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UniversityofCalifornia,Davis

MolecularPhylogeneticCharacterizationofMicrobial
CommunityImbalancesinHumanInflammatoryBowel
Diseases
AnAnalysis

AleneChang
999321777
BiologicalSciences
ProfessorEisen
EVE161
WinterQuarter2016

March18,2016

Introduction
I wanted to present about microbial phylogenomics from a pathological
standpoint.IwaslookingforsomethingsimilartotheICUpaperweread,andeventually
foundseveralonmicrobialphylogenomicsofthegutmicrobiome.Icameuponapaper
fromNormanR.Paceslabandimmediatelyfounditintriguinggivenhowoftenwehad
discussed his pioneering work in the field of metagenomics. This papers title is
Molecularphylogeneticcharacterizationofmicrobialcommunityimbalancesinhuman
inflammatoryboweldiseasesbyDNFranketalsubmittedtotheProceedingsofthe
NationalAcademyofSciencesin2007.ThisperfectlyfitwithwhatIwantedtoexplore
whilebeingrelevanttocoursematerial.
ThoughIhavenotdecidedonacareerpathyet,Iamveryinterestedinpublic
healthproblemsandhowtheycanbeanalyzedandsolvedwithmicrobialtechniques.
This paper perfectly addressed those two points of interest. The topic of the gut
microbiomeanditsimplicationsindiseaseisaveryhottopicrightnow.Manyfaddiets
arearising,aspeopleclaimthatglutenfreedietscancureautism.Butdietdoeshavean
obviouseffectonthegutmicrobiome,whichinturnclearlyinfluenceshumanhealth.
This paper attempts to address and characterize differences between microbiotas of
patients with celiac disease and ulcerative colitis and compare them with non
inflammatoryboweldiseasecontrols. Thisaddressesthequestionofwhattheactual
communitydifferencesarebetweennormalandabnormalgutmicrobiomes.Theydothis
in order to determine whether facilitating redress of the detected microbiological
imbalancescouldbeapossibletreatmentofIBD.
Ithinkthispaperhasawellsetupexperimentthatutilizesmanyofthetechniques
we have discussed in class. Our class has covered a diverse range of methods for
collection,sequencing,andanalysisofmicrobes. Thispapercoversasolidportionof
thesetopics.TheyuseQPCRtoanalyzeSSUrRNAandusetheUniFractestandother
techniquestoexaminethebiodiversityofthehumanintestine.
Theauthorshypothesizethattheinflammatoryboweldiseasesubsetandcontrol
subsetspecimensarecomposedofdistinctmicrobialcommunities.Because
inflammatorybowelsyndromemaynotbeamonolithicdisorder,theyperforma
comprehensivecultureindependentphylogeneticanalysisofmicrobesfromalarge
sampleset.Theyseektoprovethatthedifferenceinrelativedistributionofmicrobesis
significantlydifferentbetweendiseasedsamplesandcontrolsamples.
Overall,thisisaverywelldone,wellconstructedpaper.Itisnosurprisethatit
hasbeencited1,770times. Thispapersetaprecedentindoingcultureindependent
studiesofthemicrobiome,aswellashavingamoreextensivesampling. Someofthe
maingoodpointsaretheirlargesamplesize,cultureindependentanalysistosurmount
problemsfacedpreviously,andthefactthattheystratifypatientsbyGImicrobiota.
However,someoftheirsamplingandsequencingtechniquesmayintroducesome
bias. They do address some of these issues in their paper, but other ones are not
elaboratedonintheirpaperorintheirsupplementalinformation.
Iamanalyzingtheentirepaperfromresearchmethodsstandpoint.Iwillexamine
theirmethods,results,discussionandthevalidityoftheirconclusions.

Methods
Withinthepaper,theauthorsdescriptionoftheirmaterialsandmethodsisvery
vagueandjustbrieflyoutlinesthebasicsofwhattheyvedoneinlessthanahalfapage.
Most likely due to page restrictions, instead of elaborating on their methods, they
continuallyreferencetheirsupplementalmaterial. Themethodswereveryroutineand
typicalofastudyofthisgenre. Atthetime,theirmetagenomicsequencingwasvery
novelintermsofaddressinginflammatoryboweldisease.
They hadtwogroups ofsamples: patients whoexhibited inflammatorybowel
disease and control patients who were hospitalized for a variety of other diseases
includingbutnotlimitedtoabscess,chronicconstipation,pneumatosisintestinalis,and
ulcers.A1.5squarecmpiecesoftissuewerecollectedfromthesepatientsandplacedin
ethanolandshippedondryicetothePaceLabattheUniversityofColoradoatBoulder.
TheythenisolatedDNAfromtissueswithsolventandamechanicalDNAextraction
protocol.TheythenperformedPCRandamplifiedSSUrRNAgenesandgeneratedPCR
libraries. ProductswerethenusedforfurtheranalysisandBLASTofrRNAdatabases
wereusedtomakespeciesidentifications.Selectedgroupsofmicrobesweresubjectedto
QPCR. ThiswastoreducethelikelihoodthattheshiftsinOTUfrequencyinclone
librariesresultedfrombloomsordiminishedloadsofparticularmicrobialgroups.The
labproceededtoperformsequencebasecallingandcontigassembly.Theythenclustered
sequencesintorelatednessgroups(OTUs)byaveragelinkageclustering.Theyusedthe
R software package to perform cluster analysis of samples. Using the normalized
weightedUniFractestassessedthesignificance.
The methods were presented in a clear and comprehensive manner. They
explained their protocols in sufficient detail and also clearly explained how they
minimizedbias(i.e.throughfreezingandaliquotingbuffer).Theyincludedtheirspecific
primersandkitsaswellastheirPCRprotocols.Thisenableseasierreplicationoftheir
experiment.
Allofthemethodsaddressthequestionsandhypotheseswell.Thepurposewas
to use cultureindependent phylogenetic analysis to characterize the gastrointestinal
microbiotasofinflammatoryboweldiseasepatientsandnoninflammatoryboweldisease
controlpatients. Thisexperimentwaswelldesignedandtheyclearlyknewwhatthey
weredoingwhentheysetout. Everythingfromcollectiontosequencingandanalysis
helpedanalyzedisparitiesinmicrobiota.
Despitetheirdetaileddescriptionoftheirprotocols,thereareafewstepsintheir
methodsthatareunclear.However,theyfailedtomentionwhatareasofthebodytheir
sampleswerecollectedfrom.Elsewhereintheresultsofthepapertheycomparebacterial
communitiesbetweenlargeandsmallintestinalsamples.Thisleadsmetobelievethat
theycollectedsamplesfromthesmallandlargeintestinesofeachoftheirpatients.
Oneweaknessintheirmethodsistheprogressionofthediseasewhentheycollect
specimens. They used samples from CD and UC patients removed during surgery.
UsuallybythetimesomeonehassurgeryforCDorUC,itisatasignificantlyadvanced
state.Sinceallofthesamplesarefromadvancedcases,thismightskeworalterthedata.
On the other hand, a specific strength in their methods is the comprehensive

nature.Thisgivesaclearerandmorestatisticallyvalidviewofthedifferencesbetween
themicrobiotasofpatients.

Results
The authors used molecular-phylogenetic sequence analysis to identify the kinds
of microbes contained in the tissue samples. Similar sequences were clustered into
operational taxonomic units because there were few identical sequences observed
between different samples. OTUs were assembled at a range of sequence identity
thresholds so that the sample sets could be compared at multiple phylogenetic depths. At
any OTU clustering level, there were no significant differences in sampling coverage
between sample categories. This allowed them to effectively compare samples by OTU.
The majority of sequences were associated with four phyla of Bacteria; this was
true across all samples regardless of sampling site or disease state of patient. Almost half
of the sequences belonged to either the order Bacteroidales or the family
Lachnospiraceae. Small intestine samples had more sequences of the Bacillus subgroup
of the Firmicutes when compared to colon samples. In contrast, small-intestine samples
contained fewer sequences of Bacteroidetes and Lachnospiraceae when compared with
colonic samples. OTU distributions were significantly associated with disease state but
not anatomy or gross pathology. Similar results were observed when clonal prevalence
was compared across sample sets instead of frequencies.
They conducted exploratory statistical analyses to determine whether there were
any apparent structures within the sequence data set. Their data indicated that variation
along the first two principal coordinate axes was significantly influenced by disease
status rather than anatomy or pathology. When the samples were hierarchically clustered
based on component scores, they separated into two primary clusters. One cluster
contained most of the non-inflammatory bowel disease samples, and the other cluster was
dominated by celiac disease and ulcerative colitis samples. Hierarchical clustering of
samples by OTU presence/absence produced similar results with more diseased samples
clustering in the subset. To further emphasize this point, the authors performed the
UniFrac test. Their results strongly support their hypothesis that the two subsets of
diseased and control specimens are composed of distinct microbial communities.
To account for shifts in OTU frequency due to blooms or diminished loads of
particular microbial groups, Q-PCR was used to analyze selected samples. The results of
this revealed a 10-fold decrease in total bacterial load in the inflammatory bowel disease
subset and confirmed differing quantities of different bacteria between subsets of
samples.
The results are presented in a clear and comprehensive manner. They are divided
between conventional ways of analyzing OTUs and exploratory data analysis that they
have created in order to take into account the idiopathic nature of inflammatory bowel
disease. This exploratory statistical analysis helps them to be able to analyze their data in
a novel manner and assess the differences between different sample subsets more
effectively.
Though it is hard to adjust for it, one weakness of their sequence analysis is their
division into OTU by SSU rRNA. While putting these sequences through BLAST and
using alignments methods is conventional, this misses a lot of diversity. Microbes vary in
function greatly within families and that diversity is not captured. There is still a marked
difference in between the two sample subsets, but deeper analysis could serve to elucidate
community difference between disease states or differing medical treatments.

DiscussionandConclusions
Theirdiscussionadequatelyaddressestheweaknessesandpotentialbiasesoftheir
methodsandresults.TheyacknowledgethattheircensusofmicrobesreflectsSSUrRNA
genecopynumberasopposedtotruecellcounts.This isfurtherexacerbatedbythe
biasesinherentinPCRamplificationandcloning.TherRNAanalysisdoesnotdetect
functional changes in gastrointestinal microbes such as enhanced virulence, mucosal
adherence,orantibioticresistance.Theydoclaimthattheirresultsprovideaninternally
consistentcultureindependentassessmentofthegutwallassociatedmicrobiotaofthe
inflammatoryboweldiseaseandnoninflammatoryboweldiseasepatients.
Therearesignificantdifferences betweentheinflammatoryboweldiseaseand
noninflammatoryboweldiseasesamples.However,sequenceanalysisindicatesthatthe
wallofthedistalsmallboweldoesnothaveasignificantlydifferentmicrobialpopulation
thanincomparisontothelargebowel.Theresultsoftheirbacterialsurveyshowshiftsin
gastrointestinalmicrobialcommunitiesbutfailtoilluminateanyparticularspeciesasthe
causativeagent.
LevelsofEnterobacteriaceaewereuniformandconsistentthroughoutbothtypes
ofsamples,soitisnotlikelythattheyhaveskewedassessmentsofmicrobialdiversity
resulting from contamination. Medical treatment history, sampling site, and gross
pathologywerenotcorrelatedwiththemicrobiotasoftheinflammatoryboweldisease
patientsorthecontrolsubset.Theyconcludethuslythattheobserveddifferencesinthe
microbiotas are valid and not caused by trivial experimental errors, differing patient
treatmentordifferingpatientsymptoms.
Despitethegeneralvalidityoftheirdata,theirdatadonotaddressthecausal
relationships between the reported microbial community imbalances and IBD
pathophysiology. The authors do address this and try to minimize this fact. They
proposethatmicrobialimbalancescontributetodiseaseseveritywithoutidentifyingthe
causes. Theirdata,whichshowscorrelationbetweenabnormalmicrobiotaandabscess
formation in patients with celiac, as well as the association with age and abnormal
commensalmicrobiotasnonethelesssupportthisargument.Theauthorsproposethatloss
ofnormalmicrobialfloracompositioncanserveasasignformoresevereinflammatory
boweldisease.
Thediscussionsofthispaperareverythoroughinassessingthestrengthsand
weaknessesoftheexperiment.Alloftheirresultspointtotheirhypothesesbeingvalid.
Theircomparisonofsamplesfrompatientswithandwithoutinflammatoryboweldisease
revealsthatthemicrobiotasofthesepatientsaresignificantlydifferent.

OverviewandBigPicture
Thispapersmainstrengthisinitsnovelwayofanalyzingthegutmicrobiome.
Priortothispaper,thetypesofmicrobesinvolvedhadnotyetbeenadequatelydescribed.
Their exploratory data was helpful in the elucidation of the significant differences
betweensamplesofthediseasedsubsetandthecontrolsubset.Themethodstheyused
havenowbecomecommonplaceinthewayinflammatoryboweldiseasepathogenesis
hasbeendefinedandcharacterized.
Noexperimentisperfect,andthisonedoesnotescapethatrule.Thoughitiswell
constructed, there are several weaknesses that could be improved on. Firstly, their
samplingis limitedinitsscope,andthemethods andlocationofresectionwerenot
adequately described. Secondly, this study does not address disease causation.
Inflammatoryboweldiseasecouldconceivablybecausedbyrandomalterationsinthe
microbiomeduetounrelatedactivities. Furtherexperimentationordifferentsampling
methods could shed light on the pathogenesis of inflammatory bowel diseases and
identifythecauseoftheshiftinmicrobialcommunities.
WereItoperformthisexperiment,Iwoulddoseveralthingsdifferently.Their
cultureindependentsequencingrevolvedaroundusingSSU16SRNA.Theinformation
insomedatabasesisnotalwayscorrect.Furthermore,thereisnodefinitionofspeciesor
genusbasedon16SRNA.Thusfar,therearenotalternativesthatareaswidespreadas
SSURNA.Thismethodhasbecomethegoldstandard.However,manyalternativeshave
beenproposedandtested.TheseoptionsincludeRecA,rpoB,nifD,andmanyothers.
Thoughitmaynothavebeenfeasibleatthetime,longitudinalcollectionofsamples
couldalsohelpcharacterizechangesinthemicrobiomeofpatientswithintestinalbowel
diseaseduringtheprogressionorregressionoftheirdisease.
This paper is a seminal work published in a noteworthy, peerreviewed
publication.Itiswellcitedandusedmethodsthathadnotyetbeenusedinthisfield.It
isnotsurprisethatIcannoteasilyfindfaultswiththispaper.Therearemanypapersthat
havebeenpublishedsince2007thatusesimilarmethodstoanalyzeothersubsetsofthe
fieldsuchasdogintestinalmicrobiota.AquicksearchthroughGooglescholarreveals
thatthisisoneofthemosthighlycitedworksinregardstomolecularphylogeneticsand
inflammatorybowelsyndrome.Thearticlecurrentlyhas1770citations.
Thispaperhasobviouslybeeninfluential,butitalsobuiltuponinformationfrom
itspredecessors.In2005,astudyentitledDiversityoftheHumanIntestinalMicrobial
FlorawaspublishedinScience.However,thescopeofthisstudywaslimitedbyits
samplesize.Theyonlyhad3subjects,allofwhomhadintestinalboweldisease.Franks
papercanbethoughtofasafollowuptotheaforementionedpaper,withmoreresources
andmorevalidity.
Scientificresearchanswersourquestionsonlytogeneratemorequestionswith
each new finding. Although this paper provided greater insight into the community
differencescausedbyhumaninflammatoryboweldiseases,itraisesquestionsastothe
causesandpotentialtreatmentsofthecommunityshift.
Inflammatoryboweldiseaseisahottopicinthescientificcommunityrightnow,
and there had been rapid progress within the past 10 years. There has been deeper

investigation into intestinal protein formation, the influence of genetics on


gastrointestinalflora,aswellasfurthermolecularphylogeneticcharacterizationofthegut
microbiome. Theseinvestigationshavegivenusinsightintotheroleoftheimmune
systemintheinitiationandperpetuationofinflammatoryboweldisease.Thereisalotof
worktobedoneinordertofullyunderstandthepathogenesisofinflammatorybowel
diseases.
Thispaperhasaverydifferentflowtoitthanthepapersanalyzedinourcourse.
Theintroductionisfollowedbyresults,followedbythediscussion,andthenfinishes
withanoverviewoftheirmethods.Interestinglyenough,thereisnoseparateconclusions
section.Theauthorsmayhavebeenconfidentenoughintherichnessoftheirdiscussion
nottoincludeone,ortheymighthaveapproachedtheirpagelimit.Theirdiscussiondoes
summarizetheirfindingsandprovideabroadoverviewoftheimplicationsandfuture
directions.However,thelackofconclusiondoesleavethepaperseeminglikeitwascut
short.
Overall,Igreatlyenjoyedreadingthispaper.Ithinkthatthispaperhasalarge
levelofimportanceduetothedevelopmentofnewmethodsofanalysisandcomparison
ofmicrobiomesbetweenpatientswithandwithoutinflammatoryboweldisease. The
paperwaswellwritten,andtheexperimentwaswellexecuted.ThereadingIhadtodo
inordertounderstandthispaperhasdeepenedmyunderstandingofthefieldaswell.I
havemanyfriendsandcolleaguesworkingwiththegutmicrobiome,andnowIhave
greaterinsightintotheirresearch.