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Godschalk Bio Med Central 1625157249777404 Article
Transcript of Godschalk Bio Med Central 1625157249777404 Article
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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]
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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
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Conclusions
ThisstudyshowsthatbacterialGBSmarkersarelimitedinnumberandlocatedinthe
LOSbiosynthesisgenes,whichcorroboratesthecurrentconsensusthatLOSmimicry
maybetheprimeetiologicdeterminantofGBS.Furthermore,ourresultsdemonstrate
thathtAFLP,withitshighreproducibilityandresolution,isaneffectivetechniquefor
thedetectionandsubsequentidentificationofputativebacterialdiseasemarkers.
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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.
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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.
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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.
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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
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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.
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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
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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
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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----------------------------------------------------------------------------------------------------------------------------------------------------------
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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
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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
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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'--------------------------------------------------------------------------------
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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
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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
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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