High Quality Permanent Draft Genome Sequence of Chryseobacterium Bovis DSM 19482T, Isolated from Raw Cow Milk
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Polyphasic Study of Chryseobacterium Strains Isolated from Diseased Aquatic Animals Jean Francois Bernardet, M
Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals Jean Francois Bernardet, M. Vancanneyt, O. Matte-Tailliez, L. Grisez, L. Grisez, Patrick Tailliez, Chantal Bizet, M. Nowakowski, Brigitte Kerouault, J. Swings To cite this version: Jean Francois Bernardet, M. Vancanneyt, O. Matte-Tailliez, L. Grisez, L. Grisez, et al.. Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals. Systematic and Applied Microbiology, Elsevier, 2005, 28 (7), pp.640-660. 10.1016/j.syapm.2005.03.016. hal-02681942 HAL Id: hal-02681942 https://hal.inrae.fr/hal-02681942 Submitted on 1 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ARTICLE IN PRESS Systematic and Applied Microbiology 28 (2005) 640–660 www.elsevier.de/syapm Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals J.-F. Bernardeta,Ã, M. Vancanneytb, O. Matte-Taillieza, L. Grisezc,1, P. Tailliezd, C. Bizete, M. Nowakowskie, B. Kerouaulta, J. Swingsb aInstitut National de la Recherche Agronomique, Unite´ de Virologie -
Genome Analysis of Flaviramulus Ichthyoenteri Th78t in the Family
Zhang et al. BMC Genomics (2015) 16:38 DOI 10.1186/s12864-015-1275-0 RESEARCH ARTICLE Open Access Genome analysis of Flaviramulus ichthyoenteri Th78T in the family Flavobacteriaceae: insights into its quorum quenching property and potential roles in fish intestine Yunhui Zhang1, Jiwen Liu1, Kaihao Tang1, Min Yu1, Tom Coenye2 and Xiao-Hua Zhang1* Abstract Background: Intestinal microbes play significant roles in fish and can be possibly used as probiotics in aquaculture. In our previous study, Flaviramulus ichthyoenteri Th78T, a novel species in the family Flavobacteriaceae, was isolated from fish intestine and showed strong quorum quenching (QQ) ability. To identify the QQ enzymes in Th78T and explore the potential roles of Th78T in fish intestine, we sequenced the genome of Th78T and performed extensive genomic analysis. Results: An N-acyl homoserine lactonase FiaL belonging to the metallo-β-lactamase superfamily was identified and the QQ activity of heterologously expressed FiaL was confirmed in vitro. FiaL has relatively little similarity to the known lactonases (25.2 ~ 27.9% identity in amino acid sequence). Various digestive enzymes including alginate lyases and lipases can be produced by Th78T, and enzymes essential for production of B vitamins such as biotin, riboflavin and folate are predicted. Genes encoding sialic acid lyases, sialidases, sulfatases and fucosidases, which contribute to utilization of mucus, are present in the genome. In addition, genes related to response to different stresses and gliding motility were also identified. Comparative genome analysis shows that Th78T has more specific genes involved in carbohydrate transport and metabolism compared to other two isolates in Flavobacteriaceae, both isolated from sediments. -
Chryseobacterium Gleum Urinary Tract Infection
Genes Review 2015 Vol.1, No.1, pp.1-5 DOI: 10.18488/journal.103/2015.1.1/103.1.1.5 © 2015 Asian Medical Journals. All Rights Reserved. CHRYSEOBACTERIUM GLEUM URINARY TRACT INFECTION † Ramya. T.G1 --- Sabitha Baby2 --- Pravin Das3 --- Geetha.R.K4 1,2,4Department of Microbiology, Karuna Medical College, Vilayodi, Chittur, Palakkad, India 3Department of Medicine, Karuna Medical College, Vilayodi, Chittur, Palakkad, India ABSTRACT Introduction: Chryseobacterium gleum is an uncommon pathogen in humans. It is a gram negative, nonfermenting bacterium distributed widely in soil and water. We present a case of urinary tract infection caused by Chryseobacterium gleum in a patient with right lower ureteric calculi. Case presentation: This case describes a 62- year-old male admitted for ureteric calculi to the Department of Urology in a tertiary care hospital in Kerala. A strain of Chryseobacterium gleum was isolated and confirmed by MALDI-TOF MS .The bacterium was sensitive to Piperacillin-Tazobactum (100/10µg ), Cefotaxime(30µg),Ceftazidime(30 µg ) and Ofloxacin(30 µg). It was resistant to Nitrofurantoin (300µg),Tobramycin(10µg),Gentamicin(30µg),Nalidixic acid(30µg) and Amikacin(30µg). Conclusion: Chryseobacterium gleum should be considered as a potential opportunistic and emerging pathogen. Resistance to a wide range of antibiotics such as aminoglycosides, penicillin, cephalosporins has been documented. In depth studies on Epidemiological, virulence and pathogenicity factors needs to be done for better diagnosis and management. Keywords: Chryseobacterium gleum, Calculi, Flexirubin pigment, MALDI-ToF MS, Non-fermenter, UTI. Contribution/ Originality This study documents the first case of Chryseobacterium gleum associated UTI in South India. 1. INTRODUCTION Chryseobacterium species are found ubiquitously in nature. -
The Prevalence of Foodborne Pathogenic Bacteria on Cutting Boards and Their Ecological Correlation with Background Biota
AIMS Microbiology, 2(2): 138-151. DOI: 10.3934/microbiol.2016.2.138 Received: 23 April 2016 Accepted: 19 May 2016 Published: 22 May 2016 http://www.aimspress.com/journal/microbiology Research article The prevalence of foodborne pathogenic bacteria on cutting boards and their ecological correlation with background biota Noor-Azira Abdul-Mutalib 1,2,3, Syafinaz Amin Nordin 2, Malina Osman 2, Ahmad Muhaimin Roslan 4, Natsumi Ishida 5, Kenji Sakai 5, Yukihiro Tashiro 5, Kosuke Tashiro 6, Toshinari Maeda 1, and Yoshihito Shirai 1,* 1 Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan 2 Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 3 Department of Food Service and Management, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5 Laboratory of Soil Microbiology, Faculty of Agriculture, Graduate School, Kyushu University, 6- 10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 6 Laboratory of Molecular Gene Technique, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan * Correspondence: E-mail: [email protected]; Tel.: +6012-9196951; Fax: +603-89471182. Abstract: This study implemented the pyrosequencing technique and real-time quantitative PCR to determine the prevalence of foodborne pathogenic bacteria (FPB) and as well as the ecological correlations of background biota and FPB present on restaurant cutting boards (CBs) collected in Seri Kembangan, Malaysia. -
Muricauda Ruestringensis Type Strain (B1T)
Standards in Genomic Sciences (2012) 6:185-193 DOI:10.4056/sigs.2786069 Complete genome sequence of the facultatively anaerobic, appendaged bacterium Muricauda T ruestringensis type strain (B1 ) Marcel Huntemann1, Hazuki Teshima1,2, Alla Lapidus1, Matt Nolan1, Susan Lucas1, Nancy Hammon1, Shweta Deshpande1, Jan-Fang Cheng1, Roxanne Tapia1,2, Lynne A. Goodwin1,2, Sam Pitluck1, Konstantinos Liolios1, Ioanna Pagani1, Natalia Ivanova1, Konstantinos Mavromatis1, Natalia Mikhailova1, Amrita Pati1, Amy Chen3, Krishna Palaniappan3, Miriam Land1,4 Loren Hauser1,4, Chongle Pan1,4, Evelyne-Marie Brambilla5, Manfred Rohde6, Stefan Spring5, Markus Göker5, John C. Detter1,2, James Bristow1, Jonathan A. Eisen1,7, Victor Markowitz3, Philip Hugenholtz1,8, Nikos C. Kyrpides1, Hans-Peter Klenk5*, and Tanja Woyke1 1 DOE Joint Genome Institute, Walnut Creek, California, USA 2 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 3 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 4 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA 5 Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 6 HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany 7 University of California Davis Genome Center, Davis, California, USA 8 Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia *Corresponding author: Hans-Peter Klenk ([email protected]) Keywords: facultatively anaerobic, non-motile, Gram-negative, mesophilic, marine, chemo- heterotrophic, Flavobacteriaceae, GEBA Muricauda ruestringensis Bruns et al. 2001 is the type species of the genus Muricauda, which belongs to the family Flavobacteriaceae in the phylum Bacteroidetes. The species is of inter- est because of its isolated position in the genomically unexplored genus Muricauda, which is located in a part of the tree of life containing not many organisms with sequenced genomes. -
Das Mikrobiom Periimplantärer Läsionen Der Nachweis Dysbiotischer Veränderungen in Assoziation Mit Dem Schweregrad Der Erkrankung
Das Mikrobiom periimplantärer Läsionen Der Nachweis dysbiotischer Veränderungen in Assoziation mit dem Schweregrad der Erkrankung Inaugural-Dissertation zur Erlangung des Doktorgrades der Hohen Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Annika Therese Kröger aus Aachen 2020 Angefertigt mit der Genehmigung der Medizinischen Fakultät der Universität Bonn 1. Gutachter: Prof. Dr. med. dent. Moritz Kebschull 2. Gutachter: Prof. Dr. Achim Hörauf Tag der Mündlichen Prüfung: 07.10.2020 Aus der Poliklinik für Parodontologie, Zahnerhaltung und Präventive Zahnheilkunde Direktor: Prof. Dr. med. Dr. med. dent. Søren Jepsen Meinen Eltern 5 Inhaltsverzeichnis Abkürzungsverzeichnis 7 1. Einleitung 8 1.1 Periimplantitis 8 1.1.1 Dentale Implantate und umgebendes Gewebe in periimplantären gesunden Situationen 8 1.1.2 Periimplantitis 10 1.1.3 Periimplantitis versus Parodontitis 14 1.2 Das Mikrobiom bei Periimplantitis und Parodontitis 17 1.3 16s rRNA Sequenzierung 18 1.3.1 Hochdurchsatzsequenziermethodiken 18 1.3.2 Das 16s Gen 18 1.4 Hypothese und Fragestellung dieser Studie 19 2. Material und Methoden 21 2.1 Studienpopulation 21 2.1.1 Allgemeines 21 2.1.2 Ein- und Ausschlusskriterien 21 2.1.3 Dokumentierte Parameter 22 2.2 Probengewinnung und -aufbereitung 23 2.3 16s rRNA Sequenzierung und Datenaufbereitung 24 2.3.1 ‚Paired-End‘-Hochdurchsatz-Sequenzierung der V3-V4 Region des 16s rRNA Genes 25 2.3.2 Post-Sequenzierungs-Prozesse 28 2.4 Statistische Analyse 32 2.4.1 Assoziation mit PD 32 2.4.2 Netzwerkanalyse 33 2.4.3 Mikrobieller Dysbiose Index 34 2.4.4 Signifikante Unterschiede der bakteriellen Charakteristika 34 6 3. Ergebnisse 35 3.1 Studienpopulation (Tab. -
Table S5. the Information of the Bacteria Annotated in the Soil Community at Species Level
Table S5. The information of the bacteria annotated in the soil community at species level No. Phylum Class Order Family Genus Species The number of contigs Abundance(%) 1 Firmicutes Bacilli Bacillales Bacillaceae Bacillus Bacillus cereus 1749 5.145782459 2 Bacteroidetes Cytophagia Cytophagales Hymenobacteraceae Hymenobacter Hymenobacter sedentarius 1538 4.52499338 3 Gemmatimonadetes Gemmatimonadetes Gemmatimonadales Gemmatimonadaceae Gemmatirosa Gemmatirosa kalamazoonesis 1020 3.000970902 4 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas indica 797 2.344876284 5 Firmicutes Bacilli Lactobacillales Streptococcaceae Lactococcus Lactococcus piscium 542 1.594633558 6 Actinobacteria Thermoleophilia Solirubrobacterales Conexibacteraceae Conexibacter Conexibacter woesei 471 1.385742446 7 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas taxi 430 1.265115184 8 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas wittichii 388 1.141545794 9 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas sp. FARSPH 298 0.876754244 10 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sorangium cellulosum 260 0.764953367 11 Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae Sorangium Sphingomonas sp. Cra20 260 0.764953367 12 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Sphingomonas panacis 252 0.741416341 -
Diversity Structure of Culturable Bacteria Isolated from the Fildes Peninsula (King George Island, Antarctica): a Phylogenetic Analysis Perspective
RESEARCH ARTICLE Diversity structure of culturable bacteria isolated from the Fildes Peninsula (King George Island, Antarctica): A phylogenetic analysis perspective Gerardo GonzaÂlez-Rocha1, Gabriel Muñoz-Cartes1, Cristian B. Canales-Aguirre2,3, Celia A. Lima1, Mariana DomõÂnguez-YeÂvenes1, Helia Bello-Toledo1, CristiaÂn E. HernaÂndez2* a1111111111 1 Laboratorio de InvestigacioÂn en Agentes Antibacterianos. Departamento de MicrobiologõÂa, Facultad de Ciencias BioloÂgicas, Universidad de ConcepcioÂn, ConcepcioÂn, Chile, 2 Laboratorio de EcologõÂa Evolutiva y a1111111111 FiloinformaÂtica. Departamento de ZoologõÂa, Facultad de Ciencias Naturales y OceanograÂficas, Universidad a1111111111 de ConcepcioÂn, ConcepcioÂn, Chile, 3 Centro i~mar, Universidad de Los Lagos, Camino a Chinquihue 6 km, a1111111111 Puerto Montt, Chile a1111111111 * [email protected] Abstract OPEN ACCESS Citation: GonzaÂlez-Rocha G, Muñoz-Cartes G, It has been proposed that Antarctic environments select microorganisms with unique bio- Canales-Aguirre CB, Lima CA, DomõÂnguez-YeÂvenes chemical adaptations, based on the tenet `Everything is everywhere, but, the environment M, Bello-Toledo H, et al. (2017) Diversity structure selects' by Baas-Becking. However, this is a hypothesis that has not been extensively evalu- of culturable bacteria isolated from the Fildes ated. This study evaluated the fundamental prediction contained in this hypothesisÐin the Peninsula (King George Island, Antarctica): A phylogenetic analysis perspective. PLoS ONE 12 sense that species are structured in the landscape according to their local habitats-, using (6): e0179390. https://doi.org/10.1371/journal. as study model the phylogenetic diversity of the culturable bacteria of Fildes Peninsula pone.0179390 (King George Island, Antarctica). Eighty bacterial strains isolated from 10 different locations Editor: Patrick Jon Biggs, Massey University, NEW in the area, were recovered. -
Structural Basis of Mammalian Mucin Processing by the Human Gut O
ARTICLE https://doi.org/10.1038/s41467-020-18696-y OPEN Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila ✉ ✉ Beatriz Trastoy 1,4, Andreas Naegeli2,4, Itxaso Anso 1,4, Jonathan Sjögren 2 & Marcelo E. Guerin 1,3 Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness 1234567890():,; and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila,anO-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan. We determine the high-resolution X-ray crystal structures of the unliganded form of OgpA, the complex with the glycodrosocin O-glycopeptide substrate and its product, providing a comprehensive set of snapshots of the enzyme along the catalytic cycle. In combination with O-glycopeptide chemistry, enzyme kinetics, and computational methods we unveil the molecular mechanism of O-glycan recognition and specificity for OgpA. The data also con- tribute to understanding how A. muciniphila processes mucins in the gut, as well as analysis of post-translational O-glycosylation events in proteins. 1 Structural Biology Unit, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Spain. 2 Genovis AB, Box 790, 22007 Lund, Sweden. 3 IKERBASQUE, Basque Foundation for Science, 48013 ✉ Bilbao, Spain. 4These authors contributed equally: Beatriz Trastoy, Andreas Naegeli, Itxaso Anso. -
University of Oklahoma Graduate College
UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE MICROBIOLOGY OF WATER AND WASTEWATER: DISCOVERY OF A NEW GENUS NUMERICALLY DOMINANT IN MUNICIPAL WASTEWATER AND ANTIMICROBIAL RESISTANCES IN NUMERICALLY DOMINANT BACTERIA FROM OKLAHOMA LAKES A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY By Toby D. Allen Norman, Oklahoma 2005 UMI Number: 3203299 UMI Microform 3203299 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 MICROBIOLOGY OF WATER AND WASTEWATER: DISCOVERY OF A NEW GENUS NUMERICALLY DOMINANT IN MUNICIPAL WASTEWATER AND ANTIMICROBIAL RESISTANCES IN NUMERICALLY DOMINANT BACTERIA FROM OKLAHOMA LAKES A DISSERTATION APPROVED FOR THE DEPARTMENT OF BOTANY AND MICROBIOLOGY BY ____________________________ Dr. Ralph S. Tanner ____________________________ Dr. Kathleen E. Duncan ____________________________ Dr. David P. Nagle ____________________________ Dr. Mark A. Nanny ____________________________ Dr. Marvin Whiteley Copyright by Toby D. Allen 2005 All Rights Reserved “Science advances through tentative answers to a series of more and more subtle questions which reach deeper and deeper into the essence of natural phenomena” – Louis Pasteur iv ACKNOWLEDGEMENTS I consider myself fortunate to have had the opportunity to work on the projects contained in this work. I am grateful to have had the support and guidance of Dr. Ralph Tanner, who gave me the opportunity conduct research in his laboratory and to the Department of Botany and Microbiology, which has supported me in the form of teaching and research assistantships. -
Bacterial Diversity and Functional Analysis of Severe Early Childhood
www.nature.com/scientificreports OPEN Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India Balakrishnan Kalpana1,3, Puniethaa Prabhu3, Ashaq Hussain Bhat3, Arunsaikiran Senthilkumar3, Raj Pranap Arun1, Sharath Asokan4, Sachin S. Gunthe2 & Rama S. Verma1,5* Dental caries is the most prevalent oral disease afecting nearly 70% of children in India and elsewhere. Micro-ecological niche based acidifcation due to dysbiosis in oral microbiome are crucial for caries onset and progression. Here we report the tooth bacteriome diversity compared in Indian children with caries free (CF), severe early childhood caries (SC) and recurrent caries (RC). High quality V3–V4 amplicon sequencing revealed that SC exhibited high bacterial diversity with unique combination and interrelationship. Gracillibacteria_GN02 and TM7 were unique in CF and SC respectively, while Bacteroidetes, Fusobacteria were signifcantly high in RC. Interestingly, we found Streptococcus oralis subsp. tigurinus clade 071 in all groups with signifcant abundance in SC and RC. Positive correlation between low and high abundant bacteria as well as with TCS, PTS and ABC transporters were seen from co-occurrence network analysis. This could lead to persistence of SC niche resulting in RC. Comparative in vitro assessment of bioflm formation showed that the standard culture of S. oralis and its phylogenetically similar clinical isolates showed profound bioflm formation and augmented the growth and enhanced bioflm formation in S. mutans in both dual and multispecies cultures. Interaction among more than 700 species of microbiota under diferent micro-ecological niches of the human oral cavity1,2 acts as a primary defense against various pathogens. Tis has been observed to play a signifcant role in child’s oral and general health. -
Association of Coffee and Tea Intake with the Oral Microbiome: Results from a Large Cross-Sectional Study
Author Manuscript Published OnlineFirst on April 27, 2018; DOI: 10.1158/1055-9965.EPI-18-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Association of coffee and tea intake with the oral microbiome: results from a large cross-sectional study Brandilyn A. Peters,1 Marjorie L. McCullough,2 Mark P. Purdue,3 Neal D. Freedman,3 Caroline Y. Um,2 Susan M. Gapstur,2 Richard B. Hayes,1,4 Jiyoung Ahn1,4 1Division of Epidemiology, Department of Population Health, NYU School of Medicine, New York, NY, USA 2Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA 3Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA 4NYU Perlmutter Cancer Center, New York, NY, USA Running title: Coffee, tea, and the oral microbiome Corresponding author: Jiyoung Ahn (650 1st Avenue, 5th Floor, New York, NY, 10016; phone: 212-263-3390; fax: 212-263-8570; email: [email protected]) Conflict of interest statement: No conflicts of interest to disclose. Downloaded from cebp.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2018; DOI: 10.1158/1055-9965.EPI-18-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 1 ABSTRACT 2 Background: The oral microbiota play a central role in oral health, and possibly in 3 carcinogenesis. Research suggests coffee and tea consumption may have beneficial 4 health effects. We examined the associations of these common beverages with the oral 5 ecosystem in a large cross-sectional study.