The Microbiome

The Fungal Mycobiome and Human Health

Mahmoud A Ghannoum, Ph.D., MBA, FIDSA Director, Center for Medical Mycology, Professor, School of Medicine Case Medical Center and Case Western Reserve University © ESCMID Cleveland,by OH eLibraryauthor The Human Microbiome

• The human microbiome is defined as the microbial communities found at several different sites on the human body • Changes in the microbiome have been associated with different disease states including HIV, cancer and Irritable Bowl Disease • Up until recently, only the bacterial component of the microbiota have been studied © ESCMID by eLibraryauthor Change Through Personal Story!!! • In 1974, my master’s advisor handed me a paper showing that rabbits treated with antibiotics or anti-inflammatory steroids developed the fungal infection candidiasis • It made me realize that not only could fungi in the environment negatively impact our health, but fungal species also inhabit the mammalian body, alongside diverse commensal bacteria. • When one microbial community is knocked out (bacteria), another (fungi) can cause illness. • If the communities are undisturbed, however, the fungal inhabitants appear to be harmless or perhaps even beneficial© ESCMID. eLibrary This realization happenedby author 43 Years Ago!!! The Microbiome: More than Bacteria • Since the 1950s, it is well appreciated that alterations in the bacterial flora has direct impact on the fungal flora • Use of tetracycline by women often leads to vaginal candidiasis. Thus, investigations of the vaginal microbiome that focuses on bacteria only, while ignoring the fungi, is counterintuitive • Similar argument could be made for microbiome studies of other body sites (e.g. lung, GI tract, mouth, skin) • Opined: - That future human microbiome studies should be expanded beyond bacteria to include fungi, viruses, and other microbes in the same samples • Suggested: - ©That suchESCMID studies will allow a better eLibrary understanding of the role of these communities in byhealth andauthor disease Ghannoum & Mukherjee (2010). Microbe 5(11) The Fungal Community

• The scientific search engine returns only 55 papers pertaining to the “©mycobiome” ESCMID by eLibraryauthor The Scientist. 02.2016. 35 The Human Mycobiome

ORAL CAVITY LUNGS GASTRO- SKIN INTESTINAL

• Alternaria • Aspergillus • Candida • Aspergillus • Candida • Cryptococcus • Aureobasidium • Cladosporium • Aspergillus • Debaryomyces • Candida • Penicillium • Candida • Epidermophyton • Cladosporium • Cryptococcus • Cladosporium • Malassezia • Cryptococcus • Cryptococcus • Microsporum • Fusarium • Fusarium • Rhodotorula • Gibberella • Penicillium • Trichophyton • Glomus • Pneumocystis • Aspergillus • Pichia • Mucor • Chrysosporium • Saccharomyces • Saccharomyces • Epicoccum • Teratosphaeria • Leptosphaerulina • Penicillium • Phoma *Potentially pathogenic lineages • Saccharomyces • Ustilago • Surveys have revealed several pathogenic species that may increase one’s risk of disease when the healthy microbiome is disrupted. • Candida©species ESCMID are among the most common eLibrary members of the human mycobiome. • When the balance of a microbial communityby isauthor disrupted, Candida can flourish and cause disease The Scientist. 02.2016. 37 Time for a New Perspective Regarding the Role of Fungi in Health and Disease • Historically, fungi were considered passive colonizers of the microbial community that could become pathogenic as the result of a change in the environment (antibiotic use and suppression of immune defense). • Recent profiling of the fungal “Mycobiome” communities is providing a new perspective showing that: • Fungi have a complex, multifaceted role in humans and© are ESCMID active participants ineLibrary directly influencing health and disease . by author Our first endeavor to unlock the secrets of the fungal community • Profiling the fungi in the healthy mouth Oral Cavity in Health

Microbiome © ESCMID by eLibraryauthor Defining the Healthy Oral Fungal Microbiome (Mycobiome) • Study Design – 20 healthy subjects recruited after obtaining written informed consent (IRB-approved protocol) – Demographics: 21-60 years of age; 8 F, 12 M; non- smokers; non-diabetic; no antimicrobials for 3 m – Oral examination – 15 ml sterile saline rinse for 1 min, expectorate into sterile 50 mL tube, hold on ice till processing

• DNA extracted from cellular pellet • Amplified ITS1 region – designed to detect consensus sequences present in a wide range of fungi © ESCMID by eLibraryauthor Pyrosequencing-Based Microbial Detection • Pyrosequencing enables de novo sequencing of short reads of DNA • Involves three steps – PCR amplification – Template preparation – Pyrosequencing reactions. • Employs coupled enzymatic reactions to detect inorganic pyrophosphate (PPi) released as a result of nucleotide incorporation by DNA polymerase. • The released PPi is converted to ATP by ATP sulfurylase, which provides the energy for luciferase to oxidize luciferin and generate light. • This process is repeated where additional nucleotides are added • Since the added nucleotide is known, the sequence of the template can be determined. • ITS1 PCR products were sequenced • Relative peak abundance of fungal amplicons was© determined ESCMID eLibrary Ronaghi, M., and E. Elahi . 2002. Jby Chromatogr authorB Analyt Technol Biomed Life Sci 782:67- 72 • Detected 74 culturable and 11 non-culturable genera, • Total number of species identified were 101. • The© number ESCMID of species in the oral cavity of eacheLibrary individual ranged between 9 and 23 • 15 genera were present in 20% byof the participantsauthor (core mycobiome) • Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both),© Aspergillus ESCMID(35%), Fusarium (30%), eLibraryand Cryptococcus (20%). • Four of these genera are known toby be pathogenic author in humans. • Principal component (PCO) analysis revealed: - White males and Asian males clustered differently from each© other ESCMID eLibrary -Whereas both Asian and by White femalesauthor clustered together. Ghannoum, Jurevic, Mukherjee, Cui, Sikaroodi, Naqvi, Gillevet. 2010. PLoS Pathogens. 6(1):e1000713 Oral Mycobiome in Healthy Individuals: Conclusion • Surprise: Humans are colonized with numerous fungal species (85 genera), including the expected (Candida spp.), and the unexpected (Aspergillus, Fusarium, Cryptococcus spp.). • Surprise: over a 1/3 of the fungi were un-culturable. • Great individual variation in the fungal flora among 20 healthy individuals was observed. • These discoveries provide a glimpse of the complexity in the human microbial flora, which now includes numerous fungal species in addition to the bacteria. • Our findings prepare the way for studies aimed at defining the© role ESCMID of these fungi in health, diseaseeLibrary and controlling the bacterial flora (and vice versa).by author Casadevall: Faculty of 1000 Biology, 21 Jan 2010 Mycobiome: “A Tale of Two Diseases”

A Tale of Two Cities addresses the fundamentally © ESCMIDdual nature of humanityeLibrary by authorWith apologies to Mr. Dickens THE FIRST CITY: MYCOBIOME AND HIV INFECTION © ESCMID by eLibraryauthor Defining the Oral Microbiome in HIV Infection • Profile the microbes in Oral Cavity in the healthy mouth HIV Disease • Changes in the mouth in the setting of HIV disease Microbiome • Microbial interactions

in HIV setting Healthy Oral Mycobiome © ESCMID by eLibraryauthor Changes in the Oral Microbiome in HIV Setting

• Oral rinse samples were collected from 12 uninfected and 12 HIV- infected individuals following informed consent • Uninfected controls: recruited from students/staff at Case • HIV infected patients: recruited from the UH/Case Medical Center, OHARA and AIDS Research (CFAR) Units • Information collected: HAART medication, viral load, CD4 counts, smoking status, age, gender and ethnicity • Inclusion criteria: > 18 yrs, no clinical signs of oral mucosal disease • Exclusion criteria: recent use of antimicrobials/topical or systemic© ESCMID steroids, pregnancy, and insulineLibrary-dependent diabetes mellitus by author Oral Bacteriome of HIV-infected and uninfected individuals Relative Abundance (%) Abundance Relative

15.0% %Abundance

10.0%

5.0%

0.0%

Rothia Hafnia Serratia Gemella Kaistella Sneathia KingellaHallellaFilifactor Prevotella Neisseria Veillonella LeptotrichiaPasteurella Atopobium TreponemaTannerellaParvimonas Haemophilus Actinomyces Eubacterium Oribacterium SchlegelellaPaludibacterMycoplasmaLactobacillus Streptococcus Fusobacterium Granulicatella Megasphaera SolobacteriumCampylobacterActinobacillus Porphyromonas Aggregatibacter Capnocytophaga Pectobacterium • HIV-infected: – Number of taxa: 9-14 – Most common: Prevotella, Streptococcus and Rothia •©Uninfected: ESCMID eLibrary – Number of taxa: 8- 14 by author – Most common: Fusobacterium, Prevotella, and Streptococcus Core Oral Bacteriome • 14 genera constituted the core oral bacteriome in both HIV-infected and uninfected, of which 13 (93%) were common to both study groups • The core bacteriome of uninfected individuals was similar to earlier study showing 15 taxa in the oral cavity* • Capnocytophaga – unique to HIV- infcted individuals • Aggregatibacte© ESCMIDr – unique by eLibraryauthor uninfected individuals *Zaura et al. (2009). BMC Microbiol 9: 259 Oral Mycobiome of HIV-Infected and Uninfected Individuals Relative Abundance (%) Abundance Relative

70.0% MycobiomeUninfected (n=12) Elevated in Uninfected Genus vs Rank * Elevated in Uninfected 60.0% HIV-infected Healthy HIV Elevated in HIV-infected

50.0%

40.0%

30.0% %Abundance

20.0% *

10.0% *

0.0%

Pichia Valsa Yeast Candida Glomus Bullera Serpula Fusarium Claviceps Alternaria Antarctic Exophiala Lactarius Dioszegia Blumeria DavidiellaEpicoccum AspergillusClavisporaKodamaea Emericella Penicillium Gibberella Cytospora Albatrellus Nigrospora Rhodotorula Corynespora TrichosporonCryptococcusCladosporium Microbotryum Galactomyces PhaeosphaeriaAureobasidiumHanseniaspora Saccharomyces © ESCMIDCystofilobasidium eLibrary • HIV-infected: Number of taxa: 1-9 , most common: Candida, Epicoccum, and Alternaria • Uninfected: Number of taxa: 1 -9, most common:by Candida author, Pichia, and Fusarium Core Oral Mycobiome in HIV Setting • The COM of HIV-infected and uninfected individuals consisted of 5 genera • Of these, Candida and Penicillium were common between the two groups, while differing in the remaining genera • Among the Candida species detected, C. albicans was the most common (58% in uninfected and 83% in HIV-infected patients), followed by C. dubliniensis (17% in both groups) These© results ESCMID demonstrated a shift in eLibrarythe core mycobiome between HIV-infected and non- infected by controls author Correlation Between Oral Bacteriome and Mycobiome in HIV-Infected Patients

HIV-infected Uninfected

• Uninfected: 15 bacteria-fungi pairs were significantly correlated – Two (Rothia-Cladosporium and Granulicatella-Cryptococcus) negatively correlated; 13 positively correlated • HIV©-infected: ESCMID 12 bacteria-fungi correlations eLibrary were significant – 11 positive and 1 with negative bycorrelation (authorCampylobacter-Candida) Systems Biology Approach: HIV • Profile the fungal Oral Cavity in community and coined the Health and Disease term “Mycobiome” (101 fungal species detected) Microbiome • Changes in the mouth in the setting of HIV disease HIV HIV Healthy Mycobiome 1* Core Core • Microbial/metabolite Bacteriome Mycobiome interactions in HIV setting3

No major shift Shifts in HIV in HIV 2*

1. Ghannoum et al. 2010. Characterization of the oral Mycobiome in healthy individuals. PLoS Pathog. 2. Mukherjee et al. (2014) Oral Mycobiome Analysis of HIV-Infected Patients: Identification of© Pichia asESCMID an Antagonist of Opportunistic Fungi. eLibrary PLoS Pathog 3. Brown RE, et al. 2015. Quorum - Sensing by Dysbiotic authorShifts in the HIV-Infected Oral Metabiome. PLoS One. Defined Microbial Interactions in the HIV Setting • Is there an association between Candida colonization and members of the core microbiome? • Pichia and Cladosporium were detected in uninfected individuals only • Penicillium was present in both HIV- infected and uninfected individuals, at the same level (25%) • Presence of Pichia coincided with absence of Candida, and vice versa • No association with Candida colonization was found for Cladosporium or Penicillium© ESCMID eLibrary • These results suggested that by Pichia isauthor antagonistic to Candida Does Pichia Exhibit Antagonistic Interaction with Candida? • Hypothesis: Pichia inhibits Candida growth. • To test this hypothesis, we evaluated the effect of Pichia on Candida • Effect on growth, germination, and biofilm formation • Biofilms were grown in the presence or absence of spent media obtained from cultures of Pichia or Penicillium (control) • Biofilms were quantified by XTT assay • Effect© onESCMID biofilm architecture eLibrary was monitored by confocal laser scanning by microscopy author (CLSM) Effect of Pichia Spent Medium on Pathogenic Fungi

Candida Aspergillus Fusarium

• PSM inhibited growth of Candida, Aspergillus and© FusariumESCMID by eLibraryauthor Pichia Causes Stunted Filamentation

Untreated PSM-treated

• PSM induced formation of stunted filaments© ESCMID in Candida by eLibraryauthor Activity of Pichia Against Fungal Biofilms

Exposure to Pichia Cells Exposure to Spent Medium

* P ≤ .002 compared to controls

• Effect of Pichia cells on Candida • Effect of spent media from Pichia biofilm formation or Penicillium on Candida biofilms – Candida and Pichia were co- – Spent medium from Pichia (PSM) incubated [(C:P) = 3:1, 1:1, or 1:3] inhibited biofilms and biofilm formation was monitored – Spent medium from Penicillium ©– Pichia ESCMIDcells inhibited Candida eLibraryhad not effect on Candida biofilms by authorbiofilms Activity of PSM Against Fungal Biofilms: Confocal Analysis

Untreated Penicillium Pichia

• Effect of spent medium from Pichia biofilms was determined by confocal microscopy • Pichia spent medium inhibited biofilm formation • Thickness of biofilms formed in presence of Pichia spent medium was significantly reduced

compared to untreated control Thickness (µm) • Penicillium© ESCMIDspent medium had no effect on theeLibrary architecture or thickness of biofilms by author Efficacy of PSM in a Murine Model of Oral Candidiasis • Wild-type C57BL/6 mice were immunosuppressed, fed tetracycline in drinking water to prevent bacterial growth • Mice were anesthetized, scratches made on the dorsum of the tongue, and challengedwith C. albicans GDH (108 blastospores) • Mice were divided into 3 groups (n = 4): – Treated with Pichia supernatant, 100 µl in the oral cavity twice a day – Treated with topical nystatin as comparator (widely used clinically to treat oral candidiasis) – Untreated and vehicle-treated control • Treatment began on day 4 post inoculation, mice were sacrificed on day 7 • Infection score of tongues was assessed on a scale of 0 (no lesions) to 3 (wide-spread fungal plaques and mucosal erosive) • Tongues© ESCMID were harvested for determination eLibrary of fungal burden and histopathology by author PSM is Efficacious Against Oral Candidiasis in a Murine Model Infection Score Fungal Burden

Untreated PSM-Treated Nystatin-Treated

• Infection score of PSM-treated mice was significantly reduced compared to untreated mice (P = .011) • Tongue fungal CFU was significantly reduced in PSM-treated mice vs. untreated controls (P = .04) • Extensive tissue invasion by fungal hyphae and disruption of the epithelium in untreated controls and© nystatin -ESCMIDtreated mice, while PSM-treated tongues eLibrary had only superficial hyphal invasion - Mycobiome studies identified by a new wayauthor to combat oral candidiasis PSM Activity is Mediated by Protein(s) • Role of metabolites in PSM activity – Extracted metabolites from PSM – No effect on Candida biofilm formation • Role of proteins in PSM activity – Biofilms were exposed to Pichia farinosa supernatant (PSM) treated with proteinase K (PK; 100, 200 or 500 µg) – Compared to untreated PSM – Biofilm formation evaluated by confocal microscopy, ©biomass ESCMID determination and eLibrary colony forming units (CFUs) by author Effect of PSM Proteins on Candida Biofilms: Confocal Microscopy

(A) Control (B) +PSM (C) +PSM+PK (100 µg)

(D) +PSM+PK (200 µg) (E) +PSM+PK (500 µg) 1 0 0 (F) Biofilm Thickness (µm) * 8 0

6 0

4 0 * * Thickness um 2 0

0 Control PSM 100ug 200ug 500ug © ESCMID eLibraryPSM C. albicans 10341 48h Biofilm Control

by authorPK (100 µg) PK (200 µg) PK (500 µg) PSM + Effect of PSM Proteins on Candida Biofilms: Dry Weight and CFUs

7 .0 2 .0 Dry Weight CFU * 1 .5 * 6 .5 * * 1 .0 CFU

6 .0 0 .5 Dry W eight (mg)

0 .0 5 .5 Control PSM 100ug 200ug 500ug Control PSM 100ug 200ug 500ug C. albicans 10341 48h Biofilm Control C. albicans 10341 48h Biofilm Control Untreated PSM Untreated Untreated PSM Untreated PSM + PK PSM (100 µg) + PK PSM (200 µg) + PK PSM (500 µg) PSM + PK PSM (100 µg) + PK PSM (200 µg) + PK PSM (500 µg)

*significant compared to PSM alone © ESCMID by eLibraryauthor THE SECOND CITY: MYCOBIOME AND CROHN’S DISEASE © ESCMID by eLibraryauthor Historical Perspective: Can Fungi Impact Other Diseases: Association Between Fungi and GI • This association has been documented since the 18th century, with a special focus on candidiasis.1 • One of the first reports of this association was the study by Rosenstein, who described oral candidiasis that extended to the stomach and intestines. • The first reported case of GI candidiasis in an infant was described in this period. • Subsequent publications in the 19th century documented Candida infection of the stomach, colon and ileum. 2,3

1. Odds, 1988. Candida and Candidosis, Bailliere Tindall. ©2. Parrot.ESCMID Archives de Physiologie Normale eLibraryet Pathologique 3, 621–625 (1870). 3. Parrot. Archives de Physiologie byNormale authoret Pathologique 2, 579–599 (1869). Twenty First Century: Mycobiome and GI Tract in Health Two studies characterized the mycobiome of the GI tract in healthy individuals using deep sequencing: • The first study analyzed fecal samples from 10 healthy volunteers, and showed that: - the GI contains Aspergillus, Cryptococcus, Penicillium, Pneumocystis and Saccharomycetaceae yeasts (Candida and Saccharomyces).1 • The second study2, evaluated the effect of diet on the microbiota and reported the presence of 5 fungal genera • The most prevalent fungal genera detected were Saccharomyces (present in 89% of the specimens), followed© ESCMID by Candida (57%) and eLibrary Cladosporium (42%). by1. Dollive , authorS. et al. Genome Biol. 13, R60 (2012). 2. Hoffmann, C. et al. PLoS ONE 8, e66019 (2013). Mycobiome of the GI Tract in Disease

• Fungi have been linked to a number of GI diseases including: - Irritable Bowl disease (IBD) - peptic ulcers - Graft-versus-host disease (GVHD) - Antibiotic-associated diarrhea, and - Chemotherapy-induced enteric disorders. © ESCMID by eLibraryauthor Mycobiome and Crohn’s • CD is a disorder mediated by multitude of factors: - Host genotype (genetic factor), - Dysregulated immune responses, and - The intestinal microbiota • Imbalance (dysbiosis) of the intestinal flora plays a key role in modulating CD- associated inflammatory processes. © ESCMID by eLibraryauthor Mycobiome and Crohn’s • Until recently, the only study focusing on the mycobiome and IBD was by Illiev et al. using a dextran sodium sulfate (DSS)-induced colitis mouse model showing: • The gut is colonized with 10 fungal species dominated by C. tropicalis (65%), • Fungi and NOT bacteria is responsible for aggravating the severity and inflammation of IBD, rather than a cause of these symptoms. • Clearly demonstrating the contribution of the mycobiome© ESCMID to colitis. by eLibraryauthor Iliev, I. D. et al. Science 336, 1314–1317 (2012) Characterization of the Mycobiome and Bacteriome in Crohn’s Patients and Healthy Relatives in Multiplex Families Hoarau, Mukherjee , Retuerto, Poulain, Sendid, Ghannoum

Clinical Details of CD Patients

A1 (<=16 yr) 0 Subjects Demographics Age Category A2 (17-40 yr) 8 A3 (>= 40 yr) 12 L1 (Terminal Variable CD NCR NCU Total 11 Ileum) Families 9 9 4 13 L2 (Colon) 2 Individuals 20 28 21 69 Location L3 (Ileum- 6 Female 12 13 13 38 Colon) L4 (Upper GI Male 8 15 8 31 0 Age (mean, yrs) 44.5 48.4 41.3 45.1 Tract) B1 3 (Nonstenotic) CD – Crohn’s disease patients Behavior B2 (Stenotic) 4 B3 NCR – Non-Crohn’s, Related individuals 12 NCU – Non-Crohn’s, Unrelated individuals (Penetrating) © ESCMID eLibraryDisease Active 3 by authorStatus Remission 8 PCA Clustering Analysis: Crohn’s (CD, Red), non-Crohn’s Relatives (NCR, Blue), and non-Crohn’s unrelated (NCU, Green) (A) Bacteriome (B) Mycobiome

Oscillospira, P = .007 P = .0018 Prevotella

P = .034 Faecalibacterium Faecalibacterium P = .025 prausnitzii

P ≤ .002 P ≤ .001 Saccharomyces spp S. cerevisiae

Candida spp. C. tropicalis

P = .2 P = .01

CD NCR P-value Species Mean SD Mean SD Candida tropicalis 10.41% 18.59% 1.01% 1.65% 0.005 Candida glabrata 2.29% 10.23% 1.88% 10.80% 0.892 Candida albicans 0.52% 1.36% 0.67% 2.03% 0.778 Candida parapsilosis 0.34% 1.39% 0.01% 0.03% 0.17 Candida tartarivorans 0.18% 0.81% 0.00% 0.00% 0.202 Candida dubliniensis 0.06% 0.29% 0.00% 0.02% 0.23 Candida temnochilae 0.06% 0.21% 0.05% 0.21% 0.864 Candida zeylanoides 0.01% 0.04% 1.38% 7.83% 0.439 © ESCMID by eLibraryauthor Anti–Saccharomyces cerevisiae antibodies (ASCA): P = .001 - widely used in diagnosis of Crohn’s disease P = .001 - could predict CD outcome in advance of disease - Mannans are widely recognized as ASCA epitopes

• ASCA level in CD group was significantly higher than levels in both NCR and NCU groups. • There was no significant difference in ASCA level between the NCR and NCU groups (P > .05). • Importantly,© ESCMID ASCA level was correlated eLibrary with abundance of C. tropicalis. by author Sendid et al. 1998. Am. J. Gastroenterol. 93: 1306-1310 Bacterial-Bacterial Genera Correlations

(A) (B)

Number of genera = 57 Number of genera = 57 Associations among bacterial genera in: (A) CD patients and (B) Non-CD relatives (NCR).©Red circlesESCMID: negative associations, blue circleseLibrary: positive associations. Diameter of circles indicates the magnitude of theby correlation author (-1 through +1) for each bacterial pair. Fungal-Fungal Genera Correlations

CD NCR

© NumberESCMID of genera = 28 eLibraryNumber of genera = 33 Associations among fungal genera in: Red circles: negative associations, while blueby circles positiveauthor associations. Significant Correlations Between Bacterial and Fungal Genera in CD Patients

Bacterial genus Fungal genus Pearson Correlation P - value Faecalibacterium Kluyveromyces .520 0.019 Prevotella Kluyveromyces .980 < .001 Oscillospira Pichia .724 < .001 Oscillospira Ophiocordyceps .717 < .001 Oscillospira Albatrellus .717 < .001 Proteus Candida .709 < .001 © ESCMID by eLibraryauthor 6/1/2016 Crohn's Disease Study (France) 58 Significant* Correlations of C. tropicalis with Bacterial Species in CD Patients Bacterial Taxon Pearson Correlation P-value Alkalimonas amylolytica .701 .001 Aquamonas haywardensis .743 .000 Enterobacter hormaechei .808 .000 Enterobacter ludwigii .725 .000 Enterobacter pyrinus .687 .001 Erwinia chrysanthemi .577 .008 Erwinia dispersa .733 .000 Erwinia soli* .491 .028 Erwinia toletana .757 .000 Escherichia coli* .569 .009 Pantoea agglomerans .697 .001 Profftia tarda .575 .008 Serratia marcescens* .651 .002

- 13 Different bacterial species significantly correlated with C. tropicalis - S.© marcescens ESCMID and E. coli were significantly eLibrary increased in CD patients • Since microbes in the gut existby in biofilms, author we hypothesized that CT, EC and SM cooperate in CD to influence host immune response59 Confocal Microscopy (A) CT (B) CT + EC

CT EC

Side-view Thickness P < .0 0 0 1 3 0

i P = .5 5 m )  P = .9 3 2 0 ii

1 0 iii Biofilm Thickness (

iv 0

T v C M E C S SM vi + + + C CT CT E + T i. CT+EC+SM; ii. CT+SM; iii. CT+EC C O rg a n is m iv, CT; v, SM alone; vi, EC alone © ESCMID by eLibraryauthorC T - C. tropicalis, E C - E . c o li, S M - S. marcescens CT Alone

CT CT

EC EC

EC/S M

EC/SM CT

Fusion EC CT Filamen t CT

Filamen t CT © ESCMID by eLibraryauthorSM CT Collaborators • Center for Medical Mycology (Case , UH): Center for Medical Mycology: - Pranab Mukherjee • Inserm, Université Lille France: Boualem Sendid - Maurcio Retuerto Gautier Hoarau - Jyotsna Chandra Daniel Poulain - Chris Hager - Yan Sun Icahn School of Medicine • Division Gastroenterology & at Mount Sinai Jean-Frederic Colombel Liver Disease: - Fabio Cominelli - Theresa Pizarro - Alex Rodriguez