpatientsfromdifferentgeographicalareas

(TheNetherlands,Curaao,SouthAfrica).Inaddition,weanalyzedacollectionof6

GBS-associated, 1MFS-associated and6 enteritis-onlyHS:41 strains isolated from

SouthAfricanpatients[12].AfteridentificationofpotentialGBSmarkersbyhtAFLP

analysisofthesestrains,wescreenedalargercollectionof27GBS/MFS-associated

and 17control strains isolated from enteritis patientswithPCRRFLP tests for the

presenceofthesemarkers(Table1).C.jejunistrainswereculturedfor24-48hourson

bloodagarplatesinamicro-aerobicatmosphereat37oC.DNAwasisolatedusingthe

WizardGenomicDNAPurificationKit(Promega,Madison,WI).

High-throughputAFLP

AFLPanalysiswasperformedessentiallyasdescribedbyVosetal.[37].Theoptimal

enzymeandprimercombinationsforC.jejuniweredeterminedusingthepredictive

software package REcomb [38]. Digestion with MboI and DdeI (Boehringer-

Mannheim,Mannheim,Germany)wascombinedwiththeligationofaspecificlinker

oligonucleotide pair (MboI: 5-CTCGTAGACTGCGTACC-3 and 5-

GATCGGTACGCAGTCTAC-3; DdeI: 5-GACGATGAGTCCTGAG-3 and 5-

TNACTCAGGACTCAT-3). Subsequently, a non-selective pre-amplification was

performed using theMboI primer (5-GTAGACTGCGTACCGATC-3) and DdeI

primer (5-GACGATGAGTCCTGAGTNAG-3'). The selective amplifications were


19/37

-19-

performedusingdifferentlinker-specificprimercombinations.The33P-labeledMboI

primer was extended with a single nucleotide (+1),whereas theDdeI primerwas

equipped with a 3 terminal dinucleotide (+2). These nucleotides probe sequence

variation beyond that present in the restrictionsite itself.All64 possible extension

combinationswereused.Amplifiedmaterialwasanalyzedon50x30cmslabgelsand

theamplimerswerevisualizedusingphosphor-imaging.Post-AFLP,gelswerefixed,

driedandstoredatambienttemperature.

Markerselectionandidentification

MarkerbandswerescoredusingtheautomatedinterpretationsoftwarepackageAFLP

QuantarPro (Keygene N.V., Wageningen, The Netherlands), resulting in a binary

table scoring marker fragment absence (0) or presence (1). Polymorphic marker

fragmentswerevalidatedbyvisualinspectionoftheautoradiographs.Bandsdiffering

in signal intensitywere not considered to bepolymorphic.A potentialmarker for

GBSwasdefinedasanAFLPpolymorphismthatdiscernstheGBS-associatedstrains

fromtheNCTC11168isolates.PotentialGBSmarkerfragmentscaneitherbepresent

orabsentinGBS-associatedstrainsascomparedtoNCTC11168.

Relevantfragmentswereexcisedfromthegelsandre-amplifiedusingtheirmatching

AFLP consensus primer set without restriction site-specific +1 and +2 extension

sequencesattached.The amplimerswere subjected toDNAsequencingusinga96-

well capillary sequencing machine (MegaBACE; Amersham). For fragment

identification,theDNAsequencesweresubjectedtoBLASTnandBLASTxsearches

through the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/). BLAST results

enable genomic localization and gene annotation for the polymorphic marker

fragments.


20/37

-20-

DevelopmentofPCRRFLPtests

PCRRFLP testswere developed to confirm polymorphism in the differentNCTC

11168 isolates and to screen a collection of C. jejuni GBS/MFS-associated and

controlstrains.PCRRFLPtestscouldonlybedevelopedforthemarkersofwhichthe

localizationontheC.jejunigenomewasidentified.Forwardandreverseprimerswere

designed (Primer Designer 4, Sci Ed Software, North Carolina) and synthesized,

locatedapproximately50-200bpupstreamordownstreamofthehomologousregion,

respectively (Table 3). This resulted in the amplification of not only the AFLP

fragment itself, but also of their flanking sequences.Next, the PCR productswere

subjectedtoseparateorcombineddigestionswiththerestrictionenzymesusedforthe

AFLP (MboIandDdeI).The digestswereanalyzedon2% agarosegels.ThePCR

RFLPanalysiswillrevealwhetherornottheAFLPvariabilitywasduetovariationin

therestrictionsites(differentRFLPpatterns)ortoinsertionsordeletionswithinthe

AFLPfragment(sizedifferencesinPCRproducts).AFLPvariationduetodifferences

intheselectiveextensionnucleotidesoftheAFLPprimerswillnotbedetectedusing

thisapproach.


21/37

-21-

Listofabbreviations

AFLPamplifiedfragmentlengthpolymorphism

GBSGuillain-Barrsyndrome

htAFLPhigh-throughputAFLP

LOSlipo-oligosaccharide

MFSMillerFishersyndrome

ORFopenreadingframe

PFGEpulsedfieldgelelectrophoresis

Authors'contributions

PCRGanalyzedandinterpretedthedataanddraftedthemanuscript.MPBdesigned

andcarriedoutthePCRRFLPmarkeranalysis.RFJGperformedthehtAFLPanalysis

andDNAsequencing.GSparticipatedinthedesignofthestudyandhtAFLPsetup.

NVDB collected the strains, performed DNA extractions and participated in the

marker identification. AJL provided the South African C. jejuni strains and

participatedinthedesignofthestudy.HPEparticipatedinthedesignofthestudyand

writing of the manuscript. HAV participated in the design of the study. AVB

conceivedandcoordinatedthestudyandparticipatedinwritingofthemanuscript.All

authorscriticallyreadthemanuscriptandapprovedthefinalversion.


22/37

-22-

Acknowledgements

Theresearchdescribedinthiscommunicationhasbeensupportedbygrantsprovided

bytheDutchMinistryofEconomicAffairs(BTS00145),theHumanFrontierScience

Program(RPG38/2003)toAVBandHPEandbyTheNetherlandsOrganizationfor

ScientificResearch(920-03-225)toP.C.R.G.A.J.L.isindebtedtotheSouthAfrican

MedicalResearch Council and theUniversity ofCapeTown for financial support.

AFLPisaregisteredtrademarkofKeygeneNVandtheAFLPtechnologyiscovered

by patents (US006045994A, EP0534858B1) and patent applications owned by

KeygeneNV.

References

)LVKHU0$QXQXVXDOYDULDQWRIDFXWHLGLRSDWKLFSRO\QHXULWLVV\QGURPH

RIRSKWKDOPRSOHJLDDWD[LDDQGDUHIOH[LD1(QJO-0HG


23/37

-23-

3UHQGHUJDVW000RUDQ$3/LSRSRO\VDFFKDULGHVLQWKHGHYHORSPHQW

RIWKH*XLOODLQ%DUUV\QGURPHDQG0LOOHU)LVKHUV\QGURPHIRUPVRI

DFXWHLQIODPPDWRU\SHULSKHUDOQHXURSDWKLHV-(QGRWR[LQ5HV

0F&DUWK\1*LHVHFNH-,QFLGHQFHRI*XLOODLQ%DUUV\QGURPH

IROORZLQJLQIHFWLRQZLWK&DPS\OREDFWHUMHMXQL$P-(SLGHPLRO

0LVKX$OORV%$VVRFLDWLRQEHWZHHQ&DPS\OREDFWHULQIHFWLRQDQG

*XLOODLQ%DUUV\QGURPH-,QIHFW'LV6

.XURNL66DLGD71XNLQD0+DUXWD7


24/37

-24-

SRO\VDFFKDULGHWKDWDFFRXQWVIRU3HQQHUVHURW\SHVSHFLILFLW\0RO

0LFURELRO

(QGW]+3$QJ&:YDQGHQ%UDDN1'XLP%5LJWHU$3ULFH/-

:RRGZDUG'/5RGJHUV)*-RKQVRQ:0:DJHQDDU-$HWDO

0ROHFXODUFKDUDFWHUL]DWLRQRI&DPS\OREDFWHUMHMXQLIURPSDWLHQWVZLWK

*XLOODLQ%DUUDQG0LOOHU)LVKHUV\QGURPHV-&OLQ0LFURELRO

'LQJOH.(YDQGHQ%UDDN1&ROOHV)03ULFH/-:RRGZDUG'/

5RGJHUV)*(QGW]+3YDQ%HONXP$0DLGHQ0&-6HTXHQFHW\SLQJ

FRQILUPVWKDW&DPS\OREDFWHUMHMXQLVWUDLQVDVVRFLDWHGZLWK*XLOODLQ

%DUUDQG0LOOHU)LVKHUV\QGURPHVDUHRIGLYHUVHJHQHWLFOLQHDJH

VHURW\SHDQGIODJHOODW\SH-&OLQ0LFURELRO

(QJEHUJ-1DFKDPNLQ,)XVVLQJ90F.KDQQ*0*ULIILQ-:3LIIDUHWWL

-&1LHOVHQ(0*HUQHU6PLGW3$EVHQFHRIFORQDOLW\RI

&DPS\OREDFWHUMHMXQLLQVHURW\SHVRWKHUWKDQ+6DVVRFLDWHGZLWK

*XLOODLQ%DUUV\QGURPHDQGJDVWURHQWHULWLV-,QIHFW'LV

)XMLPRWR6$OORV%00LVDZD13DWWRQ&0%ODVHU0-5HVWULFWLRQ

IUDJPHQWOHQJWKSRO\PRUSKLVPDQDO\VLVDQGUDQGRPDPSOLILHG

SRO\PRUSKLF'1$DQDO\VLVRI&DPS\OREDFWHUMHMXQLVWUDLQVLVRODWHG

IURPSDWLHQWVZLWK*XLOODLQ%DUUV\QGURPH-,QIHFW'LV

/HRQDUG((,,7RPSNLQV/6)DONRZ61DFKDPNLQ,&RPSDULVRQRI

&DPS\OREDFWHUMHMXQLLVRODWHVLPSOLFDWHGLQ*XLOODLQ%DUUV\QGURPH

DQGVWUDLQVWKDWFDXVHHQWHULWLVE\D'1$PLFURDUUD\,QIHFW,PPXQ

YDQ%HONXP$YDQGHQ%UDDN1*RGVFKDON3$QJ:-DFREV%

*LOEHUW0:DNDUFKXN:9HUEUXJK+(QGW]+$&DPS\OREDFWHUMHMXQL


25/37

-25-

JHQHDVVRFLDWHGZLWKLPPXQHPHGLDWHGQHXURSDWK\1DW0HG

1DFKDPNLQ,/LX-/L08QJ+0RUDQ$33UHQGHUJDVW006KHLNK

.&DPS\OREDFWHUMHMXQLIURPSDWLHQWVZLWK*XLOODLQ%DUUV\QGURPH

SUHIHUHQWLDOO\H[SUHVVHVD*',OLNHHSLWRSH,QIHFW,PPXQ

*RGVFKDON3&5+HLNHPD$3*LOEHUW0.RPDJDPLQH7$QJ&:

*OHUXP-%URFKX'/L-


26/37

-26-

'XLP%$QJ&:9DQ%HONXP$5LJWHU$9DQ/HHXZHQ1:(QGW]

+3:DJHQDDU-$$PSOLILHGIUDJPHQWOHQJWKSRO\PRUSKLVPDQDO\VLVRI

&DPS\OREDFWHUMHMXQLVWUDLQVLVRODWHGIURPFKLFNHQVDQGIURPSDWLHQWV

ZLWKJDVWURHQWHULWLVRU*XLOODLQ%DUURU0LOOHU)LVKHUV\QGURPH$SSO(QYLURQ0LFURELRO

0HOOHV'&*RUNLQN5)-%RHOHQV+$06QLMGHUV693HHWHUV-.

0RRUKRXVH0-YDQGHU6SHN3-YDQ/HHXZHQ:%6LPRQV*

9HUEUXJK+$YDQ%HONXP$1DWXUDOSRSXODWLRQG\QDPLFVDQG

H[SDQVLRQRISDWKRJHQLFFORQHVRI6WDSK\ORFRFFXVDXUHXV-&OLQ

,QYHVW

3DWWRQ&0:DFKVPXWK,.(YLQV*0.LHKOEDXFK-$3OLND\WLV%'

7URXS17RPSNLQV//LRU+(YDOXDWLRQRIPHWKRGVWRGLVWLQJXLVK

HSLGHPLFDVVRFLDWHG&DPS\OREDFWHUVWUDLQV-&OLQ0LFURELRO

&DUULOOR&'7DERDGD(1DVK-+(/DQWKLHU3.HOO\-/DX3&

9HUKXOS50\N\WF]XN26\-)LQGOD\:$HWDO*HQRPHZLGH

H[SUHVVLRQDQDO\VHVRI&DPS\OREDFWHUMHMXQL1&7&UHYHDOV

FRRUGLQDWHUHJXODWLRQRIPRWLOLW\DQGYLUXOHQFHE\IOK$-%LRO&KHP

*D\QRU(&&DZWKUDZ60DQQLQJ*0DF.LFKDQ-.)DONRZ61HZHOO

'*7KHJHQRPHVHTXHQFHGYDULDQWRI&DPS\OREDFWHUMHMXQL1&7&

DQGWKHRULJLQDOFORQDOFOLQLFDOLVRODWHGLIIHUPDUNHGO\LQ

FRORQL]DWLRQJHQHH[SUHVVLRQDQGYLUXOHQFHDVVRFLDWHGSKHQRW\SHV-

%DFWHULRO

*LOEHUW0*RGVFKDON3&5.DUZDVNL0)$QJ&:YDQ%HONXP$/L

-:DNDUFKXN::(QGW]+3(YLGHQFHIRUDFTXLVLWLRQRIWKH

OLSRROLJRVDFFKDULGHELRV\QWKHVLVORFXVLQ&DPS\OREDFWHUMHMXQL*%D

VWUDLQLVRODWHGIURPDSDWLHQWZLWK*XLOODLQ%DUUV\QGURPHE\

KRUL]RQWDOH[FKDQJH,QIHFW,PPXQ


27/37

-27-

YDQGHU0HFK)*$9LVVHU/+-DFREV%&(QGW]+30HXOVWHH-

9DQ'RRUQ3$*XLOODLQ%DUUV\QGURPHPXOWLIDFWRULDOPHFKDQLVPV

YHUVXVGHILQHGVXEJURXSV-,QIHFW'LV6

&KLED$.XVXQRNL62EDWD+0DFKLQDPL5.DQD]DZD,6HUXPDQWL

*4E,J*DQWLERG\LVDVVRFLDWHGZLWKRSKWKDOPRSOHJLDLQ0LOOHU)LVKHU

V\QGURPHDQG*XLOODLQ%DUUV\QGURPHFOLQLFDODQG

LPPXQRKLVWRFKHPLFDOVWXGLHV1HXURORJ\

-DFREV%&9DQ'RRUQ3$6FKPLW]3,07LR*LOOHQ$3+HUEULQN3

9LVVHU/++RRLMNDDV+9DQGHU0HFK)*$&DPS\OREDFWHUMHMXQL

LQIHFWLRQVDQGDQWL*0DQWLERGLHVLQ*XLOODLQ%DUUV\QGURPH$QQ

1HXURO

$QJ&:/DPDQ-':LOOLVRQ+-:DJQHU(5(QGW]+3GH.OHUN0$

7LR*LOOHQ$3YDQGHQ%UDDN1-DFREV%&YDQ'RRUQ3$6WUXFWXUH

RI&DPS\OREDFWHUMHMXQLOLSRSRO\VDFFKDULGHVGHWHUPLQHV

DQWLJDQJOLRVLGHVSHFLILFLW\DQGFOLQLFDOIHDWXUHVRI*XLOODLQ%DUUDQG

0LOOHU)LVKHUSDWLHQWV,QIHFW,PPXQ

3DUNKLOO-:UHQ%:0XQJDOO..HWOH\-0&KXUFKHU&%DVKDP'

&KLOOLQJZRUWK7'DYLHV50)HOWZHOO7+ROUR\G6HWDO7KHJHQRPH

VHTXHQFHRIWKHIRRGERUQHSDWKRJHQ&DPS\OREDFWHUMHMXQLUHYHDOV

K\SHUYDULDEOHVHTXHQFHV1DWXUH

9RV3+RJHUV5%OHHNHU05HLMDQV0YDQGH/HH7+RUQHV0

)ULMWHUV$3RW-3HOHPDQ-.XLSHU0HWDO$)/3DQHZWHFKQLTXHIRU'1$ILQJHUSULQWLQJ1XFOHLF$FLGV5HV

5HLMDQV0/DVFDULV5*URHQHJHU$2:LWWHQEHUJ$:HVVHOLQN(YDQ

2HYHUHQ-:LW(G%RRUVPD$9RHWGLMN%YDQGHU6SHN+

4XDQWLWDWLYHFRPSDULVRQRIF'1$$)/3PLFURDUUD\VDQGJHQHFKLS

H[SUHVVLRQGDWDLQ6DFFKDURP\FHVFHUHYLVLDH*HQRPLFV


28/37

-28-

Figurelegends

Figure1-ThebasisoftheAFLPpolymorphisminthecomplementarymarkers5and6

Marker5representsafragmentwithalengthof198bpthatwaspresentintheht

AFLPGBSstrains,whereasmarker6,afragmentof253bp,wasonlypresentinthe

NCTC11168isolates.DNAsequenceanalysisoftheGB11andNCTC11168AFLP

fragmentsandsubsequentBLASTsearchesshowedthatbothmarkerswerelocatedin

genecj0615,encodingapossibleperiplasmicprotein.Furthermore,DNAsequence

analysisrevealedthatapointmutationintheGBSstrainshadresultedinanadditional

MboIrestrictionsite,resultingtheamplificationofashorterAFLPfragmentinthe

GBSstrains.BecausetheselectivenucleotideflankingtheMboIrestrictionsitewas

identicalfortheGB11andNCTC11168fragment,theGBSfragmentswere

amplifiedwiththesameprimerpairastheNCTC11168fragments.DNAsequences

ofmarkers5(cj0615_GB11_AFLP)and6(cj0615_NCTC11168_AFLP)aregiven,

aswellastheNCTC11168genomesequenceofthesamearea(cj0615_genomeseq).

TheAFLPrestrictionsitesareindicatedwithboxes,theselectivenucleotidesare

underscoredandthepointmutationisindicatedinbold.

Figure2-DetailofthehtAFLPpatternoftheHS:41strains

AfragmentofthebandingpatternsoftheSouthAfricanHS:41strainsisshown.Lane

1-6GBS-associatedstrains,lane7MFS-associatedstrain,lane8-13enteritisstrains.

PolymorphicbandsrepresentingplasmidalDNAsequencesareindicatedwitharrows.


29/37

-29-

Tables

Table1-C.jejunistrainsusedinthisstudy

-----------------------------------------------------------------------------------------------------------------------------------------------------------------strain associateddisease origin usedfor-----------------------------------------------------------------------------------------------------------------------------------------------------------------

NCTC11168-1 enteritis ID-DLO,Lelystad,TheNetherlands htAFLP,PCRRFLP(obtainedfromNCTC,UK)

NCTC11168-2 enteritis ID-DLO,Lelystad,TheNetherlands htAFLP,PCRRFLP(obtainedfromM.B.Skirrow,PHLS,UK)

NCTC11168-3 enteritis NRC,Ottawa,Canada htAFLP,PCRRFLPNCTC11168-4 enteritis UMCUtrecht,TheNetherlands htAFLP,PCRRFLPNCTC11168-5 enteritis LMGculturecollection,Gent,Belgium htAFLP,PCRRFLPNCTC11168-6 enteritis CCUGculturecollection,Gteborg,Sweden htAFLP,PCRRFLPGB1 GBS TheNetherlands PCRRFLPGB2 GBS TheNetherlands PCRRFLPGB3 GBS TheNetherlands PCRRFLPGB4 GBS TheNetherlands PCRRFLPGB5 GBS TheNetherlands PCRRFLPMF6 MFS TheNetherlands PCRRFLPMF7 MFS TheNetherlands PCRRFLP

MF8 MFS TheNetherlands PCRRFLPGB11 GBS TheNetherlands htAFLP,PCRRFLPGB13 GBS TheNetherlands PCRRFLPGB14 GBS TheNetherlands PCRRFLPGB15 GBS TheNetherlands PCRRFLPGB16 GBS Belgium PCRRFLP


30/37

-30-

GB17 GBS TheNetherlands PCRRFLPGB18 GBS TheNetherlands PCRRFLPGB19 GBS TheNetherlands PCRRFLPMF20 MFS TheNetherlands PCRRFLPGB21 GBS TheNetherlands PCRRFLPGB23 GBS TheNetherlands PCRRFLPGB24 GBS TheNetherlands PCRRFLPGB25 GBS TheNetherlands PCRRFLP

GB26 GBS TheNetherlands PCRRFLPGB27 GBS TheNetherlands PCRRFLPGB29 GBS TheNetherlands PCRRFLPGB30 GBS TheNetherlands PCRRFLPE97-0737 enteritis TheNetherlands PCRRFLPE97-0747 enteritis TheNetherlands PCRRFLPE97-0796 enteritis TheNetherlands PCRRFLPE97-0873 enteritis TheNetherlands PCRRFLPE97-0903 enteritis TheNetherlands PCRRFLPE97-0921 enteritis TheNetherlands PCRRFLPE97-0974 enteritis TheNetherlands PCRRFLPE97-0980 enteritis TheNetherlands PCRRFLPE97-0998 enteritis TheNetherlands PCRRFLPE97-1013 enteritis TheNetherlands PCRRFLPE98-623 enteritis TheNetherlands PCRRFLP

E98-624 enteritis TheNetherlands PCRRFLPE98-682 enteritis TheNetherlands PCRRFLPE98-706 enteritis TheNetherlands PCRRFLPE98-1033 enteritis TheNetherlands PCRRFLPE98-1087 enteritis TheNetherlands PCRRFLPcura7 GBS Curaao,NetherlandsAntilles htAFLP,PCRRFLPcura69 GBS Bonaire,NetherlandsAntilles PCRRFLP


31/37

-31-

cura276 GBS Curaao,NetherlandsAntilles htAFLP,PCRRFLP260.94 GBS SouthAfrica(HS:41) htAFLP,PCRRFLP233.94 GBS SouthAfrica(HS:41) htAFLP233.95 GBS SouthAfrica(HS:41) htAFLP308.95 GBS SouthAfrica(HS:41) htAFLP367.95 GBS SouthAfrica(HS:41) htAFLP370.95 GBS SouthAfrica(HS:41,samepatientas367.95) htAFLP242.98 MFS SouthAfrica(HS:41) htAFLP

378.96 enteritis SouthAfrica(HS:41) htAFLP386.96 enteritis SouthAfrica(HS:41,samepatientas378.96) htAFLP199.97 enteritis SouthAfrica(HS:41) htAFLP250.97 enteritis SouthAfrica(HS:41) htAFLP242.97 enteritis SouthAfrica(HS:41) htAFLP21.97 enteritis SouthAfrica(HS:41) htAFLP----------------------------------------------------------------------------------------------------------------------------------------------------------


32/37

-32-

aTheindividualmarkershavebeengivenanumericalcode.

bThestrainsarenumberedaccordingtoTable1.Theplussesandminusesindicate

thepresenceandabsence,respectively,oftheAFLPfragments.cThefragmentlengths,basedonfragmentpositionintheAFLPgels.dThebegin

andtheendpositionsintheNCTC11168genomearegivenforallBLASThits,aswellasthecorrespondinggenesandtheirfunction.

Table2

PolymorphichtAFLPmarkersforNCTC11168strainsofdiverseorigin

markera 1 2 3 4 5 6 lengthc

begin e nd geneandfunction

1 + + + + - + 469 28753 28317 Cj0023,purB,probableadenylosuccinatelyase

2 - - + - - + 252 67665 67858 Cj0046,probabletransmembranetransportproteinpseudogene

3 - - + - - - 1 10 1 22 00 4 1 22 08 3 Cj 01 17 , pf s, p ro ba bl e5 '- me th yl th io ad en os in e\ S- ad en osy lh om oc ys te in e nu cl eo si da se

4 - - + + + + 118 153984 154075 Cj0150c,probableaminotransferase

5 - - + + + + 86 212438 212383 C j0227,argD,probableacetylornithineaminotransferase

6 + + + + + - 420 573124 572971 Cj0612c,cft, probableferrit in;Cj0613,pstS ,possibleperiplasmicphosphatebindingprotein

7 - - + + + + 4 9 5 89 04 8 5 89 39 3 C j06 29 , po ssi bl e li po pr ot ei n; Hi gh ly si mi la r to Cj1 67 8; C j1 67 8, p oss ibl e li po pr ot ei n

8 - - + + + + 382 788562 788468 C j0840c,fbp,probablefructose-1,6-bisphosphatase

9 - - + - - - 463 1047891 104 8322 Cj 1116c,ftsH,probablemembraneboundzincmetallopepti dase

10 - - - + + - 228 1227296 1227125 Cj1295,unknown

11 + - - + - - 226 1227296 1227117 Cj1295,unknown

12 + + - + + + 191 1270291 1270131 Cj1339c,flaA,flagellinA

1 3 - - + - + + 1 26 1 51 02 86 1 51 03 81 Cj 15 80 c, p ro bab le p ep ti de ABC- tr an sp ort s ys te m AT P- bi ndi ng p ro te in

NCTC11168strainsb

11168hitsequenced


33/37

-33-

aTheindividualmarkershavebeengivenanumericalcode.

bTheplussesandminusesindicate thepresenceandabsence,respectively,ofthe

AFLPfragments.

c

TheestimatedfragmentlengthsontheAFLPgelsaregiven.

d

ThebeginandtheendpositionsintheNCTC11168genomeare

givenforallBLASThits,aswellasthecorrespondinggenesandtheirfunction.Notethatmarkers5and6arelargelyoverlappingandhavea

complementarypatternofpresenceandabsence(seealsoFigure1).

Table3

PutativeGBS-markersdetectedbyhtAFLP

markera

11168 GB11 cura7 cura276 O:41 lengthc

be gi n en d ge ne a nd f un ct io n

1 - + + + + 112 324441 324363 Cj0355c,probabletwo-componentregulator

2 + - - - - 265 400499 400264 Cj0432c,murD,probableUDP-N-acetylmuramoylalanine--D-glutamateligase

3 - + + + + 115 575807 575893 Cj0615,pstA,probablephosphatetransportsystempermeaseprotein

4 - + + + + 152 576061 576182 Cj0615,pstA,probablephosphatetransportsystempermeaseprotein

5 - + + + + 198 904760 904594 Cj0967,possibleperiplasmicprotein

6 + - - - - 253 904817 904596 Cj0967,possibleperiplasmicprotein7 - + + + + 241 1070741 1070662 Cj1136,probablegalactosyltransferase

8 + - - - - 138 1073190 1073082 Cj1138,probablegalactosyltransferase

9 - + + + + 97 1074261 1074320 Cj1139c,probablegalactosyltransferase

10 + - - - - 169 1090751 1090612 Cj1157,dnaX,probableDNApolymeraseIIIsubunitgamma

11 + - - - - 89 1092245 1092185 Cj1161c,probablecation-transportingATPase

12 + - - - - 181 1236007 1236080 Cj1306c,unknown

13 + - - - - 137 1236406 1236298 Cj1306c,unknown,similartoCj1310c,Cj1305c,Cj1342c,Cj0617/0618

14 + - - - - 438 1264182 1264586 Cj1336,unknown,identicaltoCj1318(excepttheC-term)

15 + - - - - 163 1328311 1328184 Cj1394,probablefumaratelyase;Cj1393,metC',probablecystathioninebeta-lyase

16 + - - - - 203 1334951 1334779 Cj1400c,fabI,probableenoyl-[acyl-carrier-protein]reductase[NADH]

17 + - - - - 102 Cj1101,probableATP-dependentDNAhelicase

strainsb 11168hitsequenced


34/37

-34-

Table4-PrimersequencesusedinthePCRRFLPanalysisforthevalidationofpotentialGBSmarkers

--------------------------------------------------------------------------------Markernr primers

2 5-CCTGATCATCTTTCTTGGCATGG-3' 5-AAGATCTACACCCTTATCATCTCC-3'3 5-AGAAGTGTATTAACAACCTTGC-3' 5-ATCATACCGATAATCATCAAAGG-3'5,6 5-AGCCACTCAAGCAAATACTAC-3' 5-AATAAGGAGCACCATTTAAGG-3'7 5-AAGATTATTGGCGATAATCC-3' 5-ATAGATACTATCAGCACTCGC-3'8 5-GTTATTTCAAGCATCATAGTCG-3' 5-ATTTGTCAAAGAATTAGCTCG-3'

9 5-GCATTAGAAAGTTGCATTAACC-3' 5-TTCTTCGCAAGCATTAAGTTC-3'11 5-GATGGAGCCAAAGAGCTTGTG-3' 5-CACTTGCAGCAGATAAAGCCG-3'12 5-GTCAAAGGCGTTCGGATG-3' 5-AGCATTGATATGATCAATAGC-3'13 5-GCTATTGATCATATCAATGCT-3' 5-ATCTTCTTTACTATGATAACTCAC-3'14 5-GGGTGATATTTCATATCTTGG-3' 5-GCATAAGCTAAATCCTGTCC-3'--------------------------------------------------------------------------------


35/37

-35-

Table5-ResultsofthePCRRFLPanalysisfor11potentialGBSmarkersidentifiedbyhtAFLPanalysis

2 3 5,6 7 8 9 11 12 13 14

straina

disease (cj0432) (cj0615) (cj0967) (cj1136) (cj1138) (cj1139c) (cj1161c) (cj1306) (cj1306) (cj1336)

GB1 GBS 1 1 1 1 1 0 1 2 2 1

GB2 GBS 2 2 2 - - 1 2 4 3 -

GB3 GBS 2 2 2 - - 1 2 3 3 -

GB4 GBS 1 2 2 - - 2 2 1 1 -

GB5 GBS 1 2 1 - - 3 0 5 4 -

MF6 MFS 2 2 2 - - 4 2 6 0 -

MF7 MFS 1 2 1 - - 3 1 5 4 -

MF8 MFS 1 2 2 - - 0 2 6 1 -

GB11 GBS 1 2 2 - - 4 2 7 5 -

GB13 GBS 1 2 1 1 0 0 2 5 1 -

GB15 GBS 1 1 3 1 - 0 2 5 4 2

GB16 GBS 1 1 1 - - 3 2 1 1 -GB17 GBS 1 2 1 - 0 0 1 5 4 -

GB18 GBS 2 2 2 - - 4 2 4 6 -

GB19 GBS 1 1 1 - - 5 2 3 7 -

MF20 MFS 1 2 3 - - 5 1 5 8 -

GB21 GBS 2 2 2 - - 4 2 0 5 3

GB23 GBS 1 2 1 - - 4 0 1 1 4

GB24 GBS 2 1 2 - - - 1 1 4 -

GB25 GBS 1 2 - - - 3 1 5 8 -

GB26 GBS 1 1 1 - - 3 1 5 1 -

GB29 GBS 1 2 1 1 - - 1 10 10 3

GB30 GBS 3 1 3 1 - 3 1 1 1 5

cura7 GBS 2 2 2 - - 4 2 8 5 3

cura69 GBS 2 2 2 - - 4 2 3 9 -

cura276 GBS 2 2 2 - - 4 2 8 5 3

260.94 GBS 2 2 2 - - 4 4 9 5 -

E97-0737 enteritis 1 2 1 1 nd 0 1 2 1 -

E97-0747 enteritis 1 1 1 1 nd 0 2 5 10 5

E97-0796 enteritis 1 2 1 1 - 0 1 5 4 6

E97-0873 enteritis 2 3 2 - - 0 2 5 nd 4

E97-0903 enteritis 1 2 1 1 - 2 1 11 8 -

E97-0921 enteritis 1 2 3 - - 6 0 11 6 -

E97-0974 enteritis 1 2 1 1 1 0 nd 5 1 4

E97-0980 enteritis 1 1 1 - - 5 1 5 1 -

E97-0998 enteritis 2 2 2 - - 7 2 11 nd -

E97-1013 enteritis 1 1 3 - - 5 1 5 10 -

E98-623 enteritis 2 2 2 - - 2 2 5 nd 4

E98-624 enteritis 2 2 2 1 - 2 2 2 nd 7

E98-682 enteritis 1 1 1 1 1 0 1 2 10 -

E98-706 enteritis 1 1 3 - - 0 2 1 4 -

E98-1033 enteritis 1 2 3 - - 1 1 1 1 -

E98-1087 enteritis 1 2 1 1 1 0 1 1 4 -NCTC11168-1 enteritis 1 1 1 1 1 0 1 5 1 4

markernr(genomiclocalization)b


36/37

10203040

------------------+-------------------+-------------------+-------------------+-

1TAAATTATTATGCTAAGAGTTCTAAACAATATATTAACAAcj0615_genomeseq

1AGAGTTCTAAACAATATATTAACAAcj0615_11168_AFLP

1TAAGAGTTCTAAACAAGATATTAACAAcj0615_GB11_AFLP

DdeI

50607080

------------------+-------------------+-------------------+-------------------+-

41TATTACTTTAATATATAACAATCCTAAACCAAATATTACAcj0615_genomeseq

26TATTACTTTAATATATAACAATCCTAAACCAAATATTACAcj0615_11168_AFLP

28TATTACTTTAATATATAACAATCCTAAACCAAATATTACAcj0615_GB11_AFLP

90100110120

------------------+-------------------+-------------------+-------------------+-

81AATATAAACGATTTAAATTTTAAACATTATTTACTTACTCcj0615_genomeseq

66AATATAAACGATTTAAATTTTAAACATTATTTACTTACTCcj0615_11168_AFLP

68AATATAAACGATTTAAATTTTAAACATTATTTACTTACTCcj0615_GB11_AFLP

130140150160

------------------+-------------------+-------------------+-------------------+-

121CTGATATGAGAGAAGATGAAGTTCTTTCTTTTAAAGCAAGcj0615_genomeseq

106CTGATATGAGAGAAGATGAAGTTCTTTCTTTTAAAGCAAGcj0615_11168_AFLP

108CTGATATGAGAGAAGATGAAGTTCTTTCTTTTAAAGCAAGcj0615_GB11_AFLP

170180190200

------------------+-------------------+-------------------+-------------------+-

161GCATGGTGTTAATACAGCCGGTCATAGTATAAAAACAGTTcj0615_genomeseq

146GCATGGTGTTAATACAGCCGGTCATAGTATAAAAACAGTTcj0615_11168_AFLP

148GCATGGTGTTAATACAGCCGATCcj0615_GB11_AFLP

MboI

210220230240

------------------+-------------------+-------------------+-------------------+-

201AGAGTGCTTCCTTTTTTAATCACAGCAAAAACCGATCATGcj0615_genomeseq

186AGAGTGCTTCCTTTTTTAATCACAGCAAAAACCGATCcj0615_11168_AFLP

MboI

Figure1


37/37

1

Figure2

1 2 3 4 5 6 7 8 9 10 11 12 13

260.94233.94233.95308.95367.95370.95242.98378.96386.96199.97250.97242.9721.97

godschalk bio med central 1625157249777404 article

Documents