《人体的社交网络——共生微生物》课外文献阅读：Changes in the Eye Microbiota Associated with Contact Lens Wearing

RESEARCH ARTICLE MCITY mBIO Changes in the Eye Microbiota Associated with Contact Lens Wearing (D Hakdong Shin,a Kenneth Price, a, b Luong Albert, a Jack Dodick, Lisa Park, b Maria Gloria Dominguez-Belloa Division of Translational Medicine and Department of Ophthalmology. New York University School of Medicine, New York, New York, USA lentified as a risk factor for the development of eye conditions such as giant papil lary conjunctivitis and keratitis. We hypothesized that wearing contact lenses is associated with changes in the ocular microbi- ota. We compared the bacterial communities of the conjunctiva and skin under the eye from 58 subjects and analyzed samples from 20 subjects(9 lens wearers and 11 non-lens wearers)taken at 3 time points using a 16S rRNA gene-based sequencing tech- nique(V4 region; Illumina MiSeq). We found that using anesthetic eye drops before sampling decreases the detected ocular mi- crobiota diversity Compared to those from non-lens wearers, dry conjunctival swabs from lens wearers had more variable and skin-like bacterial community structures(Uni Frac; P value 3.0). The results indicate that wearing contact lenses alters the microbial g structure of the ocular conjunctiva, making it more similar to that of the skin microbiota. Further research is needed to deter mine whether the microbiome structure provides less protection from ocular infections. IMPORTANCE As in other body sites(i.e, the gut, skin, and mouth), the eye has a normal community of bacteria which are ex pected to confer resistance that provides protection from invaders. However, the eye microbiome has been largely neglected and t is relevant to eye health and understanding eye diseases and to discovery of its functions. This report of a baseline study shows differences in the eye microbiome of contact lens wearers in relation to those of non-lens wearers and has the potential to help future studies explore novel insights into a possible role of the microbiome in the increased risk for eye infections in contact lens wearers. eceived 9 February 2016 Accepted 25 February 2016 Published 22 March 2016 Citation Shin H, Price K, Albert L, Dodick J, Park L Dominguez-Bello MG 2016. Changes in the eye microbiota associated with contact lens wearing. mBio 7(2): e00198-16 doi 10.128/mBo0019816. Editor Janet K Jansson, Pacific Northwest National Laboratory Copyright o 2016 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license Address correspondence to Maria G Dominguez-Bello, Maria. Dominguez-Belloenyumcorg This article is a direct contribution from a Fellow of the American Academy of Microbiology. External solicited reviewers: Jonathan Eisen, UC Davis Genome Center, Anthony St eger, NIH/NEE Jack A Gilbert, Argonne National Laborator n culture-dependent studies, over 50% of swabs from the con- supporting the hypothesis of microbial involvement and interaction junctiva showed growth of skin-like bacteria(mostly coagulase- with host conjunctival epithelium. negative staphylococci, Propionibacterium, and Corynebacterium While several studies have used culture-dependent approaches (1). Ocular bacterial communities have been studied using to demonstrate bacterial contamination of contact lenses(13, 14) culture-dependent methods(2, 3)and, more recently, with 16s little is known about the impact of contact lenses on the structure rRNA gene sequencing in healthy subjects(4)and in people with and function of the microbiota on the ocular surface. In this work, such as Pseudomonas, Bradyrhizobium, Acinetobacter, Brevundi. we compare the microbiota of the ocular surface of lens wearers eye diseases(5, 6). Using sequencing methods, additional bacteria, we using 16s rRNA gene sequencing monas, Aquabacterium, Sphingomonas, Streptococcus, Strepto- surveys phyta, Methylobacterium, Enhydrobacter, Bacillus, and Ralstonia spp, were detected(4-6). As in other body sites, the ocular mi- RESULTS robiota is expected to play a defensive role against colonization of We obtained 7,010,096 sequences(paired end; Phred = Q20)with pathogens in the eye(7). Despite being important in ophthalmol- an average of 21, 569 reads per sample, yielding 11,750 operating ogy, the eye microbiome has been largely neglected, and its func- taxonomic units(OTUs)(11, 700 OTUs without singletons)(see tions remain unknown Table SI in the supplemental material) In the United States, over 30 million people wear contact lenses, The bacterial alpha diversity of the conjunctiva was signifi nearly one-third of the -100 million worldwide(8). Wearing contact cantly higher in subjects sampled at the laboratory without anes- lenses has been identified as a risk factor for eye conditions such as thetic than was seen in the ophthalmologic practice, which used an giant papillary conjunctivitis(9)and keratitis(10-12). The conjunc- anesthetic eye drop (whole-tree phylogenetic diversity [PD] tiva of patients with vernal keratoconjunctivitis(inflammation of the number of observed species; P value =<0.001 [nonparametric eye that involves both the cornea and conjunctiva)showed increased Students t test))(see Fig. SIA in the supplemental material). US- Toll-like receptor 4(TLR-4)levels in relation to those of controls(7), ing an anesthetic eye drop significantly altered microbial commu March/April 2016 Volume 7 Issue 2 e00198-16 msio° mbio. asm. org

Changes in the Eye Microbiota Associated with Contact Lens Wearing Hakdong Shin,a Kenneth Price,a,b Luong Albert,a Jack Dodick,b Lisa Park,b Maria Gloria Dominguez-Belloa Division of Translational Medicinea and Department of Ophthalmology,b New York University School of Medicine, New York, New York, USA ABSTRACT Wearing contact lenses has been identified as a risk factor for the development of eye conditions such as giant papil￾lary conjunctivitis and keratitis. We hypothesized that wearing contact lenses is associated with changes in the ocular microbi￾ota. We compared the bacterial communities of the conjunctiva and skin under the eye from 58 subjects and analyzed samples from 20 subjects (9 lens wearers and 11 non-lens wearers) taken at 3 time points using a 16S rRNA gene-based sequencing tech￾nique (V4 region; Illumina MiSeq). We found that using anesthetic eye drops before sampling decreases the detected ocular mi￾crobiota diversity. Compared to those from non-lens wearers, dry conjunctival swabs from lens wearers had more variable and skin-like bacterial community structures (UniFrac; P value 3.0). The results indicate that wearing contact lenses alters the microbial structure of the ocular conjunctiva, making it more similar to that of the skin microbiota. Further research is needed to deter￾mine whether the microbiome structure provides less protection from ocular infections. IMPORTANCE As in other body sites (i.e., the gut, skin, and mouth), the eye has a normal community of bacteria which are ex￾pected to confer resistance that provides protection from invaders. However, the eye microbiome has been largely neglected and is relevant to eye health and understanding eye diseases and to discovery of its functions. This report of a baseline study shows differences in the eye microbiome of contact lens wearers in relation to those of non-lens wearers and has the potential to help future studies explore novel insights into a possible role of the microbiome in the increased risk for eye infections in contact lens wearers. Received 9 February 2016 Accepted 25 February 2016 Published 22 March 2016 Citation Shin H, Price K, Albert L, Dodick J, Park L, Dominguez-Bello MG. 2016. Changes in the eye microbiota associated with contact lens wearing. mBio 7(2):e00198-16. doi: 10.1128/mBio.00198-16. Editor Janet K. Jansson, Pacific Northwest National Laboratory Copyright © 2016 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Maria G. Dominguez-Bello, Maria.Dominguez-Bello@nyumc.org. This article is a direct contribution from a Fellow of the American Academy of Microbiology. External solicited reviewers: Jonathan Eisen, UC Davis Genome Center; Anthony St. Leger, NIH/NEI; Jack A. Gilbert, Argonne National Laboratory. I n culture-dependent studies, over 50% of swabs from the con￾junctiva showed growth of skin-like bacteria (mostly coagulase￾negative staphylococci, Propionibacterium, and Corynebacterium) (1). Ocular bacterial communities have been studied using culture-dependent methods (2, 3) and, more recently, with 16S rRNA gene sequencing in healthy subjects (4) and in people with eye diseases (5, 6). Using sequencing methods, additional bacteria, such as Pseudomonas, Bradyrhizobium, Acinetobacter, Brevundi￾monas, Aquabacterium, Sphingomonas, Streptococcus, Strepto￾phyta, Methylobacterium, Enhydrobacter, Bacillus, and Ralstonia spp., were detected (4–6). As in other body sites, the ocular mi￾crobiota is expected to play a defensive role against colonization of pathogens in the eye (7). Despite being important in ophthalmol￾ogy, the eye microbiome has been largely neglected, and its func￾tions remain unknown. In the United States, over 30 million people wear contact lenses, nearly one-third of the ~100 million worldwide (8). Wearing contact lenses has been identified as a risk factor for eye conditions such as giant papillary conjunctivitis (9) and keratitis (10–12). The conjunc￾tiva of patients with vernal keratoconjunctivitis (inflammation of the eye that involves both the cornea and conjunctiva) showed increased Toll-like receptor 4 (TLR-4) levels in relation to those of controls (7), supporting the hypothesis of microbial involvement and interaction with host conjunctival epithelium. While several studies have used culture-dependent approaches to demonstrate bacterial contamination of contact lenses (13, 14), little is known about the impact of contact lenses on the structure and function of the microbiota on the ocular surface. In this work, we compare the microbiota of the ocular surface of lens wearers with that of non-lens wearers using 16S rRNA gene sequencing surveys. RESULTS We obtained 7,010,096 sequences (paired end; Phred Q20) with an average of 21,569 reads per sample, yielding 11,750 operating taxonomic units (OTUs) (11,700 OTUs without singletons) (see Table S1 in the supplemental material). The bacterial alpha diversity of the conjunctiva was signifi- cantly higher in subjects sampled at the laboratory without anes￾thetic than was seen in the ophthalmologic practice, which used an anesthetic eye drop (whole-tree phylogenetic diversity [PD], number of observed species; P value 0.001 [nonparametric Student’s t test]) (see Fig. S1A in the supplemental material). Us￾ing an anesthetic eye drop significantly altered microbial commu￾RESEARCH ARTICLE crossmark March/April 2016 Volume 7 Issue 2 e00198-16 ® mbio.asm.org 1 mbio.asm.org on June 29, 2016 - Published by mbio.asm.org Downloaded from

hin et al PD Whole Tree Observed Species Non-lens wearers Conjunctiva +- 3o8:中 Non-lens wearers Skin under he eye Lens wearers g Contact lens 10 20 30 100 Phylogenetic diversity Number of observed specie rarefication to 2,090 reads per sample. The nonparametric P values were calculated using 999 Monte Carlo permutations. + outlier samples excluded from the G FIG 1 Bacterial diversity in the conjunctiva, skin, and contact lens between non-lens wearers and lens wearers. Box plots of alpha diversity were generated with nity composition(linear discriminant analysis [LDA] score= mental material). However, there was no clustering by subject or >3.0; see Fig SIB)and structure(permutational analysis of vari- sampling time points( Fig 2A and B). Moreover, the conjunctival ance PERMANOVA] P value = 3.0; 3.0; Fig. 4). In non-lens wearers, these were de- bacterial diversity in the skin of the face reported by Bouslimani et tected at a higher relative abundance in skin samples than in the al.(16)(see Fig S3A). We consider these good positive controls conjunctiva(except for Lactobacillus)(Fig. 4), suggesting that and are confident in the diversity found in the conjunctiva and these bacteria could be classified as skin bacteria Levels of Haemoph kin under the eye lus, Streptococcus, Staphylococcus, and Corynebacterium were depleted There were no significant differences in bacterial alpha diver- in the ocular microbiota of lens wearers compared to non-lens wear- 4. y between the conjunctiva of lens wearers and that of non-lens ers(LDa score=>3.0; Fig 4). Contact lenses demonstrated ahigher wearers(nonparametric Student's t test; Fig. 1). The microbial relative abundance of Acinetobacter and Methylobacterium than the structures of the conjunctiva and skin under the eye were more conjunctiva(lda score=>3.0; Fig 4 dissimilar in non-lens wearers than in lens wearers(unweighted We compared the conjunctival microbiota with that UniFrac, nonparametric t test P value = 3.0; Fig. 4B) 2 mBio mbio. asm. org March/April 2016 Volume 7 Issue 2 800198-16

nity composition (linear discriminant analysis [LDA] score 3.0; see Fig. S1B) and structure (permutational analysis of vari￾ance [PERMANOVA] P value 0.001; see Fig. S1C), as mea￾sured by unweighted/weighted UniFrac distances. In addition, the use of contact lenses caused only minor changes in the conjuncti￾val microbiota of subjects at the ophthalmology practice (see Fig. S2), supporting the idea of an effect of anesthetic. Due to the apparent perturbation caused by the eye drops, we performed further analyses solely on the 20 subjects sampled in the laboratory without anesthesia. A total of 250 samples obtained in the laboratory (116 conjunc￾tiva, 114 skin under the eye, and 20 contact lenses) were rarefied at 2,090 sequences per sample (see Table S2 in the supplemental material). Notably, the conjunctival samples showed higher alpha diversity than the skin under the eye or contact lenses (P value 0.05 [nonparametric Student’s t test]; Fig. 1). In the same se￾quencing run in which we sequenced the eye project samples, we sequenced samples of the vaginal microbiota from different sub￾jects. The alpha diversity of the vaginal microbiota was similar to that reported by the HMP Consortium (15), and the bacterial diversity in the skin under the eye in our study was similar to the bacterial diversity in the skin of the face reported by Bouslimani et al. (16) (see Fig. S3A). We consider these good positive controls and are confident in the diversity found in the conjunctiva and skin under the eye. There were no significant differences in bacterial alpha diver￾sity between the conjunctiva of lens wearers and that of non-lens wearers (nonparametric Student’s t test; Fig. 1). The microbial structures of the conjunctiva and skin under the eye were more dissimilar in non-lens wearers than in lens wearers (unweighted UniFrac, nonparametric t test P value 0.001; Fig. 2A and B). Consistently, the conjunctival microbiota of lens wearers was more similar to the microbiota of the skin under the eye than was the case with the non-lens wearers (unweighted UniFrac distance, P value 0.001) (Fig. 2A and B; see also Fig. S4 in the supple￾mental material). However, there was no clustering by subject or sampling time points (Fig. 2A and B). Moreover, the conjunctival microbiota in lens wearers was more similar to human skin mi￾crobiota from a previous study (15) than to that in non-lens wear￾ers (Fig. 3). Weighted UniFrac distances depicted on principal coordinate analysis (PCoA) plots also supported these results (see Fig. S4). Lens wearers also had higher interindividual variability in their ocular microbiota than non-lens wearers (unweighted Uni￾Frac, nonparametric P value 0.001; Fig. 2C and D). The conjunctival microbiota of non-lens wearers was influ￾enced by gender (PERMANOVA P 0.001; see Fig. S5A and B in the supplemental material). Numbers of Acinetobacter organisms and of members of family Enterobacteriaceae were increased and those of Anaerococcus were depleted in the ocular microbiota of female subjects compared to male subjects (LDA score 3.0; see Fig. S5C and D). Regardless of gender, lens wearers had a skin-like conjunctival microbiota compared to non-lens wearers (see Fig. S5E and F). Compared to levels seen with non-lens wear￾ers, the ocular microbiota of lens wearers was enriched in Pseu￾domonas, Acinetobacter, Methylobacterium, and Lactobacillus (LDA score 3.0; Fig. 4). In non-lens wearers, these were de￾tected at a higher relative abundance in skin samples than in the conjunctiva (except for Lactobacillus) (Fig. 4), suggesting that these bacteria could be classified as skin bacteria. Levels ofHaemophi￾lus, Streptococcus, Staphylococcus, andCorynebacteriumwere depleted in the ocular microbiota of lens wearers compared to non-lens wear￾ers (LDA score3.0; Fig. 4). Contact lenses demonstrated a higher relative abundance of Acinetobacter and Methylobacterium than the conjunctiva (LDA score 3.0; Fig. 4). We compared the conjunctival microbiota with that in the skin under the eye. In relation to the skin, the conjunctiva of normal eye (non-lens wearers) had higher abundances of Haemophilus, Neisseria, Streptococcus, Staphylococcus, Rothia, and Corynebacte￾rium and lower abundances of Pseudomonas, Acinetobacter, Sph￾ingobium, and Methylobacterium (LDA score 3.0; Fig. 4B). FIG 1 Bacterial diversity in the conjunctiva, skin, and contact lens between non-lens wearers and lens wearers. Box plots of alpha diversity were generated with rarefication to 2,090 reads per sample. The nonparametric P values were calculated using 999 Monte Carlo permutations. , outlier samples excluded from the analyses; **, P value 0.05. Shin et al. 2 ® mbio.asm.org March/April 2016 Volume 7 Issue 2 e00198-16 mbio.asm.org on June 29, 2016 - Published by mbio.asm.org Downloaded from

Altered Eye Microbiota by Wearing Contact Lenses A Non-lens wearers Time points O Conjunctiva O skin under the eye争 3o8:中 Pc1(12% B Non-lens wearers vs Lens wearers D g farers) Non-lens wearer Conjunctiva ]: n-s wEarers Non-ens wearers Lens wearers FIG 2 Beta diversity in conjunctiva, skin, and contact lens samples between non-lens wearers and lens wearers. Unweighted Unifrac distances were used to evaluate diversities between samples. (A)PCoA plot of bacterial communities by body site, time, and subject in non-lens wearers(left)and lens wearers(right) PCI Box plots of intragroup and intergroup di in the ocular microbiota of non-lens wearers and lens wearers. *+, Pvalue= <0.001 However, regardless of wearing or not wearing lenses, Source O Conjunctiva(Non-lens wearers)(n=64) Tracker analyses using the v4 region from data generated in a O Conjunctiva( Lens wearers)(n=52) previous study of the skin microbiome(16)showed that the con- Skin under the eye(n=114) junctival microbiota of both lens wearers and non-lens wearers had higher proportions of hand -like bacteria than of face-like bac teria(P<00001; see Fig. S3B in the supplemental material) O Skin HMP(n=2, 199 There were no significant differences in bacterial diversity and O Stool HMP(n=437) composition between the conjunctival microbiota and skin mi- O Vaginal HMP(n=564) crobiota at different time points(see Fig. S6 in the supplemental material) Predictive functional profiling showed that the conjunctiva of lens wearers had a higher proportion of bacterial genes related to ell motility and xenobiotic biodegradation than the conjunctiva of non-lens wearers. On the other hand, the conjunctival micro- biota of non-wearers had higher relative content of genes related to genetic processing and nucleotide metabolism(see Fig S7 in lemental material DISCUSSION Wearing contact lenses is a known risk factor for development of microbial keratitis and other inflammatory eye conditions(17, 18). Several researchers have tried to identify the causative organ- PC1(16%)ism of these contact lens-related diseases using culture-dependent methods(1, 18). To the best of our knowledge, this was a pioneer n, and contact lens samples with ing study in identifying the structure of the microbiota from con- weighted Un ots (green, purple, cyan, and pink) indi- tact lenses and conjunctiva of contact lenses wearers using 16S and large dots(red, orange, yellow, and rRNA gene sequencing. While mostly illustrated by results of blue) indicate studies of the gut, the concept of colonization resistance, a protec March/April 2016 Volume 7 Issue 2 e00198-16 msio° mbio. asm. org3

However, regardless of wearing or not wearing lenses, Source￾Tracker analyses using the V4 region from data generated in a previous study of the skin microbiome (16) showed that the con￾junctival microbiota of both lens wearers and non-lens wearers had higher proportions of hand-like bacteria than of face-like bac￾teria (P 0.0001; see Fig. S3B in the supplemental material). There were no significant differences in bacterial diversity and composition between the conjunctival microbiota and skin mi￾crobiota at different time points (see Fig. S6 in the supplemental material). Predictive functional profiling showed that the conjunctiva of lens wearers had a higher proportion of bacterial genes related to cell motility and xenobiotic biodegradation than the conjunctiva of non-lens wearers. On the other hand, the conjunctival micro￾biota of non-wearers had higher relative content of genes related to genetic processing and nucleotide metabolism (see Fig. S7 in the supplemental material). DISCUSSION Wearing contact lenses is a known risk factor for development of microbial keratitis and other inflammatory eye conditions (17, 18). Several researchers have tried to identify the causative organ￾ism of these contact lens-related diseases using culture-dependent methods (1, 18). To the best of our knowledge, this was a pioneer￾ing study in identifying the structure of the microbiota from con￾tact lenses and conjunctiva of contact lenses wearers using 16S rRNA gene sequencing. While mostly illustrated by results of studies of the gut, the concept of colonization resistance, a protec￾FIG 2 Beta diversity in conjunctiva, skin, and contact lens samples between non-lens wearers and lens wearers. Unweighted UniFrac distances were used to evaluate diversities between samples. (A) PCoA plot of bacterial communities by body site, time, and subject in non-lens wearers (left) and lens wearers (right). PC1, first principal component; PC2, second principal component. (B) Box plots of intragroup and intergroup distances in bacterial communities between conjunctiva and skin under the eye. (C) PCoA plot of ocular microbiota in non-lens wearers and lens wearers with bacterial communities of contact lenses. (D) Box plots of intragroup and intergroup distances in the ocular microbiota of non-lens wearers and lens wearers. ***, P value 0.001. FIG 3 Beta diversity in conjunctiva, skin, and contact lens samples with samples from the HMP database. A PCoA plot was generated using un￾weighted UniFrac distances. Small dots (green, purple, cyan, and pink) indi￾cate samples from the HMP database, and large dots (red, orange, yellow, and blue) indicate samples from this study. Altered Eye Microbiota by Wearing Contact Lenses March/April 2016 Volume 7 Issue 2 e00198-16 ® mbio.asm.org 3 mbio.asm.org on June 29, 2016 - Published by mbio.asm.org Downloaded from

hin et al A Enterobacteriaceae: g f Comamonadaceae: g Methylobacterium g Non-lens wearers Lens wearers Conjunctiva Conjunctiva Contact lens n=52 P Proteobacteria: F. Firmicutes: C, Cyanobacteria: A, Actinobacteria Conjunctiva Non-lens wearers oniunctia vs Contact ans Pseudomonas 旦_ Haemophilus g Streptococcus Sphingobium FIG 4 Differences in relative abundances of phylotypes in ocular microbiota between non-lens wearers and lens wearers. Each phylotype(>1% of average relative abundance in groups)is indicated by a different color at the genus level. (A)Bacterial taxon plots at the genus level. (B)Histogram of unique biomarker bacteria in each group. The LDA effect size(>3.0-fold)was used to detect unique biomarkers. tive effect shown by microbiota against invasions of foreign bac- phyta spp (chloroplast DNA, possibly an environmental contami- teria, has been suggested for the eye(7). This would imply that nant) showed a nonsignificant tendency(nonparametric t test; P some impacts on the eye microbiota, such as those related to wear- 0.26)to be higher in the conjunctiva of lens wearers than in that of ing contacts, might decrease the resilience or self-restoring capac- non-lens wearers(Fig 4; see also Fig. S5 in the supplemental mate ity of the ecosystem. Although the data represent only 3 time rial). We would not have detected differences in the conjunctival mi- points, we found relatively high stability of the eye microbiome. crobiota between lens wearers and non-lens wearers in this study if we The healthy eye is constantly wetted by tears, which may provide a hadincluded samples collected from the ocular surface after the use of constant chemical environment topical proparacaine hydrochloride anesthetic, highlighting the im- The bacterial diversity of the conjunctiva is higher than that in portance of sampling microbiome sites with minimal perturbations. the skin. Moreover, conjunctiva had an alpha diversity similar to The anesthetic may have diluted or washed away bacteria from the that of the oral microbiota(see Fig S3 in the supplemental mate- ocular surface rial). These results are remarkable considering the antimicrobial By comparing the microbiota of ocular conjunctiva to that of effects of tear compound skin under the eye, we found that normal Corynebacterium and This study showed that the eye microbiota of lens wearers is dif- Staphylococcus skin bacteria were detected at higher relative abun ferent from that of non-wearers, resembling more closely the micro- dance in the eye than in the skin, consistent with a recent study biota of the skin. The results are consistent with those previously showing the distribution of skin microbiome by body sites(16) found with culture-dependent methods(20-22). Consistently, the Tear lactoferrin plays a role in reducing levels of Staphylococcus presence of Staphylococcus, Propionibacterium, Corynebacterium, Ba- epidermidis biofilms(23). However, in this study, we found that illus, Micrococcus, Rothia(previously assigned to Stomatococcus), and Staphylococcus levels were lower in lens wearers than in non-lens Pseudomonas in cultures from contact lenses has been reported(1). wearers. There are not many reports of studies using molecular We also detected members of other taxa in contact lenses, such as methods in the diseased eye. Lee et al. (6)studied blepharitis using families Oxalobacteraceae and Enterobacteriaceae at a relative abun- and platforms(Vi to V3 16S rRNA genes with Roche-454). 9 Streptococcus, Methylobacterium, and Acinetobacter, and members of different sampling sites(eyelashes and tears), sequencing region dance of >1% in more than half of the samples. In addition, Strepto- results showed that blepharitis patients(n=7) had increased 4 mBio mbio. asm. org March/April 2016 Volume 7 Issue 2 e00198-16

tive effect shown by microbiota against invasions of foreign bac￾teria, has been suggested for the eye (7). This would imply that some impacts on the eye microbiota, such as those related to wear￾ing contacts, might decrease the resilience or self-restoring capac￾ity of the ecosystem. Although the data represent only 3 time points, we found relatively high stability of the eye microbiome. The healthy eye is constantly wetted by tears, which may provide a constant chemical environment. The bacterial diversity of the conjunctiva is higher than that in the skin. Moreover, conjunctiva had an alpha diversity similar to that of the oral microbiota (see Fig. S3 in the supplemental mate￾rial). These results are remarkable considering the antimicrobial effects of tear compounds (19). This study showed that the eye microbiota of lens wearers is dif￾ferent from that of non-wearers, resembling more closely the micro￾biota of the skin. The results are consistent with those previously found with culture-dependent methods (20–22). Consistently, the presence of Staphylococcus, Propionibacterium, Corynebacterium, Ba￾cillus,Micrococcus,Rothia (previously assigned toStomatococcus), and Pseudomonas in cultures from contact lenses has been reported (1). We also detected members of other taxa in contact lenses, such as Streptococcus, Methylobacterium, and Acinetobacter, and members of families Oxalobacteraceae and Enterobacteriaceae at a relative abun￾dance of 1% in more than half of the samples. In addition, Strepto￾phyta spp. (chloroplast DNA, possibly an environmental contami￾nant) showed a nonsignificant tendency (nonparametric t test; P 0.26) to be higher in the conjunctiva of lens wearers than in that of non-lens wearers (Fig. 4; see also Fig. S5 in the supplemental mate￾rial). We would not have detected differences in the conjunctival mi￾crobiota between lens wearers and non-lens wearers in this study if we had included samples collected from the ocular surface after the use of topical proparacaine hydrochloride anesthetic, highlighting the im￾portance of sampling microbiome sites with minimal perturbations. The anesthetic may have diluted or washed away bacteria from the ocular surface. By comparing the microbiota of ocular conjunctiva to that of skin under the eye, we found that normal Corynebacterium and Staphylococcus skin bacteria were detected at higher relative abun￾dance in the eye than in the skin, consistent with a recent study showing the distribution of skin microbiome by body sites (16). Tear lactoferrin plays a role in reducing levels of Staphylococcus epidermidis biofilms (23). However, in this study, we found that Staphylococcus levels were lower in lens wearers than in non-lens wearers. There are not many reports of studies using molecular methods in the diseased eye. Lee et al. (6) studied blepharitis using different sampling sites (eyelashes and tears), sequencing regions, and platforms (V1 to V3 16S rRNA genes with Roche-454). Their results showed that blepharitis patients (n 7) had increased FIG 4 Differences in relative abundances of phylotypes in ocular microbiota between non-lens wearers and lens wearers. Each phylotype (1% of average relative abundance in groups) is indicated by a different color at the genus level. (A) Bacterial taxon plots at the genus level. (B) Histogram of unique biomarker bacteria in each group. The LDA effect size (3.0-fold) was used to detect unique biomarkers. Shin et al. 4 ® mbio.asm.org March/April 2016 Volume 7 Issue 2 e00198-16 mbio.asm.org on June 29, 2016 - Published by mbio.asm.org Downloaded from

Altered Eye Microbiota by Wearing Contact Lenses Staphylococcus, Streptophyta, Corynebacterium, and Enhydrobacter ophthalmology office after utilization of a 50-pl drop of topical 0.5% levels in their eyelashes and tears in relation to controls(n= 4), proparacaine hydrochloride anesthetic Negative-control swabs(n=3 none of which were increased in our lens wearers. In addition to with no sample)were also included. the different sites sampled, their patients were older than ours(59 DNA extraction and sequencing. DNA extraction was carried out 4.5]-year-old patients in our study). More studies are needed to according to the instructions provided by the manufacturer. Extracted g understand which are the opportunistic pathogens associated DNA from samples was stored at-"C until sequencing. The V4 region with infections in lens wearers. Other skin bacteria were enriched of 16s rRNA gene was amplified by PCR using barcoded primers, as pre in contact lenses and in the conjunctiva of lens wearers, which viously described ( 29). To rule out and control for possible reagent con- a considered to be opportunistic pathogens in conjunctivitis, kera- equimolar ratios and purified using a QIAquick PCR purification kit 8 titis, and endophthalmitis(24-26). Enrichment of skin bacteria Illumina Mise platform( Genome Technology Center of NYU Medical y wearing contact lenses suggests that contact lenses could Cente function as a medium to transfer skin bacteria to the ocular sur- Dar NY) using a paired-end technique(2 150-cycle runs) face. Alternatively, contact lenses may be exerting selective pres- the QUIME suite of software tools(v1. 8)(31). The filtered sequence reads sure on ocular bacterial communities in favor ofskin-like bacteria. (Phred 2 Q20)were used to pick the operational taxonomic units Given that people use their fingers(no matter how well (OTUs), with an open-reference OTU picking method based on 97% washed)to put in contact lenses, we tested whether conjunctival identity to entries in the Greengenes database( v13_8). Negative-control. g acterial OTUs in lens wearers originate from hands, and results derived OTUs were discarded from the OTU table using a filtration script suggested that they do not. Consistently, evidence suggests that (filter_otus_ from_otu_table.py)in QIIME. After the chimeric sequences ransplantation of microbiota between body sites changes the bac- were removed using UCHIME (32), all communities were rarefied to terial population structure only temporarily( 27). An experiment 2,090 reads per sample. For comparison of levels of beta diversity between in which lens wearers use or do not use sterile gloves to insert their communities, the unweighted/weighted UniFrac distances (33)were cal- lenses would clarify the origin of the bacteria in contact lenses. culated and PERMANOVA(34)was used to test significance. Linear dis The different taxa in the conjunctiva of lens wearers are re- criminant analysis effect size(LEfSe)(35)was used to detect unique bio- flected in the predicted bacterial gene content, so the differences markers by determinations of the relative abundances of the members of terial gene profiles(mean accuracy =0.99+0.03 standard devia- using PICRUSt(28)with Kegg Orthology(KO)classification(36) tion [SD] for core/bacterial housekeeping functions; minimum To determine the overlap between OTUs of the eye samples and OTUs accuracy=0.82 for membrane-associated functions)(28); fur- found in other skin sites, we used Source Tracker(37)with a previously reported 16S rRNA gene data set derived from samples from human face whether bacterial gene functions confer reduced protection The HMP data set(15)of 16S rRNA (regions V3 to V5)sequences was against ocular infections. downloaded from the NIH HMP website(hmpdacc org) and was Previous studies have suggested that the commensal microbi- trimmed to contain only the v4 region of the 16S rRNA gene using otaoftheocularsurfacecouldinteractwiththehostinimmuneBioperl(http://www.bioperlorg/wiki/main_page).thE16srrNa(V4 system to suppress microbial pathogenicity (4, 6, 7), and the im- region)sequences from the skin of the face(16)were downloaded from pact of wearing contact lenses may affect this protective function. EMBL EBI database(ERP005182). The sequences from the Bouslimani Our report provides novel insights into possible mechanisms by study and the V4-trimmed hMP data set were merged with sequences hich wearing contact lenses increases eye infection risks Further from this study. The QIIME suite(v1. 8)was used to pick OTUs from the research is required to determine if the risk is related to contami- merged sequences using the closed-reference method. Then, all commu- ating the lenses with bacteria from the skin of the finger or if nities were rarefied to 1,000 sequences per sample to calculate bacterial contact lenses exert selective pressures on the eye bacterial com- diversity munity in favor of skin bacteria. Nucleotide sequence accession number. The raw sequences support ing the results of this article are available in the European Nucleotide MATERIALS AND METHODS Archiverepositoryunderaccessionno.PrjEb12498(htTp://www.ebi Sample collection. The study was performed under IRB protocol S12 ac uk/ena/data/view/PRJEB12498) 03905 of the NYU Medical Center. Subjects seeking routine eye care at the ophthalmology practice at the NYU Medical Center from October 2013 to SUPPLEMENTAL MATERIAL June2014weresampledfromtheirleftandrighteyesAtotalof58subSupplementalmaterialforthisarticlemaybefoundathttp://mbio.asm.org/ cts(40 females and 18 males) provided samples; 20 of those subjects lookup/suppl/doi: 10. 1128/mBio. 00198-16/-/DCSupplemental including 9 lens wearers and 11 non-lens wearers) Figure S1, PDF file, 0.4 MB udinally, with biweekly samples collected for 6 weeks(see Table Sl and Figure S2, PDF file, 1.5 MB Table S2 in the supplemental material). Dry sterile cotton swabs(Fishe Figure S3, PDF file, 0.1 MB Scientific)used to swab samplin laced in sterile Figure S4, PDF file, 0.4 MB cryogenic tubes(Thermo Scientific). Sites sampled included right and left Figure S5, PDF file, 1.3 MB. onjunctivas, skin under the right and left eyes, and contact lens(250 total Figure S6, PDF file, 0.7 MB es, including 116 conjunctiva samples, 114 skin samples, and 20 Figure S7, PDF file, 0.1MB contact lens samples). Samples were immediately transported to the lab Table s1. PDF file. 0.1 MB. oratory on ice and frozen at -80C. A total of 38 additional subjects were Table S2. PDF file. 0.1 MB. led once(including 16 lens wearers; n=75)(see Table S3)in an Table S3, PDF file. 0.1 MB. March/April 2016 Volume 7 Issue 2 e00198-16 msio° mbio. asm. org5

Staphylococcus, Streptophyta,Corynebacterium, and Enhydrobacter levels in their eyelashes and tears in relation to controls (n 4), none of which were increased in our lens wearers. In addition to the different sites sampled, their patients were older than ours (59 [ 16.6]-year-old patients in the study by Lee et al. and 26 [ 4.5]-year-old patients in our study). More studies are needed to understand which are the opportunistic pathogens associated with infections in lens wearers. Other skin bacteria were enriched in contact lenses and in the conjunctiva of lens wearers, which have increased relative abundances of Pseudomonas, Acinetobac￾ter, and Methylobacterium. Representatives of these genera are considered to be opportunistic pathogens in conjunctivitis, kera￾titis, and endophthalmitis (24–26). Enrichment of skin bacteria caused by wearing contact lenses suggests that contact lenses could function as a medium to transfer skin bacteria to the ocular sur￾face. Alternatively, contact lenses may be exerting selective pres￾sure on ocular bacterial communities in favor of skin-like bacteria. Given that people use their fingers (no matter how well washed) to put in contact lenses, we tested whether conjunctival bacterial OTUs in lens wearers originate from hands, and results suggested that they do not. Consistently, evidence suggests that transplantation of microbiota between body sites changes the bac￾terial population structure only temporarily (27). An experiment in which lens wearers use or do not use sterile gloves to insert their lenses would clarify the origin of the bacteria in contact lenses. The different taxa in the conjunctiva of lens wearers are re- flected in the predicted bacterial gene content, so the differences are not redundant in gene content. We could reliably predict bac￾terial gene profiles (mean accuracy 0.99  0.03 standard devia￾tion [SD] for core/bacterial housekeeping functions; minimum accuracy 0.82 for membrane-associated functions) (28); fur￾ther metagenomic/transcriptomic research is needed to elucidate whether bacterial gene functions confer reduced protection against ocular infections. Previous studies have suggested that the commensal microbi￾ota of the ocular surface could interact with the host in immune system to suppress microbial pathogenicity (4, 6, 7), and the im￾pact of wearing contact lenses may affect this protective function. Our report provides novel insights into possible mechanisms by which wearing contact lenses increases eye infection risks. Further research is required to determine if the risk is related to contami￾nating the lenses with bacteria from the skin of the finger or if contact lenses exert selective pressures on the eye bacterial com￾munity in favor of skin bacteria. MATERIALS AND METHODS Sample collection. The study was performed under IRB protocol S12- 03905 of the NYU Medical Center. Subjects seeking routine eye care at the ophthalmology practice at the NYU Medical Center from October 2013 to June 2014 were sampled from their left and right eyes. A total of 58 sub￾jects (40 females and 18 males) provided samples; 20 of those subjects (including 9 lens wearers and 11 non-lens wearers) were followed longi￾tudinally, with biweekly samples collected for 6 weeks (see Table S1 and Table S2 in the supplemental material). Dry sterile cotton swabs (Fisher Scientific) used to swab sampling sites were immediately placed in sterile cryogenic tubes (Thermo Scientific). Sites sampled included right and left conjunctivas, skin under the right and left eyes, and contact lens (250 total samples, including 116 conjunctiva samples, 114 skin samples, and 20 contact lens samples). Samples were immediately transported to the lab￾oratory on ice and frozen at  80°C. A total of 38 additional subjects were sampled once (including 16 lens wearers; n 75) (see Table S3) in an ophthalmology office after utilization of a 50-l drop of topical 0.5% proparacaine hydrochloride anesthetic. Negative-control swabs (n 3, with no sample) were also included. DNA extraction and sequencing. DNA extraction was carried out using MoBio (CA, USA) PowerSoil-htp 96 well soil DNA isolation plates according to the instructions provided by the manufacturer. Extracted DNA from samples was stored at 20°C until sequencing. The V4 region of 16s rRNA gene was amplified by PCR using barcoded primers, as pre￾viously described (29). To rule out and control for possible reagent con￾tamination, reagents for DNA extraction and for PCR amplification were also sequenced as controls (30). The amplicons were then pooled in equimolar ratios and purified using a QIAquick PCR purification kit (Qiagen Inc., CA, USA). The pooled amplicons were sequenced on an Illumina MiSeq platform (Genome Technology Center of NYU Medical Center, NY) using a paired-end technique (2 150-cycle runs). Data analysis. The 16S rRNA sequence analyses were performed with the QIIME suite of software tools (v1.8) (31). The filtered sequence reads (Phred Q20) were used to pick the operational taxonomic units (OTUs), with an open-reference OTU picking method based on 97% identity to entries in the Greengenes database (v13_8). Negative-control￾derived OTUs were discarded from the OTU table using a filtration script (filter_otus_from_otu_table.py) in QIIME. After the chimeric sequences were removed using UCHIME (32), all communities were rarefied to 2,090 reads per sample. For comparison of levels of beta diversity between communities, the unweighted/weighted UniFrac distances (33) were cal￾culated and PERMANOVA (34) was used to test significance. Linear dis￾criminant analysis effect size (LEfSe) (35) was used to detect unique bio￾markers by determinations of the relative abundances of the members of the bacterial taxonomies. Predictive functional analysis was performed using PICRUSt (28) with Kegg Orthology (KO) classification (36). To determine the overlap between OTUs of the eye samples and OTUs found in other skin sites, we used SourceTracker (37) with a previously reported 16S rRNA gene data set derived from samples from human face and hand (16). The HMP data set (15) of 16S rRNA (regions V3 to V5) sequences was downloaded from the NIH HMP website (hmpdacc.org) and was trimmed to contain only the V4 region of the 16S rRNA gene using BioPerl (http://www.bioperl.org/wiki/Main_Page). The 16S rRNA (V4 region) sequences from the skin of the face (16) were downloaded from EMBL EBI database (ERP005182). The sequences from the Bouslimani study and the V4-trimmed HMP data set were merged with sequences from this study. The QIIME suite (v1.8) was used to pick OTUs from the merged sequences using the closed-reference method. Then, all commu￾nities were rarefied to 1,000 sequences per sample to calculate bacterial diversity. Nucleotide sequence accession number. The raw sequences support￾ing the results of this article are available in the European Nucleotide Archive repository under accession no. PRJEB12498 (http://www.ebi .ac.uk/ena/data/view/PRJEB12498). SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at http://mbio.asm.org/ lookup/suppl/doi:10.1128/mBio.00198-16/-/DCSupplemental. Figure S1, PDF file, 0.4 MB. Figure S2, PDF file, 1.5 MB. Figure S3, PDF file, 0.1 MB. Figure S4, PDF file, 0.4 MB. Figure S5, PDF file, 1.3 MB. Figure S6, PDF file, 0.7 MB. Figure S7, PDF file, 0.1 MB. Table S1, PDF file, 0.1 MB. Table S2, PDF file, 0.1 MB. Table S3, PDF file, 0.1 MB. Altered Eye Microbiota by Wearing Contact Lenses March/April 2016 Volume 7 Issue 2 e00198-16 ® mbio.asm.org 5 mbio.asm.org on June 29, 2016 - Published by mbio.asm.org Downloaded from

Shin et al ACKNOWLEDGMENTS 18. Szczotka-Flynn LB, Pearlman E, Ghannoum M. 2010. Microbi We thank Denni Catalano for her technical support in preparation of tamination of contact lenses, lens care solutions, and their access samples for sequencing. We also thank Keith A. Martin teraturereviewEyeContactLens36:116-129.http://dx.doi.org/ 10.1097/CL0b0l3e3l8ld20ca 19. McDermott AM. 2013. Antimicrobial compounds in tears. Exp Eye Res g FUNDING INFORMATION l17:53-61.htp:dx. doi org10.1016/exer2013.0701 20. Stapleton F, willcox MD, Fleming CM, Hickson S, Sweeney DF, This work, including DNA sequencing and the efforts of Jack Dodick and Holden BA. 1995. Changes to the ocular biota with time in extended-and sa Park, was funded by the research to Prevent Blindness Foundation laily-wear disposable contact lens use. Infect Immun 63: 4501-4505 21. Larkin DF, Leeming JP. 1991. Quantitative alterations of the commensal REFERENCES eyebacteriaincontactlenswearEye(lond)5:70-74.http://dx.doiorg/o 1. 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ACKNOWLEDGMENTS We thank Denni Catalano for her technical support in preparation of samples for sequencing. We also thank Keith A. Martinez II for language editing. FUNDING INFORMATION This work, including DNA sequencing and the efforts of Jack Dodick and Lisa Park, was funded by the Research to Prevent Blindness Foundation. REFERENCES 1. Willcox MD. 2013. Characterization of the normal microbiota of the ocular surface. Exp Eye Res 117:99 –105. http://dx.doi.org/10.1016/ j.exer.2013.06.003. 2. Locatelli CI, Kwitko S, Simonetti AB. 2002. Conjunctival endogenous microbiota in patients submitted to cataract surgery. Braz J Microbiol 34:203–209. http://dx.doi.org/10.1590/S1517-83822003000300004. 3. Graham JE, Moore JE, Jiru X, Goodall EA, Dooley JS, Hayes VE, Dartt DA, Downes CS, Moore TC. 2007. Ocular pathogen or commensal: a PCR-based study of surface bacterial flora in normal and dry eyes. Invest Ophthalmol Vis Sci 48:5616–5623. http://dx.doi.org/10.1167/iovs.07-0588. 4. 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