Individual and household attributes influence the dynamics of the personal skin microbiotaand its association network

Leung, Marcus H.Y.; Tong, Xinzhao; Wilkins, David; Cheung, Hedwig H.L.; Lee, Patrick K.H.

Published in:Microbiome

Published: 01/01/2018

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Published version (DOI):10.1186/s40168-018-0412-9

Publication details:Leung, M. H. Y., Tong, X., Wilkins, D., Cheung, H. H. L., & Lee, P. K. H. (2018). Individual and householdattributes influence the dynamics of the personal skin microbiota and its association network. Microbiome, 6(1).https://doi.org/10.1186/s40168-018-0412-9

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RESEARCH Open Access

Individual and household attributesinfluence the dynamics of the personal skinmicrobiota and its association networkMarcus H. Y. Leung, Xinzhao Tong, David Wilkins, Hedwig H. L. Cheung and Patrick K. H. Lee*

Abstract

Background: Numerous studies have thus far characterized the temporal dynamics of the skin microbiota of healthyindividuals. However, there is no information regarding the dynamics of different microbial association networkproperties. Also, there is little understanding of how living conditions, specifically cohabitation and household occupancy,may be associated with the nature and extent (or degree) of cutaneous microbiota change within individuals over time.In this study, the dynamics of the skin microbiota, and its association networks, on the skin of urban residents over fourseasons were characterized.

Results: Similar to western cohorts, the individuals of this cohort show different extents of variations in relative abundanceof common skin colonizers, concomitant with individual- and household-associated changes in differential abundances ofbacterial taxa. Interestingly, the individualized nature of the skin microbiota extends to various aspects of microbialassociation networks, including co-occurring and excluding taxa, as well as overall network structural properties.Household occupancy is correlated with the extent of variations in relative abundance of Propionibacterium, Acinetobacter,and Bacillus over multiple skin sites. In addition, household occupancy is also associated with the extent of temporalchanges in microbial diversity and composition within a resident’s skin.

Conclusions: This is the first study investigating the potential roles household occupancy has on the extent of change inone’s cutaneous microbiota and its association network structures. In particular, we show that relationships between theskin microbiota of a resident, his/her cohabitants, and those of non-cohabitants over time are highly personal and arepossibly governed by living conditions and nature of interactions between cohabitants within households over 1 year.This study calls for increased awareness to personal and lifestyle factors that may govern relationships between the skinmicrobiota of one individual and those of cohabitants, and changes in the microbial association network structures withina person over time. The current study will act as a baseline for future assessments in comparing against temporaldynamics of microbiota from individuals with different skin conditions and for identifying residential factors that arebeneficial in promoting the dynamics of the skin microbiota associated with health.

BackgroundA community of microbial life forms, constituting ofbacteria, fungi, viruses, and parasites make up thecommensal skin microbiota responsible for modulatingthe host immunity responses and preventing colonizationand invasion by pathogens [1]. Various medical and aller-gic conditions are associated with alterations in one’soverall cutaneous microbial community and dysbiosisbetween specific skin colonizers [2–4], highlighting the

importance of the microbiota in cutaneous health. Thanksto advances in sequencing technology, the scientificcommunity has gained a wealth of information regardingthe skin microbiota, including the biogeography of theskin microbiota, roles of host properties, activities, andthe environment on cutaneous microbial communities,co-abundance and co-exclusion correlations between taxa,and changes of microbiota associated with health anddisease [4–15].Recent investigations have also reported on the temporal

dynamics of the cutaneous microbiota [2, 10, 16–21],highlighting the highly personal nature of the skin

* Correspondence: patrick.kh.lee@cityu.edu.hkSchool of Energy and Environment, City University of Hong Kong,B5423-AC1, Tat Chee Avenue, Kowloon, Hong Kong

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Leung et al. Microbiome (2018) 6:26 DOI 10.1186/s40168-018-0412-9

microbiota across healthy individuals over weeks andmonths. Changes in the microbiota may also be associatedwith cutaneous or immunological conditions, or asresponses to clinical interventions [7, 19]. Altogether, thesestudies reinforce the importance of understanding potentialrelationships between the environment, host characteristics,and microbiota variations over time, and whether micro-biota changes over time is related to temporal variations indisease phenotypes and prognosis [7].As with other ecosystems, microorganisms found on

skin engage in co-abundance and exclusion relationships[22]. While correlation does not equate causation,microbial association networks can be analyzed to inferpotential ecological relationships between communitytaxa, as well as relationships between taxa and theenvironment. Recent works have performed correlationnetwork analyses to characterize potential relationshipsbetween specific taxa within the skin microbiota [5, 6, 22].However, understanding association networks at thestructural level provides additional information on howmicrobial associations as a whole respond to differenthuman or temporal variables, potentially revealing howthis global association framework changes, and the factorsthat may drive such changes over time.Previous studies have illustrated the effects of

cohabitation on the skin microbiota within individuals.Specifically, our previous work [5] recapitulates thediscoveries of other studies [19, 23] that the cutaneousbacterial assemblage of cohabitants are more similarcompared to non-cohabitants. While these works comple-ment earlier reports describing the effects of lifestyles andliving conditions on occupant skin microbiota [13, 15], nostudy has extensively and directly evaluated the potentialroles of cohabitation and household occupancy in shapingthe nature and extent of changes in the skin microbiotaand its association networks within individuals over time.Given that cohabiting individuals and their microbiotamay facilitate the transmission of microorganisms [24], agreater insight into the potential roles of household prop-erties such as occupancy on an individual’s skin microbiotaand its dynamics may be of clinical significance.Therefore, following our previous skin microbiota studies

involving a single time point [5, 6], we now provide anaccount of the skin microbiota (while the term microbiotacommonly encompasses all microbial domains, the term ishereafter referred to as the total bacterial and archaealcommunity) and its dynamics, of 24 individuals withinHong Kong (HK) households over the period of 1 year.Different aspects of the nature and extent of microbiotadynamics, with a focus on the roles of cohabitation andhousehold occupancy, are described. We show that whileour observations are consistent with the individuality ofthe dynamics of cutaneous microbiota shown in previousworks [10, 20], we extend this personalized property to the

dynamics of microbial association networks. At the sametime, we suggest that the extent of changes in differentcharacteristics of the skin microbiota may be associatedwith the number of cohabitants an individual resides with.By focusing on the changes of multiple aspects of the cuta-neous microbiota at individual and household levels, thisstudy sets the stage for comparative analyses pertaining tomicrobiota dynamics between healthy individuals and thosewith various skin conditions, thereby potentially identifyingpersonal, household, and related factors associated withhealthy microbiota dynamics.

Results and discussionIn this study, we first characterize and discuss the natureand degree of variability of various facets of the skinmicrobiota within healthy individuals. This is followedby a focus of how effects of cohabitation and residentialoccupancy may play roles in shaping the observed differ-ences in the degree of temporal variability in one’s skinmicrobiota over time.

Taxonomic and sub-genus overview of the skin microbiotaover timeTop genera (i.e., those with average relative abundanceof ≥ 1% across the entire dataset) are detected across allindividuals and skin sites and comprise under 60% ofthe skin microbial community, reflecting the overalltaxonomic diversity of the cutaneous microbiota(Additional file 1: Figure S1 and Additional file 2:Table S1) [25]. The top genera include Enhydrobacter,which has been detected in previous reports and isconsidered to be enriched on skin surfaces of Chinese indi-viduals [5, 26–28], suggesting that this genus is a prevalentand stable skin colonizer in subjects of this cohort.Information is currently lacking on the ecological andphysiological bases for the apparent enrichment ofEnhydrobacter in the Chinese. However, an associationbetween relative abundance of Enhydrobacter on skin andfacial sebum level and hydration has been reported in arecent Asian study [28]. Further examination of geneticand/or lifestyle factors that may affect skin physiology(such as living conditions, environmental exposure, foodintake, and use of cosmetic and sanitary products [11, 25,29–31]) may help elucidate the physiology of this colonizeron skins of Chinese and other host populations.We sought to examine the extent of change in

relative abundance of a given genus within an indi-vidual over the four seasons. Here, the extent ofchange in relative abundance for a particular genusis expressed as its coefficient of variation (CV). Re-gardless of skin site, each individual presents a dis-tinctive signature of CV for each of the top genera(Additional file 2: Table S1 and Additional file 3:

Leung et al. Microbiome (2018) 6:26 Page 2 of 15

Figure S2). Previously, it was revealed that the rela-tive abundance and the variations (as expressed bythe standard deviation) of microbial taxa within theskin microbiota of a western cohort exhibit asecond-order relationship, where low- and high-abundance microbial taxa vary less than those thatare moderately abundant [10]. In contrast, our HKcohort shows strong linear correlative relationshipsacross individuals (Additional file 4: Figure S3). Asno genus in our dataset represents an average ofover 45% of any individual’s microbiota, our correla-tive relationship between relative abundance andstandard deviation over time is expected to be differ-ent from the previous study, where the second-orderrelationship observed appears to be strongly drivenby taxa detected in over 50% of a subject [10].Differential abundance (Fig. 1 and Additional file 5:

Table S2), negative binomial mixedmodel (Additional file 5:Table S2), and oligotype (Additional file 6: Figure S4)analyses reveal that genera are comprised of multiplespecies/strains, and specific taxa may be enriched indifferent seasons, independent of household or individualfactors (for example, OTU_27 and OTU_746 in Fig. 1a, b;mixed model results are shown in Additional file 5:Table S2 and also oligotypes of genera in Additional file 6:Figure S4a-d). At the same time, taxa may be enrichedin specific households/individuals within a season (forexample, OTU_27 and OTU_20 within WKS, andOTU_24 within TK in Fig. 1a, c, d, and also oligo-types of genera in Additional file 6: Figure S4e-g).

Cohort-wide variations in taxa distribution in differentseasons may suggest general temporal trends in theemergence of particular species or strains on skin[10]. However, individual- or household-specific pas-sive exposure may contribute to certain subjects orhouseholds carrying a particular species or strain at agiven time point. It is possible that particular taxamay be picked up by cohabiting members via physicalcontact with residential surfaces, given the overlap be-tween occupant skin and surface microbiota withinhouseholds [21, 32, 33]. However, it is not knownhow these individual- or household-specific taxa canstably colonize the skin, as the fluctuations in relativeabundance of taxa at different time points within anindividual may also represent its poor adaptability andcompetitiveness with co-colonizers [10]. Whether ataxon presents population-wide or individual/house-hold-associated temporal variation in abundance maybe dependent on the taxon and how it interacts withthe host, the environment, and co-colonizing mi-crobes. As different species and strains may show var-iations in physiologies [34, 35], future temporalanalyses of taxon-level variation in subjects may provevaluable in linking individual, household, and tem-poral factors with detection of taxa associated withincreased virulence and resistance. Compared to themarker-based analyses, multi-omics strategies mayprovide more detailed genomic and metabolic assess-ments of strain variations between individuals, house-holds, skin sites, and over time [9, 10].

Fig. 1 DeSeq2 differential abundance analysis of OTUs between pairwise seasonal comparison within individuals. OTUs showing cohort-wide differentialabundances between seasons include a OTU 27 of Acinetobacter and b OTU 746 of Caldithrix. In addition, OTUs with household-specific differentialabundance patterns include c OTU 20 of Roseomonas (household WKS) and d OTU_24 of Deinococcus (household TK). Note that despite a OTU 27showing cohort-wide patterns, OTU 27 in occupants of WKS shows an opposite trend, showing a household-specific pattern in differential abundance.Occupants are color-coded according to households. A full list of OTUs with strong and significant differential abundances, which is defined by DeSeq2log-fold difference of at least three and with an adjusted p value of ≤ 0.05, is presented in Additional file 5: Table S2

Leung et al. Microbiome (2018) 6:26 Page 3 of 15

Microbial diversity variations within individuals over timeShannon diversity, taking into account both communityrichness and evenness, was calculated to determine thenature and range of temporal variability in microbialdiversity across individuals. When all samples wereincluded for analysis, significant differences in Shannondiversity are observed between individuals, seasons, andskin sites (FDR-adjusted p ≤ 0.02 for all, Additional file 7:Table S3). Within each season, individuals and house-holds are the major factors explaining significant differencesin diversity. Linear mixed model also suggests that each ofthese major factors is potential drivers for differences inShannon diversity (p < 0.001 for the three factors of season,individuals, and households. For each test, the other twofactors, as well as sequencing batches, were considered asrandom variables). Significant differences in forehead andforearm diversities are observed between households, whileforearms also exhibit significant differences in diversitybetween individuals (Additional file 7: Table S3).To understand the personal rate of change in microbial

diversity over the course of 1 year, we calculated the CVof Shannon diversity at a given site within an individual.Considering all samples, the Shannon diversity CVs aredifferent between individuals, households, and occupancy(Kruskal-Wallis (KW) FDR-adjusted p < 0.005 for all). Incontrast, CVs are not significantly different betweensamples of different skin sites, age group, and gender(FDR-adjusted p > 0.05, KW for skin site and age group,and Mann-Whitney (MW) for gender). Communities onleft forearm sites varied in CVs between individuals andhouseholds (KW FDR-adjusted p < 0.04 for both,Additional file 8: Figure S5a-b). Shannon diversityCVs in forearms are also significantly higher in adults(average within-individual CV = 0.145) compared tothe elderly (CV = 0.0871, KW post hoc pairwise p < 0.05,Additional file 8: Figure S5c).

Microbial compositional variations within individuals overtimeWe analyzed variations in microbial community composi-tions between subjects, households, and within individualsover time using weighted UniFrac distances, taking intoaccount both the presence and abundance of OTUs. Skinmicrobiota significantly cluster by season, individual,household, and age groups overall, as well as withinseparate skin sites, as shown by both analysis of similar-ities (ANOSIM) and permutational multivariate analysisof variance (PERMANOVA) analyses (Additional file 9:Table S4). Microbiota do not appear to cluster strongly byskin sites within each season in our cohort (and only mar-ginally in summer).Within an individual, the microbiota of forearm and

palms are more similar within than between seasons(Additional file 9: Table S4 and Additional file 10:

Figure S6. Forehead sites were not analyzed, as thereis only one forehead sample per individual per sea-son). Also, within an individual, the extent of micro-biota dissimilarity on a skin site between any twotime points is significantly and positively correlatedwith that of another skin site (Table 1), consistentwith the work of Flores et al. [20]. Furthermore, ex-tremity sites within individuals with greater extent oftemporal change in microbial diversity also vary greater inthe change in community composition over the year(Additional file 11: Figure S7). While correlations betweentemporal community variation and average community di-versity (i.e., beta-diversity versus alpha-diversity) within anindividual over time have been reported previously[10, 20], here, we demonstrate that the extent of changesin the two community properties (beta-diversity versusCV of alpha-diversity) are also correlated.As with previous investigations [10, 17], the microbiota

of the same site within some individuals between seasonscloser together are more similar, as suggested by thesignificantly negative time-decay relationships(Additional file 9: Table S4). As samples for this studywere collected within a single year, it is currently notknown whether the decay relationships observed willpersist for longer time frames as in other ecosystems[36], or whether an annual or seasonal cyclicalpattern will emerge over longer periods of time, asseen in the gut microbiota of different mammals [37] andpotentially in humans [38]. A greater understanding oftime-decay relationships of cutaneous microbiota hasforensic implications, where the identifiability of a person’smicrobiota on a surface may decrease over time [21].Given that time-decay relationships are more likely to be apersonal trait rather than one shared by members of acohort or household level (based on our results here andthose reported previously [20]), additional works will berequired to understand why some subjects present time-decay relationships and some do not, and how inter-indi-vidual time-decay variability affects the feasibility of usingmicrobiota data in forensic applications.Overall, our observations are congruent with previous

western studies looking at the temporal dynamics of theskin microbiota [10, 17, 20]: (1) individuals experienceranges in the extents of temporal changes in relative

Table 1 Spearman’s correlation based on pairwise weightedUniFrac distance within individual over time

Forehead Leftforearm

Rightforearm

Leftpalm

Rightpalm

Foreheada 0.479 0.268 0.314 0.372

Left forearm 0.611 0.542 0.454

Right forearm 0.525 0.467

Left palm 0.539aAll correlations statistically significant (FDR-adjusted p < 0.001)

Leung et al. Microbiome (2018) 6:26 Page 4 of 15

abundance of genera and overall microbial diversity andcommunity composition, (2) time-decay relationships inmicrobiota are present but not a ubiquitous property onskin, and (3) within the same individual, multiple skin sitespresent significant correlations in the extent of microbiotavariations at any two time points, and (4) there exists sig-nificant correlations between the extent of temporal varia-tions between different skin sites. Therefore, despiteknown differences in microbial community compositionsacross continental populations [5, 11, 13–15, 30], sometemporal properties of cutaneous microbiota may be moreuniversal. Such knowledge regarding the behaviors of mi-crobial communities within and between individuals overtime may provide fundamental basis for subsequent andmore focused microbiota analyses in special populationsaround the globe, such as those adopting different lifestylesand are living in drastically different environments [13].

Dynamics of microbial networks are individualized inassociating taxa and overall network structureWhile the dynamics of microbial diversity and communitycomposition within individuals have been examined aboveand in previous works [7, 10, 17, 20], little information iscurrently available on the temporal properties of microbial

correlative association networks. As correlative associationmay represent ecologically significant relationships betweenmicrobial constituents, tracking correlations over time ineach individual will allow the assessment on the factorsgoverning network structure property at a personal level.Here, insights into SParse InversE Covariance Estimationfor Ecological Association Inference (SPIEC-EASI) [39]were used to (1) identify co-abundance and co-exclusionrelationships between specific taxa within individuals and(2) determine whether within-individual network structuredynamics can potentially be explained by individual, house-hold, or temporal factors.Within an association network for each individual

over the course of 1 year (networks of individuals inWKS are presented as examples in Fig. 2; networksof remaining individuals in cohort are presented inAdditional file 12: Figure S8, and Additional file 13:Table S5), positive correlations represent an averageof 82.2% (71.0–92.2% for 24 individuals) of all associ-ations, consistent with previous works demonstratingthat the majority of significant correlations betweenskin sites are positive [22, 40]. Intra-genus correla-tions OTU pairs are more likely to be positive,whereas OTUs of different genera may be involved in

Fig. 2 Microbial association network for individuals of household WKS over the period of 1 year. Significant correlative associations between OTUs(represented by nodes) within each individual determined based on the SPIEC-EASI pipeline. Correlations between OTUs can be positive (representedby blue edges) or negative (represented by red edges). SPIEC-EASI correlations with magnitude of < 0.05 are not represented in figure. OTUs belongingto one of top 20 taxonomic families are color-coded, whereas OTUs of other families are grouped into “ minor/unclassified” group, represented by darkgray nodes. Networks of individuals within WKS presented here as a visual example of the differences in network structures. Network representationsof remaining individuals are included in Additional file 12: Figure S8, and a full list of significant correlative associations is presented in Additional file 13:Table S5

Leung et al. Microbiome (2018) 6:26 Page 5 of 15

both positive and negative correlative associations(Fig. 2, Additional file 12: Figure S8, Additional file 13:Table S5, and Additional file 14: Figure S9). A taxo-nomically diverse collection of OTUs are involved in sig-nificantly strong associations, suggested by the largenumber of OTU nodes classified as “minor/unclassified.”Moreover, most of the highly connected nodes (i.e., keyhub OTUs) are not necessarily the most abundant taxa(Additional file 13: Table S5). Relatively minor and raremicrobial members within the microbiota have been ap-preciated as possibly important drivers of communitycomposition dynamics [41]. Here, we complement ourprevious works demonstrating that these rare taxa are notmerely present on skin [5], but may engage in correlativerelationships with other microbial members, and are po-tentially central members in the maintenance of associ-ation network structure [42]. Individuals within aresidential unit do not share similar hub OTUs, suggestingthat microbial members central to association structuresare not strongly influenced by household factors.While strong positive associations are detected within

OTUs of Enhydrobacter across individuals of differenthouseholds (QB3z and SW3z), this genus is also involvedin positive associations with OTUs of other genera (OTU_4of Enhydrobacter with OTUs of Chryseobacterium andAcinetobacter within households of STW and TMb,respectively Additional file 14: Figure S9). Consistent withour previous work [5], Enhydrobacter is negatively associ-ated with OTUs of Streptococcus (HFC3z, Additional file 14:Figure S9). OTUs of Enhydrobacter and Corynebacteriumcan be involved in both positive and negative associations(within individual TMb3x), demonstrating that species andstrains of Enhydrobacter and other genera deserve furtheranalyses in not only its physiology on skin but also theirpotential interactions within the skin microbiota.With the exception of individual MOS3x, most of the as-

sociations between OTUs of Propionibacterium andStaphylococcus are positive correlations. Propionibacteriumacnes and Staphylococcus epidermidis (the dominantStaphylococcal species on skin) can be mutually inhibitory,but genetic variations between strains influence the extentsto which one species affect the physiology and survival ofthe other [43]. Previous species-level analysis ofStaphylococcus in our cohort reveals that as detected insome healthy individuals in the western world [18], mostOTUs of this genus are in fact Staphylococcus aureus [5].Intriguingly, S. aureus has been shown to respond to mole-cules secreted by P. acnes at particular conditions [44].While correlation analyses act as gateways for identifyinginteractive relationships between microbial members, itprovides no information on the environmental and strainphysiological states necessary for such interactions tooccur. Coupled with the recent identification of separateevolutionary patterns between continental strains of S.

aureus [45], the results here again beckon the need forshotgun metagenomics sequencing to examine microbialassociations at higher resolution, not only to understandspecies- and strain-level patterns in microbial associativerelationships but also the metabolic and physiological basesfor such relationships [10].Following the description of taxa within association net-

works, we assessed the dynamics of association networkstructures within individuals over the four seasons by gen-erating a network for each season within every individual.Graphlet correlation distance information between networkpairs was used to visualize similarities in graphlet proper-ties between association networks in a multidimensionalscaling (MDS) plot (Fig. 3). Graphlet MDS ordination sug-gests that the dynamics of the association networks arehighly personal and do not appear to be governed by sea-sonal or household factors considered in the study. Simi-larly, the dynamics of the association network structure, asmeasured by node degree distribution (Additional file 15:Figure S10a) and natural connectivity (Additional file 15:Figure S10b-c), also appear to be individualized. Microbialassociation network structures have been analyzed andcompared between different natural and human ecosys-tems [5, 22, 39, 40, 46], but not between individuals, norover multiple time points within individuals. Here, we sur-mise that the temporally personal nature of the human skinmicrobiota is not merely limited to its community diversityand composition [10, 20] but extends to the potential eco-logical interactions within its members within this micro-biota. Whether these personal differences in associationnetwork dynamics play roles in differences in susceptibil-ities to conditions associated with skin microbiota dysbiosisis unclear, but our association network findings here neces-sitate a more individual and egocentric approach in thefuture by linking personal attributes to microbial associa-tions, and whether the nature of changes in association net-works over time can act as indicators or predictors forcutaneous health and disease. Our observations here alsoraise the question of, despite the diversity observed acrossindividuals and over time, whether a core microbial associ-ation network for the skin microbiota exist across healthyindividuals at a time and across time within an individual[22], and how this core network is affected by the over-growth of opportunistic pathogens associated with diseaseonsets [18]. These valuable questions should be addressedin future large-scale skin microbiota investigations.

Roles of cohabitation on microbiota changes withinindividuals over timeMicrobiota within individuals are more similar than thosebetween non-cohabitants regardless of season (Fig. 4 andAdditional file 16: Table S6), which is similar to ourprevious study [5] based on a single time point andconglomerating all subjects as a unit. However, depending

Leung et al. Microbiome (2018) 6:26 Page 6 of 15

on the individual and/or time point, the similarity inmicrobiota between a resident and his/her cohabitant maynot be significantly greater than that between that residentand non-cohabiting individuals (by post hoc test indicatingno significant differences in average pairwise weightedUniFrac distances between cohabitants and betweenhouseholds for a particular individual, Additional file 16:Table S6). Conversely, in other instances, the microbialassemblage within a resident is not significantly moresimilar than that between his/her cohabitants (by post hoctest indicating no significant differences in average pairwiseweighted UniFrac distances between within individual andbetween cohabitants, Additional file 16: Table S6).Therefore, the effects of cohabitation on microbiotasimilarities between individuals may be more personal andcomplicated than previously appreciated. Song et al. [23]report that cohabiting couples share more similarmicrobiota compared to between parents and theirchildren and that dogs may be a factor in homogenizingskin microbiota between cohabitants. In contrast, a morerecent study shows lack of microbiota differences betweencohabitants of different relationships [19]. Our cohort donot present enough individuals to assess how particular

relationships between household members affect individualmicrobiota over time, and how domesticated animalscontribute to the overlapping of microbiota betweencohabitants over time. Nonetheless, given the lack ofconsistency over seasons in how more or less similarcohabitants’ microbiota are between different individualsand households in our cohort, we hypothesize that similar-ity between microbiota of cohabitants is more complexthan the cohabitants’ relationships alone. Alternatively, spe-cific interactions between cohabitants, differences in skinphysiologies, and lifestyle [23, 25] between different occu-pants within a household likely play more prominent rolesin determining each occupant’s microbiota, how variablethe microbiota is within the occupant, how similar themicrobiota of his/her cohabitants are, and whether suchsimilarities change over time.The contribution of cohabitants’ skin microbiomes as

potential sources of an individual’s microbial assemblagein each season was assessed using Bayesian SourceTrackeranalysis [47]. For individuals with cohabitants (i.e.,excluding households ADMa, FH, HFC, and HHawhere no cohabitant microbiota data is available), the pro-portions of each subject’s microbiome coming from

Fig. 3 Multidimensional scaling plot (MDS) of graphlet correlation distances between microbial association networks of individuals over fourseasons. Network structures (each network represented by a data point) in each season that are closer together along the MDS axes have moresimilar association graphlet structures. The MDS result is separated into four plots for visual ease, and MDS axes are identical in the four plots, asordination was performed with data points from all four plots included for analysis. Points are color-coded by household and shaped accordingto individuals within the households

Leung et al. Microbiome (2018) 6:26 Page 7 of 15

cohabitants range from 7.0% (SW3y sink in winter) to94.2% (WKS3x sink in summer) (Fig. 5). No one seasonappears to be associated with an increased sourcing of in-dividuals’ microbiomes from cohabitants across house-holds, suggesting that the potential effects of cohabitantsin sourcing one’s skin microbiome has minimal temporaleffect within the year of sampling. In contrast, the overallproportions and dynamics of cohabitants’ microbiome aspotential sources of each individual’s skin microbial com-munity are personal in nature. In addition, there is anoverall significant and positive correlation betweenthe proportion of one’s skin microbiome potentiallysourced from cohabitants and the number of cohabitantsthat sink subject is living with (Kendall’s τ = 0.210, p = 0.01).However, this can merely be due to the increased numberof cohabitant samples as sources available for analysis.Nonetheless, in all households, the proportion of unknownsources is low (average proportion of 3.3% within an indi-vidual at a given time point). This suggests that the micro-bial assemblages across our subjects overall are relativelysimilar, and any individual- and household-based differ-ences may be due to microbial taxa that are low inabundances. Given that this cohort is a relatively small col-lection of cohorts within a single city, a similar analysis canbe performed in cohorts across continents to testwhether global skin microbiota, which are known topresent community differences [5, 11, 13–15, 30],present similar extents of source overlap over time,so as to potentially infer the size of the global skinpan-microbiota [5, 48].

Roles of occupancy on degree of microbiota changeswithin individuals over timeMembers within a household do not appear to sharesimilar signatures of CV in relative abundance of specificgenera, depending on the genus and skin site. However,significant correlations can be observed between CVs ofrelative abundance and household occupancy. Specifically,individuals within households of higher occupancy are as-sociated with greater CVs of Propionibacterium andAcinetobacter over the period of the year (Additional file 17:Figure S11a-c). Conversely, a significantly negative correl-ation, as shown for Bacillus on palms, suggests that indi-viduals with fewer cohabitants tend to have greater ratesof changes in relative abundance of this genus(Additional file 17: Figure S11d). In addition to changes inrelative abundance of specific genera, individuals residingin households with higher occupancy also present higherCVs of Shannon diversity on left forearm but not for theright forearm and other sites (Additional file 17: FigureS11e). The significance on the left forearm is intriguingand may perhaps be related to the combined effects ofhandedness (since all but one individual in this study areright-handed) and environmental exposure. However, thisis speculative, and additional investigations in controlledsettings will be required to assess the roles of these effects,and with a focus on how cohabitation, handedness, andenvironmental exposure affect the temporal dynamics ofskin microbiota. Thus, it is currently not knownwhether the different correlation trends in differentskin sites are ecologically significant. Metagenomic

Fig. 4 Density plots of pairwise UniFrac distances between samples of the same individuals (green), between cohabitants (orange), and betweennon-cohabitants (purple) across seasons. Density plots are faceted by household and season. Households with data from only one occupant(ADMa, FH, HFC, HHa) do not have cohabitant curves

Leung et al. Microbiome (2018) 6:26 Page 8 of 15

shotgun sequencing may prove beneficial inexplaining any potential ecological and physiologicalrationale for the correlative observations here.To examine whether the number of cohabitants within

a household is also associated with variations in commu-nity composition of the skin microbiota of each occupantand site, Kendall τ correlation was performed between themean UniFrac distances for a particular site within anindividual between any two seasons, and the number ofoccupants present in his/her household. Forehead and leftforearm sites from individuals living in residences ofhigher occupancy show more temporally variable micro-bial communities over the course of the year(Additional file 17: Figure S11f).Overall, the number of residents within a household ap-

pears to be associated with changes in the dynamics of thepersonal skin microbiota over 1 year at multiple levels: (1)the extent of variation in the relative abundance of specificcolonizers (CVs of Propionibacterium, Acinetobacter, and

Bacillus relative abundance across multiple skin sites), (2)the variation in community diversity (as determined byCVs of Shannon diversity within each individual on fore-arm sites), and (3) the variation in community structure(through average weighted UniFrac distances betweenforehead and left forearm sites of the same individualsover seasons). Our households are limited to a maximumnumber of six occupants, and it is currently not knownhow residences with more individuals, or other built en-vironment settings (such as workplaces and other publicspaces of higher occupancy) would affect one’s skin micro-biota and its dynamics.However, it is unlikely that occupancy directly shapes

microbiota changes within occupants, as individuals livingin different households of the same occupancy still havedifferent microbiota and variable CVs. Instead, we proposethat the number of residents within a household indirectlyaffects an occupant’s microbiota dynamics by how anoccupant interacts with the cohabitants and their

Fig. 5 Estimated proportions of cohabitants’ skin microbiota as potential sources of an individual’s skin microbiota. Source prediction are groupedby individual and season. For a given individual, a microbiota source can be from cohabitants (red) or from non-cohabitants (green). Individualsfrom households ADMa, FH, HFC, and HHa either live alone or do not have cohabitant microbiota data. Microbiota that cannot be sourced withconfidence are indicated as “Unknown Sources” (blue). Source proportion estimated using the Bayesian SourceTracker analytic tool [47]

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microbiota. A household with more people may facilitateincreased contact, which may facilitate direct transfer ofpersonal skin taxa and potentially pathogenic microorgan-isms [12, 24]. Alternatively, occupants may releasepersonal microbiota into the residential air and ontosurfaces, and microbial members may subsequently bepicked up by its cohabitants through passive exposure oractive surface touching [21, 32]. We suggest that over singleor multiple time points, the relationships between one’sown microbiota and that of its cohabitants and others (thatis, how similar or different their microbiota are, Fig. 4 andAdditional file 16: Table S6) depend on the biogeographical(across skin sites within a time point) and temporal (withina site over multiple time points) variability of a person’smicrobiota (which is in itself now known to be highlypersonal [10, 20]) and the nature of interactions betweenthe person and his/her cohabitants. Individual factors, inaddition to household occupancy and cohabitation, may asa result influence the temporal stability of one’s baselinemicrobiota over time, which is consistent with our pairwiseUniFrac (Additional file 16: Table S6) and SourceTracker(Fig. 5) observations. Therefore, occupancy and cohabit-ation represent part of a collection of household properties,along with living conditions and lifestyles [15] that mayinfluence the nature and the extent of change in one’s skinmicrobiota over time. In addition, given that populationdensity in HK is among the highest in the world, it iscurrently not known how household properties that takeinto account the crowdedness of households (such asoccupant density or privacy indexes as defined previouslyfor microbiota of residences [49]) will influence skinmicrobiota of residents. We believe that separating thesehousehold properties, perhaps via controlled chamber andinoculation experiments [7, 17], will be crucial in specifyingthe relative proportion of contribution of each householdfactor in shaping an individual’s microbiota dynamics.

ConclusionWhile our findings corroborate previous western studiesin identifying the personalized properties of skin micro-biota dynamics, we have highlighted in this study pos-sible associations between occupancy to multiple aspectsof microbiota dynamics within individuals, including forthe first time, the changes of microbial association net-work structures within every individual over 1 year. Ourfindings underscore the need for close examination onindividual physiological and lifestyle properties inexplaining changes in cutaneous microbiota but also anin-depth assessment of the person’s living environment,including number of cohabitants and their interactionsover time. Also, while a daunting task, a more individu-alistic approach may therefore be beneficial to determinefactors governing changes in microbial association net-work structures within an individual, thereby gaining

understanding on properties associated with network dy-namics associated with adverse skin conditions.Finally, there is little information regarding whether

individuals suffering from cutaneous ailments presentdifferent temporal patterns on their skin microbiotafrom that of healthy individuals, in ways similar to howgut microbiomes of individuals suffering from variousgut conditions have distinctive microbiota temporaltraits [50]. Data presented here therefore can act ascommunity-level baseline in understanding how themicrobiota dynamics of healthy individuals comparewith those with various skin conditions. Future longitu-dinal and comparative microbiota works will potentiallyprovide perspectives on whether disease onset, progres-sion, and recovery are related to changes in microbiota-host relationships over time and identify host and otherfactors that may be associated with these changes.

MethodsCohort characteristics and samplingA total of 480 skin samples from this study are part of alarger seasonal analysis of skin, air, and surface micro-biomes of Hong Kong residences [21], and a continuationof previous single-season skin microbiota works [5, 6].Ethics approval for subject sampling was granted by theCity University of Hong Kong Ethics Committee(reference number 3-2-201,312 (H000334)). Afterbeing informed about the nature of the study, as wellas their roles and responsibilities as subjects, writteninformed consent was given by all individuals.From 24 healthy individuals (coded one of 3u to 3z) of

11 households (ADMa, FH, HFC, HHa, MOS, QB, STW,SW, TK, TMb, WKS), samples were collected in January(winter), April/May (spring), August (summer), andNovember/December (autumn) of 2014 (Additional file 18:Table S7). All samples were collected between 19:00 and20:00, as individuals were most likely to be at home duringthose times and to minimize the disruption caused to theindividuals due to sampling. For each individual, five skinsamples (forehead, left/right forearm, left/right palm) werecollected at his/her residence by samplers wearing glovessterilized with 70% ethanol to minimize contamination orskin taxa transfer between sampler and the subject. Allsubjects included this study are ethnically Chinese and arelong-term residents of Hong Kong. Subjects of this studyhad not taken antibiotics and antifungals at least 3 monthsprior to each sampling episode. The individuals in thisstudy were living in households throughout rural andurban areas of Hong Kong to cover a broad localgeographical scope. Individuals and households wereselected to cover a range of age and lifestyle choices suchas smoking, pet ownership, and allergies. All householdsinvolved in this study did not use pesticide or havepurchased new furniture during the course of the

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sampling periods. While antibiotic ingestion may or maynot have an effect on skin microbiota dynamics [19, 20],we note that all subjects had not taken antibiotics 1 monthprior and after each sampling period. Subjects wereinstructed to not wash their hands or shower immediatelyprior to sampling. Samples were collected as previouslydescribed [5]. Briefly, skin samples were collected usingsterile cotton swabs moistened with 100 μL swab solution(0.15 M NaCl and 0.1% Tween 20) [25], and each samplewas collected using a back-and-forth swabbing motion for15 s. Samples were stored in − 80 C within 1 h of samplingand were stored until genomic DNA (gDNA) extraction.

Genomic DNA extraction, 16S rRNA gene amplificationand sequencingFollowing sampling, gDNA was extracted using thePowerSoil DNA Isolation Kit (MO BIO Laboratories,Inc., Carlsbad, CA, USA), with modifications asdescribed previously [5]. Sterile cotton swabs notexposed to skin surfaces were included in the extractionprocess as negative controls to account for possible con-tamination. Purified gDNA samples, including negativecontrols, were sent to Health Genetech Corporation(Taoyuan City, Taiwan) for PCR, library preparation, andsequencing. PCR, library preparation, and IlluminaMiSeq sequencing were prepared as described [5, 51].Briefly, the 515f/806r primer pair was used to target andamplify the V4 hypervariable region of the 16S rRNAgene [29]. PCR amplification was performed in a 20-μLreaction volume containing 10 μl 2× Phusion HF mastermix (New England BioLabs, Ipswich, MA, USA), 0.5 μMeach forward and reverse primer, consisting of customizedbarcodes present on both primers for multiplex sequen-cing, and 50 to 150 ng DNA template. The PCR condi-tions consisted of an initial of 98 °C for 30 s, followed by30 cycles of 98 °C for 10 s, 54 °C for 30 s, and 72 °C for30 s, as well as a final extension of 72 °C for 5 min. Positiveamplification was verified by agarose gel electrophoresis.Amplicons in triplicates were pooled and purified usingAMPure XP beads (Agencourt, Brea, CA, USA) and quan-tified using a Qubit double-stranded DNA HS assay kit ona Qubit fluorometer (Invitrogen, Carlsbad, CA, USA), allaccording to the respective manufacturers’ instructions.For library preparation, Illumina adapters were attached toamplicons using the Illumina TruSeq DNA samplepreparation kit v2. Purified libraries were applied forcluster generation and sequencing on the MiSeq platformusing paired-end 150-bp reads.

Sequence analysisA total of 12,212,096 forward sequences in .fastq formatunderwent quality filtering using the “fastq_filter” com-mand in USEARCH [52], based on a maximum expectederror of 1 error/read, reads trimmed to a length of

139 bp and shorter reads removed. Eighty percent of reads(9,792,397 reads) were of acceptable quality. Filtered readswere then de-multiplexed and binned into OTUs usingUPARSE with 97% sequence identity threshold [53].OTUs were provided with taxonomic information usingthe “assign_taxonomy.py” command in QIIME [54] basedon the Greengenes reference database (May 2013 version,99,322 sequences). Chimeras were detected in UCHIME2(high-confidence mode) against the Greengenes databaseto minimize risk of identifying false-positive chimeraOTUs [55]. OTU lineages present in an average of > 5% ofreads in negative controls (each control averages 20,000reads and 600 OTUs) were considered contaminants andwere removed from all samples. OTUs classified as chlo-roplasts and mitochondria were also removed. Followingread filtering and OTU removal, a total of 8,093,026 readsare retained for community analyses described below. Fol-lowing quality control and OTU taxonomic classification,archaeal OTUs represent ~ 0.3% of reads detected.

Bioinformatics analysesThe coefficient of variation (CV) of relative abundancefor top genera for each individual over the four seasonsis calculated by the standard deviation of the generawithin that individual, divided by the mean of the relativeabundance of that genera in the individual. A low CV rep-resents a relatively stable relative abundance for that givengenus on a given individual, and high CV represents lessstable relative abundance. Total Shannon (alpha-) diversityfor each sample was estimated using the “breakaway”package (version 4) in R available from CRAN [56].Shannon diversity values presented in Additional file 7:Table S3 are average values following 999 reiterations.Similar to previous skin microbiota works [10, 20], the CVfor Shannon diversity within each individual, representingthe change of within-sample microbial diversity of an indi-vidual over the four seasons, is calculated by the standarddeviation of the Shannon diversity values for that individ-ual, divided by the mean of the Shannon diversity of thesame individual. Weighted UniFrac distances were com-puted using the QIIME script “beta_diversity.py.” Analysesof similarities (ANOSIM) were computed on based onβ-diversity data using “vegan” package in R. ThePERMANOVA pseudo-F statistic and significance werecalculated using QIIME’s “compare_categories.py” script(with 999 permutations) based on the weighted UniFracdistance matrix generated from QIIME. Differentialabundance of OTUs between sample groups wereperformed with DeSeq2 using the QIIME script“differential_abundance.py,” performed separately foreach individual and each season pair. OTUs that showedsignificant DeSeq2 results in more than half of the individ-uals for a given season pair were included for negativebinomial and zero-inflated negative binomial mixed model

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analyses (“glmmADMB” package in R) to confirm signifi-cant differences in abundance for these OTUs, usingseason as a fixed effect and count data as response variable.Variations between individuals, skin sites, and sequencingbatches were considered as random effects. OTUs that ap-pear in less than 20% of samples, and OTUs with less than100 reads were not included for the analysis. OTUs of topgenera that were detected at an average of ≥ 1% relativeabundance and were present across all samples were sub-jected to oligotyping (version 2.1 from http://merenlab.org/software/oligotyping/) [57]. To remove oligotypeswith low read counts, a minimum substantive abundance(M) of 10 was adopted. A linear mixed effects model usingthe R package “lme4” was used to determine whetherShannon diversity differences can possibly be driven byseasonal, individual, and household factors, with individ-ual, site, household, and/or sequencing batch differencesas random effects when not considered as a predictor ef-fect. The analysis of variance (ANOVA) on the hypothe-sized and null models was performed to determinestatistical significance in Shannon diversity differences foreach predictor factor.

Microbial source-tracking analysisTo examine the roles of cohabitants as potential microbialsources of an occupant’s skin in each residence and timepoint, Bayesian SourceTracker [47] analysis was used toestimate the proportional contribution of the cohabitants’skin microbiome to the individual microbiome at thegiven season. OTUs present in less than 10% of thesamples were excluded for the analysis. SourceTrackerpredictions were generated for each individual across fourseasons, with the skin samples of each individual in oneseason as the sinks, and the skin samples of the remainingindividuals in the same season as potential sources. Theresults were presented as the source proportions fromcohabitants, non-cohabitants, and unknown sources basedon whether the sink and source communities can besourced from the same household.

Microbial association network analysisSParse InversE Covariance Estimation for EcologicalAssociation Inference (SPIEC-EASI) was used to assesspotential ecological associations between microbial taxa.SPIEC-EASI has been recently applied to other microbiotainvestigations [58, 59] and is currently one of the preferredmethods for association network analysis, as it is robust toissues of compositional bias, conditional independence,and dimensionality, all properties commonly encounteredin microbiota data [39]. Two sets of association networkanalyses were performed, where (1) microbiota data for allseasons within an individual was combined (to assess foroverall microbial associations within an individual, set of24 networks), and (2) where microbiota data for each

season within each individual was analyzed separately (toassess for dynamics of association networks within indi-viduals, set of 96 networks). For each group, abundancedata over different samples underwent centered-log ratiotransformation. OTUs with less than 150 and 10 readswere not included in the first and second analysis, respect-ively. Based on transformed data, the Stability Approachto Regularization Selection (StARS) method [60], suitablefor high-dimensional data, was used to infer the networkstructure, employing the node-based neighborhood selec-tion procedure, with a minimum lambda ratio of 0.01 andreiteration of 50 times as recommended [39]. Networks asshown in Fig. 2 were constructed with Cytoscape(version 3.4.0) [61].Using SPIEC-EASI, general network structural attributes,

including degree distribution (distribution of the numberof significant associations (i.e., edges) per each OTU (i.e.,node), and natural connectivity (a proxy for networkstability and robustness to targeted node removal, byassessing the extent of alternative paths present betweenany two nodes) for each network were also determined.Natural connectivity for network within each individualand season was assessed based on node removal of eitherdecreasing node betweenness centrality, which is ameasure of the paths between a node to all other nodes,or decrease node degree, which is a measure of the numberof edges a particular node contains. Natural connectivity ispresented as the proportion of the total network size, and ahigher natural connectivity represents a more stable androbust association network upon node removal. Graphletcorrelation distance approach as described by Yaveroğluand colleagues [62] was also performed on the set of 96networks to compare the roles of season, household, andindividuals in shaping association network structure.Briefly, each network was broken down into subgraphs(graphlets), each consisting of up to four nodes, and per-formed the graphlet frequency distribution of each nodeacross networks. A graphlet correlation matrix wasgenerated for each of the 96 networks based on pairwiseSpearman correlations of the 11 non-redundant orbits ofall nodes. This matrix is then used to calculate the graphletcorrelation distances, and relationships between networkswhich were then visualized into multidimensional scalingplots constructed using R. Each network is represented bya position on the plot, and networks that are closer to eachother in the MDS plot space are considered to be moresimilar in network structure.

Statistical analysesThe nonparametric Mann-Whitney (MW) and Kruskal-Wallis (KW) tests were employed to determine signifi-cance when comparing between two or morecomparison groups, respectively. p values were adjustedfor multiple comparisons using the false-discovery rate

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(FDR) algorithm in R. Potential batch effect between se-quencing runs were considered as a possible confound-ing factor when performing multiple comparisonsthroughout the study (Additional file 19: Table S8) andhave been treated as an extra sample variable along withother sample variables for FDR-multiple comparisonusing “p.adjust” in R. Where indicated in the main text,post-hoc KW pairwise comparison tests for significancebetween individual groups were performed using thekruskalmc function in R package pgirmess (http://cran.r-project.org/web/packages/pgirmess/index.html),following significant KW observations (adjusted p ≤ 0.05).Spearman’s and Kendall’s τ ranked correlations werecomputed using the “cor.test” function in R.

Additional files

Additional file 1: Figure S1. Heat maps based on relative abundance(RA) of top genera across four seasons within each individual, groupedby anatomical site. Top genera with average RA of ≥1% in the datasetare represented. Note the different relative abundance ranges providedfor each genus. Individuals are color-coded on the y-axis according totheir households. (PDF 6992 kb)

Additional file 2: Table S1. Relative abundance and coefficients ofvariations of top genera. Top genera with average relative abundance of≥1% across the dataset are represented. For each genus, individual, andskin site, the CV is represented by the standard deviation of the relativeabundance of that genus divided by its mean relative abundance overthe four seasons. (XLSX 322 kb)

Additional file 3: Figure S2. Coefficient of variation (CV) measurementsbased on relative abundance (RA) of top genera on a) forehead, b) leftand c) right forearm, and d) left and e) right palm sites for eachindividual. CV is indicated by color gradient, and RA for each individual,site, and genus indicated by point size. Top genera are those withaverage RA of ≥1% in the dataset. Individuals are color-coded accordingto households. (PDF 7184 kb)

Additional file 4: Figure S3. Correlation between standard deviationand average relative abundance of genera grouped by individuals.Standard deviation and average relative abundance of the 100 mostabundant genera are plotted, with Spearman’s ρ correlation significantfor all individuals (adjusted-p < 0.05 for all). Each point represents aparticular genus found on either forehead (red), forearm (green), or palm(blue) sites within each individual. Spearman’s correlation and linearregression line calculated and constructed in R. (PDF 2767 kb)

Additional file 5: Table S2. Deseq2 and negative binomial mixedmodel analyses of OTUs showing differential abundance between seasonpairs. (XLSX 203 kb)

Additional file 6: Figure S4. Cohort-wide seasonal differences in relativeproportions of oligotypes for top genera. Oligotypes 2, 1, 1, and 1 of a)Sphingomonas, b) Bacillus, c) Propionibacterium, and d) Staphylococcus,respectively, show cohort-wide differences in relative abundance of differentoligotypes at specific seasons across cohort and skin sites. At the same time,oligotypes 3 and 4, 9, and 5 of e) Enhydrobacter, f) Sphingomonas, and g)Chryseobacterium, respectively, show household-specific differences inrelative abundance of different oligotypes at different seasons across cohortand skin sites. All relative abundance comparisons statistically significantbetween seasons or households (KW p < 0.05 for all oligotypes focused).(PDF 7042 kb)

Additional file 7: Table S3. Shannon diversity across sites, seasons, andindividuals, and diversity comparison between sample groups. Shannondiversity calculated based on “breakaway” package in R [56]. (XLSX 51 kb)

Additional file 8: Figure S5. Coefficient of variation (CV) of Shannondiversity between a) individuals, b) households, and c) age groups,

grouped by anatomical sites. The Shannon diversity CV of particular skinmicrobiota is represented by sizes of circles. For a) individuals arecolor-coded to represent the households as shown in b). (PDF 1863 kb)

Additional file 9: Table S4. Community composition, seasonal, andtime-decay analyses. (XLSX 35 kb)

Additional file 10: Figure S6. Density plots of pairwise weightedUniFrac distances between samples of the same (red) and different (blue)seasons. Density plots faceted according to each individual. Onlywithin-individual pairwise comparisons were included in analysis. (PDF3915 kb)

Additional file 11: Figure S7. Correlations between the pairwiseweighted UniFrac within-site distances and Shannon diversity CV of skin sitesover time within individuals. Spearman’s correlation and linear regressiondetermined and constructed in R. P-values adjusted using false-discovery ratemethod. (PDF 2015 kb)

Additional file 12: Figure S8. Microbial association network for eachindividual over the period of one year. Significant correlative associationsbetween OTUs (represented by nodes) within each individual determinedbased on the SPIEC-EASI pipeline. Correlations between OTUs can bepositive (represented by blue edges) or negative (represented by red edges).SPIEC-EASI correlations with magnitude of < 0.05 are not representedin figure. OTUs belonging to one of top 20 taxonomic families arecolor-coded, whereas OTUs of other families are grouped into“Minor/Unclassified” group, represented by dark gray nodes. (PDF5774 kb)

Additional file 13: Table S5. Significant SPIEC-EASI correlations betweenpairs of OTUs and taxonomic information of hub OTUs. Absolutecorrelations of ≥0.05 are included in the table. (XLSX 232 kb)

Additional file 14: Figure S9. SPEIC-EASI density plot of positive andnegative correlations involving OTUs of the same (orange shade) ordifferent (purple shade) genera, and heatmap plots of pairwise significantcorrelations of OTUs of the top genera within each individual. Onlysignificant correlations with an absolute SPIEC-EASI correlation magnitudeof ≥0.05 are included. (PDF 6984 kb)

Additional file 15: Figure S10. Network structure properties perindividual over four seasons. a) Node degree distribution is plotted foreach individual for winter (blue), spring (green), summer (red), andautumn (black). In households with multiple occupants, distributions fromeach individual are combined into single plots. b-c) Natural connectivityof microbial association network of each individual in winter (blue),spring (green), summer (red), and autumn (black) upon sequential noderemoval in order of decreasing b) node betweenness centrality (i.e. nodeshaving the shortest paths to other nodes removed first) and c) nodedegree (i.e. nodes having the highest number of edges to other nodesremoved first). Natural connectivity, as a measure of a network’s robustnessand stability to node removal, is plotted against removal of up to 80% ofnodes of a given network. Natural connectivity is expressed as the relativeproportion of the size of the original network prior to node removal. (PDF6137 kb)

Additional file 16: Table S6. Pairwise weighted UniFrac distancesbetween samples from the same individual, cohabitants, andnon-cohabitants. (XLSX 49 kb)

Additional file 17: Figure S11. Effect of household occupancy on theextents of changes in skin microbiota within individuals over time.Kendall’s correlation between household occupancy and CVs of relativeabundance for a) Propionibacterium and b) Acinetobacter on forehead, c)Acinetobacter on forearm, and d) Bacillus on palm within an individual.Correlation between household occupancy and e) Shannon diversity CVand f) pairwise weighted UniFrac distances between communities on thesame individual and site over any two seasons. Kendall’s correlation andlinear regression determined and constructed in R. Significant correlationsfollowing false-discovery rate adjustment are in bold (adjusted-p < 0.05).(PDF 4782 kb)

Additional file 18: Table S7. Sample metadata. (XLSX 73 kb)

Additional file 19: Table S8. Weighted UniFrac distance Global Ranalysis for effect of sequencing batch on microbial communitycomposition within a season. (DOCX 43 kb)

Leung et al. Microbiome (2018) 6:26 Page 13 of 15

AcknowledgementsWe thank William Chan for sample processing, staffs of the Health GeneTechCorporation for their expertise in sequencing, and members of the samplingteam (Ellen Li, Fred Kong, Wai Shan Chow, Catherine Chung, Ka Yan Ng, YuetYing Wong, and Flora Yeh) for their assistance. We are grateful for theparticipation of the cohort in this study.

FundingThis research was supported by the Research Grants Council of Hong Kongthrough Project 11211815.

Availability of data and materialsRaw reads in fastq format and metadata file for all samples (includingnegative controls) included in this study have been deposited in the NCBISRA archive as BioProject PRJNA390040. In-house scripts for data analysishave been uploaded onto FigShare (https://figshare.com/articles/Skin_16-S_temporal_microbiome/3410134).

Authors’ contributionsMHYL collected samples, analyzed the data, developed and wrote themanuscript. XT analyzed the data and wrote the manuscript. DW wrote thein-house scripts and provided bioinformatics and technical support. HHLCprocessed samples for sequencing. PKHL guided and assisted in study designand analysis, as well as provided support for writing the manuscript. All au-thors read and approved the final manuscript in its current form.

Ethics approval and consent to participateEthics approval for subject sampling was granted by the City University ofHong Kong Ethics Committee (reference number 3-2-201,312 (H000334)).After being informed about the nature of the study, as well as their rolesand responsibilities as subjects, written informed consent was given by allindividuals.

Consent for publicationConsent was given by subjects to release personal data for publication asneeded.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Received: 11 April 2017 Accepted: 19 January 2018

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