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• Vaginal bacterial communi0es are thought create and maintain a low environmental pH that restricts the growth of non‐indigenous popula0ons. • We sought to assess the stability of vaginal bacterial communi0es and model temporal dynamics. • 33 asymptoma0c, reproduc0ve‐age women self‐collected mid‐vaginal swabs every third day for 16 weeks as part of a vaginal douching cessa0on study. (Brotman et al. Am J Obstet Gynecol 2008) • Bacterial community composi0on was determined based on 16S rRNA gene sequences (V1‐V2 region). • A dataset of more than 2.97 million 16S rRNA gene sequences was obtained from 988 samples (29.9 ± 1.6 (SD) samples per subject) with an average of 3,005 ± 1257 (SD) sequences per sample. Taxonomic assignments were performed as described by Ravel et al. (PNAS 2010) using a combina0on of the RDP classifier and SpeciateIT. • A linear mixed effect model for the log of Jensen‐Shannon divergence rate of community change was used to inves0gate the role of menstrua0on and sexual ac0vity on the constancy of the vaginal microbiota. The model accounted for correla0ons between samples from the same par0cipant and was adjusted for 0me‐varying confounders including vaginal douching, contracep0ve and normalized menstrual cycle 0me. • COMMUNITY STATE TYPE: A state type is comprised of samples that have similar bacterial species composi0on. In this study five community state types were iden0fied. Profiles of community state types were determined for each woman. • COMMUNITY STATE CLASS: A community state class is formed of similar community state profiles. • The species composi+on of bacterial communi+es in some women changed markedly over short periods of +me while in others it was rela+vely stable. • Modeling of vaginal bacterial community dynamics indicates that community stability was associated with +me in the menstrual cycle, and varied among communi+es that differed in composi+on. • A be?er understanding of factors that lead to the development and maintenance of specific and stable vaginal communi+es is needed so strategies can be developed to promote and maintain reproduc+ve health. • In future work we will determine if habits and prac+ces (including use of lubricants, feminine hygiene products, and specific sexual behaviors) influence community dynamics. 1 Department of Biological Sciences, University of Idaho, Moscow, Idaho, USA, 2 Ini+a+ve for Bioinforma+cs and Evolu+onary Studies (IBEST), University of Idaho, Moscow, Idaho, USA, 3 Department of Epidemiology and Public Health, University of Maryland School of Medicine, Bal+more, Maryland, USA, 4 Ins+tute for Genome Sciences, University of Maryland School of Medicine, Bal+more, Maryland, USA, 5 Department of Mathema+cs, University of Idaho, Moscow, ID, USA, 6 Department of Sta+s+cs, University of Idaho, Moscow, ID, USA, 7 Department of Microbiology and Immunology, University of Maryland School of Medicine, Bal+more, Maryland, USA Roxana J. Hickey 1,2 , Rebecca M. Brotman 3,4 , Pawel Gajer 4 , Joyce Sakamoto 4 , Sara S.K. Koenig 4 , Li Fu 4 , Xia Zhou 1,2 , Zaid Abdo 2,5,6 , Jacques Ravel 4,7 , Larry J. Forney 1,2 Temporal dynamics of vaginal bacterial communities Figure 1. Heatmap of phylotype rela0ve abundance from all 998 samples longitudinally collected from 33 women. Each ver0cal line represents one sample. Three of the community state types were dominated by Lactobacillus sp. and represented 17% (community state type I), 6% (community state type II) and 41% (community state type III) of the samples. A total of 36% of the samples did not contain significant number of Lactobacillus species (community state type IV‐A and IV‐B). Figure 2. Dynamics of vaginal community state types in 33 women over 16 weeks. A. Dendrogram of distances between propor+ons of the five community state within a woman over +me. B. Heatmap of propor+ons of community state types measured within a woman over +me. C. Color bar indica+ng community class (DA, LC, LG, DB, and LI) as defined by clusters of propor+ons of community state types within a woman over +me. D. Temporal profiles of community state types for 33 women over 16 weeks. Each +me point is represented by a color‐coded community state type assigned to the corresponding community state. E. Box plot of Nugent scores for each woman over 16 weeks. Figure 3. Vaginal community dynamics. Panels (A) and (C). Subject 21 and 2 ‐ Interpolated bar graphs of phylotypes abundance. Metadata and profile of community states types are shown below. Panels (B) and (D). Schema+cs of the dynamics of vaginal communi+es of subject 21 and 2 in the vaginal community space as defined by Ravel et al. (PNAS 2010). Figure 4. Examples of vaginal bacterial community that exhibit low dynamics. Community dynamics are represented by a heatmap (top) and an interpolated bar graph of phylotypes abundance (middle). The bo?om panel shows the rate of change of the Jensen‐Shannon divergence, and selected metadata, including menses and sexual ac+vi+es. Figure 5. Examples of vaginal bacterial community that exhibit high dynamics. (See legend Fig. 4) This work was supported by grants UH2 AI083264 and K01 AI080974 from the Na+onal Ins+tute of Allergy and Infec+ous Diseases, Na+onal Ins+tutes of Health. • It is the most common member of vaginal communi+es among women of all ethnic groups. • L. iners has the smallest genome (~1.3 Mb) of all known Lactobacillus sp.; appears to have undergone dras+c genome reduc+on (Macklaim et al. PNAS 2010). The genomes of other vaginal Lactobacillus sp. range from ~1.6 – 2.3 Mb. • Genomic features of L. iners: incomplete TCA cycle; missing genes include those required for synthesis of vitamins, cofactors, amino acids and purines. • Is L. iners dependent on their neighbors for growth and survival – is there an ecological network based on nutri+onal dependencies? What disrupts these community networks? • Are differences in community composi+on relevant to differences in community stability? Why is L. iners of interest? Community networks, ecological resistance and resilience Walker’s “Driver‐Passenger Model” • One or more species “drive” the community; the remainder are not essen+al for community performance and may vary. • We postulate that lac+c acid bacteria drive the vaginal ecosystem L .iners Species 1 Species 2 Species 3 Species 4 Species 5 ? RESEARCH AIMS 1. Gain a be?er understanding of the strain diversity of L. iners in the vagina. Are there differences in the metabolic capabili+es or ecological func+ons of strains? 2. Iden+fy and characterize ecological networks of bacteria that L. iners may depend on for survival and persistence. Frequency or Dura0on Disturbed Ecosystem Intensity

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Page 1: Temporal dynamics of vaginal bacterial communities

•  Vaginal bacterial communi0es are thought create and maintain a lowenvironmental pH that restricts the growth of non‐indigenouspopula0ons.

•  We sought to assess the stability of vaginal bacterial communi0es andmodeltemporaldynamics.

•  33 asymptoma0c, reproduc0ve‐age women self‐collected mid‐vaginalswabs every third day for 16 weeks as part of a vaginal douchingcessa0onstudy.(Brotmanetal.AmJObstetGynecol2008)

•  Bacterial community composi0on was determined based on 16S rRNAgenesequences(V1‐V2region).

•  A dataset of more than 2.97 million 16S rRNA gene sequences wasobtainedfrom988samples(29.9±1.6(SD)samplespersubject)withanaverage of 3,005 ± 1257 (SD) sequences per sample. Taxonomicassignmentswere performed as described by Ravel et al. (PNAS 2010)usingacombina0onoftheRDPclassifierandSpeciateIT.

•  A linearmixed effectmodel for the log of Jensen‐Shannon divergencerate of community change was used to inves0gate the role ofmenstrua0on and sexual ac0vity on the constancy of the vaginalmicrobiota.Themodelaccountedforcorrela0onsbetweensamplesfromthe same par0cipant and was adjusted for 0me‐varying confoundersincluding vaginal douching, contracep0ve and normalized menstrualcycle0me.

•  COMMUNITYSTATETYPE:Astatetypeiscomprisedofsamplesthathavesimilarbacterialspeciescomposi0on.Inthisstudyfivecommunitystatetypes were iden0fied. Profiles of community state types weredeterminedforeachwoman.

•  COMMUNITYSTATECLASS:Acommunitystateclassisformedofsimilarcommunitystateprofiles.

•  Thespeciescomposi+onofbacterialcommuni+esinsomewomenchangedmarkedlyovershortperiodsof+mewhileinothersitwasrela+velystable.

•  Modelingofvaginalbacterialcommunitydynamicsindicatesthatcommunitystabilitywasassociatedwith+meinthemenstrualcycle,andvariedamongcommuni+esthatdifferedincomposi+on.

•  Abe?erunderstandingoffactorsthatleadtothedevelopmentandmaintenanceofspecificandstablevaginalcommuni+esisneededsostrategiescanbedevelopedtopromoteandmaintainreproduc+vehealth.

•  Infutureworkwewilldetermineifhabitsandprac+ces(includinguseoflubricants,feminine hygiene products, and specific sexual behaviors) influence communitydynamics.

1DepartmentofBiologicalSciences,UniversityofIdaho,Moscow,Idaho,USA,2Ini+a+veforBioinforma+csandEvolu+onaryStudies(IBEST),UniversityofIdaho,Moscow,Idaho,USA,3DepartmentofEpidemiologyandPublicHealth,UniversityofMarylandSchoolofMedicine,Bal+more,Maryland,USA,4Ins+tuteforGenomeSciences,UniversityofMarylandSchoolofMedicine,Bal+more,Maryland,USA,5DepartmentofMathema+cs,UniversityofIdaho,

Moscow,ID,USA,6DepartmentofSta+s+cs,UniversityofIdaho,Moscow,ID,USA,7DepartmentofMicrobiologyandImmunology,UniversityofMarylandSchoolofMedicine,Bal+more,Maryland,USA

RoxanaJ.Hickey1,2,RebeccaM.Brotman3,4,PawelGajer4,JoyceSakamoto4,SaraS.K.Koenig4,LiFu4,XiaZhou1,2,ZaidAbdo2,5,6,JacquesRavel4,7,LarryJ.Forney1,2

Temporal dynamics of vaginal bacterial communities

Figure1. Heatmapofphylotyperela0ve abundance from all 998samples longitudinally collectedfrom 33 women. Each ver0callinerepresentsonesample.

Three of the community state typeswere dominated by Lactobacillus sp.andrepresented17%(communitystatetype I), 6% (community state type II)and 41% (community state type III) ofthe samples. A total of 36% of thesamples did not contain significantnumber of Lactobacillus species(communitystatetypeIV‐AandIV‐B).

Figure 2. Dynamics of vaginalcommunity state types in 33womenover16weeks.

A.  Dendrogram of distancesbetweenpropor+onsof thefivecommunity state within awomanover+me.

B.  Heatmap of propor+ons ofcommunity state typesmeasuredwithin awoman over+me.

C.  Color bar indica+ng communityclass (DA, LC, LG,DB, and LI) asdefined by clusters ofpropor+ons of community statetypeswithinawomanover+me.

D.  Temporal profiles of communitystate types for 33 women over16 weeks. Each +me point isrepresented by a color‐codedcommunity state type assignedtothecorrespondingcommunitystate.

E.  Box plot of Nugent scores foreachwomanover16weeks.

Figure3.Vaginalcommunitydynamics.

Panels(A)and(C).Subject21and2‐Interpolatedbargraphsofphylotypesabundance.Metadataandprofileofcommunitystatestypesareshownbelow.

Panels(B)and(D).Schema+csofthedynamicsofvaginalcommuni+esofsubject21and2inthevaginalcommunityspaceasdefinedbyRaveletal.(PNAS2010).

Figure 4. Examples of vaginalbacterial community that exhibitlowdynamics.

Communitydynamicsarerepresentedbyaheatmap(top)andaninterpolatedbargraphofphylotypesabundance(middle).The bo?om panel shows the rate ofchange of the Jensen‐Shannondivergence, and selected metadata,includingmensesandsexualac+vi+es.

Figure5.Examplesofvaginalbacterialcommunitythatexhibithighdynamics.(SeelegendFig.4)

ThisworkwassupportedbygrantsUH2AI083264andK01AI080974fromtheNa+onalIns+tuteofAllergyandInfec+ousDiseases,Na+onalIns+tutesofHealth.

•  Itisthemostcommonmemberofvaginalcommuni+esamongwomenofallethnicgroups.

•  L. iners has the smallest genome (~1.3Mb) of all known Lactobacillus sp.;appears to have undergone dras+cgenomereduc+on(Macklaimetal.PNAS2010). The genomes of other vaginalLactobacillus sp. range from ~1.6 – 2.3Mb.

•  Genomic featuresofL. iners: incompleteTCA cycle; missing genes include thoserequired for synthesis of vitamins,cofactors,aminoacidsandpurines.

•  IsL.inersdependentontheirneighborsforgrowthandsurvival–isthereanecologicalnetworkbasedonnutri+onal dependencies?Whatdisrupts thesecommunitynetworks?

•  Aredifferencesincommunitycomposi+onrelevanttodifferencesincommunitystability?

WhyisL.inersofinterest? Communitynetworks,ecologicalresistanceandresilience

Walker’s“Driver‐PassengerModel”•  One or more species “drive” the community; the

remainder are not essen+al for communityperformanceandmayvary.

•  We postulate that lac+c acid bacteria drive thevaginalecosystem

L.iners

Species1

Species2

Species3

Species4

Species5

?

RESEARCHAIMS1.  Gain a be?er understanding of the strain

diversity of L. iners in the vagina. Are theredifferences in the metabolic capabili+es orecologicalfunc+onsofstrains?

2.  Iden+fy and characterize ecological networksof bacteria that L. iners may depend on forsurvivalandpersistence.

FrequencyorDura0on

DisturbedEcosystem

Intensity

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