pissn: 2288-0402 eissn: 2288-0410 Allergy Asthma Respir Dis 4(5):311-320, September 2016 http://dx.doi.org/10.4168/aard.2016.4.5.311 REVIEW 국내외호흡기및인체마이크로바이옴연구 최성미, 1 조상헌, 2 이하나 1,3 1 고려대학교보건과학과, 2 서울대학교의과대학내과학교실, 3 고려대학교보건과학대학바이오시스템의과학부 Human microbiome studies in Korea Sungmi Choi, 1 Sang-Heon Cho, 2 Hana Yi 1,3 1 Department of Public Health Sciences, Graduate School, Korea University, Seoul; 2 Department of Internal Medicine, Seoul National University College of Medicine, Seoul; 3 School of Biosystem and Biomedical Science, Korea University, Seoul, Korea During the second half of the 2000s, the significant impact of human microbiome on human diseases and health conditions was found. Since the Human Microbiome Project, many microbiome studies have been reported in domestic and international references. Gastrointestinal tract microbiome has been most investigated so far, and the association with illness has been demonstrated in many diseases. Recently, the range of study was extended to multiple human organs, such as the respiratory tract, skin, and urogenital tract. Given the scale and speed of research and development in recent years, the role of microbiome in many diseases would be established before long. In this review, we aimed to summarize the current status of microbiome studies in Korea and foreign countries with an emphasis on respiratory tract microbiome. The main concept and analytical methods for microbiome research, associations of microbiome and diseases, and research projects on Korean microbiome are reviewed. (Allergy Asthma Respir Dis 2016:4:311-320) Keywords: Microbiota, Gastrointestinal microbiome, Metagenomics, Respiratory airflow, Korea 서론 1990 년에시작하여 27 억달러를투입하여진행된인간게놈프로 젝트 (Human Genome Project, HGP) 가인간전장유전체분석을 완료하였을때, 1 많은학자들은인간게놈을 the blueprint for human life 라고부르며인간질병의난제들을상당부분해결해줄것 으로기대했다. 그러나인간게놈의초안을확인한학자들이직면했 던가장큰놀라움은인간게놈이예상외로너무단순하다는것이 었다. 프로젝트를시작할당시인체의복잡성을고려하여당초 10 만개정도의유전자가존재할것이라고믿었으나, 결과적으로는인 간이초파리와비슷한 2 만개정도의유전자를가지고있다는것이 밝혀졌다. 이것으로는인체의복잡한생명현상을설명하는데한 계가있었기에인체에더불어공존하고있는미생물들과그들의유 전자에의해크게영향을받고있어이런다양한유기체들의총체 를하나의생명현상으로이해해야한다는 superorganism 이라 는개념이탄생하였다. 2 Correspondence to: Hana Yi http://orcid.org/0000-0002-4910-122x School of Biosystem and Biomedical Science, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea Tel: +82-2-3290-5644, Fax: +82-2-940-2849, E-mail: hanayi@korea.ac.kr *This research was supported by a fund (code: 2015ER660300) by Research of Korea Centers for Disease Control and Prevention. Received: March 30, 2016 Revised: April 29, 2016 Accepted: May 16, 2016 인간게놈해독이끝난직후이미일부과학자들은마이크로바 이옴연구의필요성을깨닫고연구에착수하였다. Davies 3 는인간 게놈해독이 2001 년 2 월에발표된후한달뒤 Science 지에기고를 통해, 우리몸에살고있는 1,000 종이상의상재균과이들이가지고 있는 2 4 백만개의유전자를파악하고, 인체와미생물의상호작용 을밝혀야만인체의건강과질병을이해할수있다고주장하였고, 이를바탕으로인체미생물상의유전자목록 (inventory of microbial genes and genomes) 을작성하는 세컨드게놈프로젝트 (A second human genome project) 가시작되었다. 4 이후인체마이크 로바이옴에대한관심은확대되어 Nature 와 Science 지에서는 2010 년과 2012 년에핵심주제로마이크로바이옴을다루었으며, 2015 년 에는 Nature 지에마이크로바이옴특별편이게재되고, 2016 년에는 Nature 자매지로 Nature Microbiology 가발간될정도로, 마이크 로바이옴은최근생물학계의가장큰이슈로각광받고있다. 이처 럼 2000 년대후반부터본격적으로시작된마이크로바이옴연구는 주로장내마이크로바이옴을대상으로하는연구가수행되었으며, 2016 The Korean Academy of Pediatric Allergy and Respiratory Disease The Korean Academy of Asthma, Allergy and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/). 311 http://www.aard.or.kr
Allergy Asthma Respir Dis Choi S, et al. Korean microbiome studies 최근에는장이외에호흡기를비롯한여러기관에존재하는마이크로바이옴연구가진행되고있다. 따라서본논문에서는마이크로바이옴의개요와국내외마이크로바이옴관련연구들에대해개괄적으로살펴보고자한다. 마이크로바이옴의정의마이크로바이옴 (microbiome) 이라는용어는노벨생리의학상수상자인 Lederberg와 McCray 5 의 2001년 The Scientist지기고를통해최초로정의되었다. 그는 마이크로바이옴은인체에존재하며우리몸을함께공유하며살고있지만그동안건강이나질병의원인으로거의무시되어온상재균 공생균 병원균등모든미생물들의총합 ( the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease. ) 이라고정의하였으며현재는세균 (bacteriome) 뿐만아니라바이러스 (virome) 와곰팡이 (mycobiome) 까지모든미생물을포괄하는용어로사용된다. 마이크로바이옴은마이크로바이오타 (microbiota) 라는용어와혼동하여쓰이는데엄밀히말하자면마이크로바이오타는인체에서식하는미생물군집을유전자수준이아닌개체수준으로한정하여지칭할때쓰는표현이며, 마이크로바이옴은마이크로바이오타와그들이가진유전정보전체 ( the catalog of these microbes and their genes ) 를통칭한다. 6 인체를구성하는체세포의수가약 10 13 개인데반해인체에공생하는미생물은 10 14 개로 superorganism 전체세포의 90% 이상을차지한다. 7 인체마이크로바이옴의 95% 는대부분장을포함한소화기관에존재하지만호흡기, 생식기, 구강, 피부등에도널리분포한다. 8 마이크로바이옴은체중의 1% 3% 를차지하지만인간의건강에매우중요한역할을하고있는데, 8 예로마이크로바이옴은 superorganism의유전적다양성의원천이자면역작용의중요요소이며, 약물에대한반응을조절하고대사에영향을주는기능적단위이기도하다. 이런중요성에따라 2006년 O Hara와 Shanahan 9 은장내마이크로바이옴을 제2의기관 (forgotten organ) 이라고칭하기도하였다. 마이크로바이옴분석기술현대적인의미의마이크로바이옴연구는 2000년대에메타게노믹스와유전자시퀀싱기술이일반화되면서본격적으로시작되었다. 대부분의미생물이배양되지않는다는사실 ( the great plate count anomaly ) 이알려진후 10,11 배양을통해미생물상을분석하는방법은더이상쓰이지않고있다. 일반적인자연환경의경우약 1% 미만의소수의미생물들만이일반적인실험실배양조건에서 배양이가능하다고알려져있는데, 12,13 이러한난배양성은인체에살고있는미생물에게도마찬가지이며, 신체부위에따라다르지만대략 20% 60% 정도의마이크로바이옴이배양불가하다고추정된다. 14 따라서배양법의한계를극복하고자연환경에존재하는미생물군집을있는그대로유전자수준에서해독하기위해여러가지분자생물학적인실험방법들이개발되었다. 유전자지문 (DNA fingerprinting) 분석법, 마이크로어레이칩등이많이사용되었으나, 가장최근의분석방법은메타게노믹스이다. 메타게놈 (metagenome) 이란 Handelsman 등 15 이처음사용한용어로 시료에존재하는모든미생물유전체의집합 (collective genomes of soil microflora) 이라는뜻이다. 메타게노믹스 (metagenomics) 또는군유전체학연구를위해서는미생물을분리및배양하는과정없이시료로부터직접 DNA를추출한다. 추출한메타게놈에는시료에존재하던모든미생물의유전체 DNA가섞여있게되는데, 이를차세대염기서열분석법을통해염기서열을확인하여유전자수준에서군집구조를분석한다. 메타게노믹스의목적은첫째, 시료내에어떤미생물들이존재하는지를분석하는것이고둘째는시료내에서어떤대사과정과기능유전자들이존재하는지를밝히는것이다. 기능유전자를보기위해서는샷건 (whole-genome shotgun) 메타게노믹스방법을주로사용하는데, 기능성유전자들의구성을포함하여전체메타게놈그대로를볼수있다는점은장점이지만시퀀싱비용이많이들고시퀀스의동정이어려운단점이있다. 반면미생물군집분석을하여어떤미생물이존재하는지를알고싶을때는종동정을위한표지유전자만을선택적으로증폭한후그증폭산물의염기서열을분석하는앰플리콘 (amplicon) 메타게노믹스방법을사용함으로써비용및분석노력을크게절감할수있다. 세균군집분석을하기위해서는 16S rrna 유전자를마커유전자로사용하는데, 어떤시퀀싱장비를사용할것인지에따라서사용하는프라이머가달라진다. GS Junior를사용하여분석하려고하면변이부위 V1 V3 부분을증폭하는 27F와 518R 프라이머를사용하는데, 454 기술은 reverse primer로부터거꾸로시퀀스를읽기때문에 V3에서시작하여 V1 언저리에다다르는 400 450 bp 길이의염기서열한가닥이얻어진다. 이에비해 MiSeq v3를이용하여시퀀싱한다면, V3 V4 부분을증폭하는 318F 와 806R 프라이머를사용하고 paired-end sequencing 을통해 forward 와 reverse 쪽의 300 bp 길이의염기서열한쌍을하나로합쳐서 400 bp 이상의길이의염기서열을산출한다. 시퀀싱장비에서산출된원본데이터는실험및시퀀싱과정에서발생하는에러들을포함하고있다. Polymerase chain reaction 과정에서중합효소가원래가지고있는오류와이종 DNA 간의키메라시퀀스생산, 그리고시퀀싱도중발생하는호모폴리머에러 (homopolymer) 등, 오류의진원지는매우다양하고실험법이나시 312 http://dx.doi.org/10.4168/aard.2016.4.5.311
최성미외 마이크로바이옴연구현황 Allergy Asthma Respir Dis 퀀싱장비에따라오류유형도달라진다. 이렇게잘못만들어진시퀀싱리드 (read) 들은필터링을통해걸러지고, 그후남은리드들을가지고시퀀스동정 (taxonomic assignment) 과다양성분석 (diversity calculation) 을수행한다. 메타게노믹스를위한다양한생물정보학적분석플랫폼들이개발되어있는데그중가장많이사용되는프로그램으로는 QIIME, 16 MOTHUR, 17 RPD, 18 PlutoF 19 등이있다. 이들플랫폼은원본데이터로부터분석에적합한서열만을필터링하여바코드에따른분리, 동정, 다양성계산들을수행한다. 인체마이크로바이옴의역할 1992년 Bocci 20 는장내마이크로바이옴을 neglected organ 이라고표현하며사실상우리몸의하나의기관으로간주할만큼인체면역조절작용에중대한역할을한다고주장하였고, 1998년 Wold 21 는알레르기질환이증가하는원인역시마이크바이옴으로지목하면서소화기능을돕는정도로간주되었던장내마이크로바이옴은 1990년대에들어서야비로소인체건강상태를좌우하는주요인자로새로이인식되게되었다. 이후많은연구를통해서마이크로바이옴은체내에서영양분흡수, 약물대사조절, 면역체계조절, 뇌 / 행동발달조절및감염성질환예방등의역할을하고있는것으로밝혀졌다. 1. 영양분흡수장내세균은주로 Firmicutes와 Actinobacteria에속하지만사람마다장내마이크로바이옴의구조는다르다. 22 동일한영양분을섭취하더라도개체에따라영양분흡수양상에차이를보이는것은유전적차이라고만여겨졌으나최근에는그원인을장내마이크로바이옴구조에서찾기도한다. 마이크로바이옴은장내강에서의단당류의흡수에관여하고간에서의지방생성에까지영향을주는것이밝혀졌으며이를바탕으로비만의원인가운데하나로지목받았다. 22 이를기반으로장내마이크로바이옴을사전에분석하여영양실조또는비만의가능성이높은관찰군을선별하는바이오마커의연구가국내외에서진행중이다. 2. 약물대사조절마이크로바이옴은다양한대사작용및효소작용을바탕으로체내에유입된약물이나발암물질로부터인간을보호하는기능이있다. 23,24 장내마이크로바이옴에의한탈수산화, 탈카르복실화, 탈알킬화그리고탈아미노화반응과같은대사작용과효소작용등이보고된바있고 25,26 마이크로바이옴이수산염 (oxalate) 의대사에영향을끼치거나 27,28 지방대사과정에서사용되는담즙산의생성과정에도관여하는것도연구되었다. 29-31 3. 면역체계조절인체의면역시스템은고전적개념으로는외부의병원성미생물로부터인체를보호하는것이지만, 인체에는많은미생물이공생하며복잡한시스템을이루고있기때문에면역체계와미생물이상호작용을통해면역시스템을이루고있다는개념으로확장되고있다. 32 항체나면역세포가미생물의기작과개체수를조절하기도하며, 반대로마이크로바이옴이비장이나흉선과같은림프계의발달에중요한역할을하고면역세포의기능에영향을끼치기때문에 33 신생아시기에장내에마이크로바이옴이제대로형성되지않으면면역학적관용이정착되지않아알레르기가발생할가능성이높다는것이밝혀졌다. 21 아토피환자의피부에서 Gammaproteobacteria의다양성이저하되면서 interleukin (IL)-10 이감소되며, 그에반해건강인에서는 Acinetobacter의비율이높아지며동시에 IL-10 의생산량높게관찰되는등 34 아토피환자의면역관용과마이크로바이옴의상관성이보고되었다. 4. 뇌 / 행동발달조절마이크로바이옴과그생성물질은뇌의발달과신경에영향을주기도한다. 35 동물실험결과, 무균쥐에서는포유류의운동통제및분노조절과관련된뇌의 2차전령경로 (second messenger pathway) 유전자들의발현이달라지지만, 정상마이크로바이옴에노출된이후에는정상생쥐와유사한발현을보였다. 36 인간을대상으로한연구에서는장내유익균으로알려진 Lactobacillus을비롯한여러유산균을장기간섭취하였을경우, 대조군에비해우울증과분노의정도가상당수줄어들었다. 37,38 5. 감염성질환예방안정적으로정착된인체마이크로바이옴은외부병원균의침입에대항해방어막의역할을한다. 신생아시기모유수유는신생아장내 Bifidobacterium bifidus의비율을높여병원균으로부터신생아를보호하는효과가있다. 39,40 아프리카와아시아의영아에게흔히발생하는설사증의경우, 마이크로바이옴에 Lactobacillus ruminis이존재하거나그비율이높을경우설사증이발병하지않거나발병하더라도발병률과심각도가낮았으며, 41 여행자가여행중설사증에걸리는그룹과그렇지않은그룹의장내마이크로바이옴의구조도공통적인차이를보였다. 42 마이크로바이옴과질병 1. 비만, 당뇨및대사질환장내마이크로바이옴유형은크게 2가지유형, Firmicutes와 Actinobacteria가높은경우와 Bacteroidetes의비율이높은경우로나눌수있다. 43 비만인쥐는정상쥐에비해 Bacteroidetes의비율이 http://dx.doi.org/10.4168/aard.2016.4.5.311 313
Allergy Asthma Respir Dis Choi S, et al. Korean microbiome studies 작고 Firmicutess 는높은비율로존재한다. 44 정상쥐에게고지방식이를통해비만을유도하면마이크로바이옴도비만형으로변화하며, 45-47 무균의쥐에비만쥐의장내마이크로바이옴을이식하면체내지방축적이증가하며, 22 동물모델을벗어나인간쌍둥이를대상으로한실험에서도앞선실험과동일한결과가확인되었다. 46 이는미생물의변화가물질대사과정에영향을주어발생한결과로, 실제로무균쥐의장에비만쥐의마이크로바이옴을이식하면단당류의흡수가높아지고지방세포의트리글리세리드축적되었다. 46 대사작용에관여하는세균인 Akkermansia muciniphila는점액에서식하며뮤신 (mucin) 을분해하는역할을하는데, 항생제로이세균을제거할경우지방이축적되고지방세포가커졌다. 48,49 이는 A. muciniphila가비만과이형당뇨병을낮추는역할을하고있다는증거이며반대로 2형당뇨를가진환자들은장내미생물의균형이깨지는것역시확인되었다. 일반적으로존재하는 butyrate 생산세균들이줄어들고, 대신다양한기회감염균들이증가하였으며, 50 sulfate reduction 이나 oxidative stress resistance 에관련된유전자들도증가하였다. 50 따라서구조및변화분석을통해마이크로바이옴을 2형당뇨진단마커로쓸수있을것으로기대하고있다. 2. 염증성장질환 (inflammatory bowel disease) 염증성장질환 (inflammatory bowel disease, IBD) 은대장성크론병 (Crohn disease) 과궤양성대장염 (ulcerative colitis) 을포함하는만성적인염증성질환으로아직까지발생기작이정확히밝혀지지않았으나유전적요인과환경적요인을비롯하여마이크로바이옴이중요원인가운데하나로알려져있다. 51 대장성크론병은특이하게무균쥐에서는발병하지않는데 52 이것은장내마이크로바이옴을항원으로인식하여면역반응및염증이발생하기때문으로예측하였다. 2010년실시된 40쌍의쌍둥이연구에따르면회장과결장위주의대장성크론병을가진환자의장내마이크로바이옴은건강한사람의장내마이크로바이옴과차이를보였고, 53 대장성크론병환자의분변샘플에서 OTUs의수가현저히떨어지고마이크로바이옴의다양성도낮았으며, Firmicutes와 Proteobacteria가상대적으로많이분포하는경향을보였다. 53 3. 인지장애장에존재하는그람양성혐기성세균이나 Lactobacillus, Bifidobacterium 은 GABA (gamma-aminobutyric acid) 의대사에영향을주기때문에, 마이크로바이옴의조성변화가중추신경의변화를야기하여여러질환을야기함이밝혀졌다. 54 그밖에도 Cyanobacteria가생성하는 saxitoxin, anatoxin 등은신경질환을야기할수있는데특히노화로인해흡수율이높아지는것으로알려져있으며, 다른생성물질의영향으로인해파키슨질환을발생할수 있다는연구결과가보고되었다. 54 노인성치매쌍둥이연구결과에서치아손실과치매가연관이있다는보고를시작으로구강세균구조와치매의연관성이제기되었다. 55 구강세균이알츠하이머와관련이있다는것이밝혀졌는데, 56 구강마이크로바이옴이뇌, 특히치매에영향을주게된이유로는첫째, 노화로인해구강마이크로바이옴을안정화시키는침의분비가줄어들게되면서유해한균이구강내에과도하게성장하게되며둘째, 삼차신경과후각신경과같은신경들이구강세균이뇌로침입하거나세균이분비하는아밀로이드등을뇌로전달하는통로로작용할수있기때문이라고추측하고있다. 57 4. 심혈관계질환붉은육류를과도하게섭취하게되면붉은육류에풍부한 L-carnitine을장내미생물이대사하면서생기는물질이동맥경화를유발하기때문에이후에그양을줄여도동맥경화가생길확률이높아진다. 58 그러나육류를섭취하지않는채식주의자들은해당미생물이적은마이크로바이옴환경을가지고있기때문에육류를섭취해도동맥경화유발물질이생기지않아질병에걸리지않는다. 58 구강마이크로바이옴도심혈관질환에영향을줄수있는것으로알려져있으며, 따라서심혈관질환예방및완화를위해서도평소인체마이크로바이옴관리가필요하는것을시사한다. 57 국외연구동향마이크로바이옴에대한연구는 2007년 Human Microbiome Project Jumpstart가시작되면서본격적으로시작되었다. 초기에는인체마이크로바이옴연구가주를이루었으나점차동물과환경내마이크로바이옴의대규모연구로확장되었으며, 2010년부터는데이터의양이폭발적으로증가하며연구의규모가확장되었다. 2012년이후로는기존의미국및유럽연합주도의마이크로바이옴프로젝트외에한국, 프랑스, 호주, 일본등개별국가수준의연구들이시작되었으며장내마이크로바이옴뿐만아니라호흡기, 피부, 구강, 여성생식기등으로연구대상이확장되었다 (Table 1). 1. International Human Microbiome Consortium 다국적연구자간데이터공유와비교분석을위해 2008년 10월 ' 국제인체마이크로바이옴컨소시엄 (International Human Microbiome Consortium, IHMC) 이조직되었다. 2015년까지 9번의정기회의를통해각프로젝트연구내용을공유하고책자와뉴스레터를통해내용을전달하였다. 현재 full member로가입한나라는총 9개국이며 observing member 는 5개국으로, 이중우리나라는 2개기관이각각 full member 와 observing member 로활동하고있다. 314 http://dx.doi.org/10.4168/aard.2016.4.5.311
최성미외 마이크로바이옴연구현황 Allergy Asthma Respir Dis Table 1. Major international microbiome projects Country Project title Duration Objective International International Human Microbiome Consortium (IHMC) 2010 Set principles and policies to study microbiome. Mediate projects to generate a comprehensive data resource. EU Metagenomics of the Human Intestinal Tract (MetaHIT) 2008 2011 Construct large catalog of intestinal microbiome and genes of IBD and obesity. International Human Microbiome Standards (IHMS) 2011 2015 Establish a standard protocol for human microbiome studies. USA NIH Human Microbiome Project (HMP) 2008 2015 Generate resources of the human microbiome. Diagnosis and treatment through a correlation between diseases and microbiome. Home Microbiome Project 2012 Analyze bacterial community in living space. Data Analysis and Coordination Center (DACC) 2008 2013 Assist in standard of data pipeline. Hospital Microbiome Project 2012 2014 Analyze bacterial community in hospital, and find correlation between the bacteria and patient. Microbiome Quality Control Project (MBQC) 2013 Establish analytic pipeline of human fecal microbiome. American Gut Project 2013 Study of bacterial diversity of the public and construct data resource. Japan Japanese Consortium for Human Microbiome (JCHM) 2014 Study of Japanese specific intestinal microbiome and disease biomarker. Canada Canadian Microbiome Initiative (CMI) 2009 Support seven research teams to conduct a prospective study of diseases by modeling and mapping microbial diversity. France MicroObes 2008 2010 Research on obesity and human intestinal microbiome. Korea MetaGenoPolis (MGP) 2012 2019 Find the correlation of non-infectious diseases and intestinal microbiome. Korean Microbiome Diversity Using Korean Twin Cohort Project 2010 2015 Study on human diseases related microbiome using twin cohort and characterize Korean specific microbiome. Australia The Australian Jumpstart Human Microbiome Project 2009 Analyze metagenome of intestinal microbes and Australian specific microbiome. Number of publications Number of publications 160 140 120 100 80 60 40 20 0 160 140 120 100 80 60 40 20 0 Fig. 1. Number of the publications by National Institutes of Health (NIH) Human Microbiome Project (HMP), USA. Distribution of the publications depending on calendar year (A), and depending on associated organs or diseases (B). Data from the NIH HMP website (http://hmpdacc.org). 2. 미국 2009 2010 2011 2012 2013 2014 2015 Calendar year Gut Obesity IBD 1) Human Microbiome Project Vaginal Skin Oral Organs or diseases Probiotics Respiratory Immune Urine Cancer 2007 년미국국립보건연구원에서 인체마이크로바이옴프로젝 트 (Human Microbiome Project, HMP) 를발족하였다. 2007 년부 A B 터 2012년까지 1단계 (phase I) 을완료하였으며, 2016년현재 2013년부터진행된 2단계 (phase II) 의막바지연구가진행중이다. 2015년까지발표된논문은총 539편이며, 주로장내마이크로바이옴과비만관련논문이많았다 (Fig. 1). 1단계 HMP는인체에존재하는미생물의특성을규명하고건강과의상관성을설명하는것을목표로했다. 5년간 1억 7,300만달러 (1,900억원 ) 의연구비가지원된결과, 마커유전자및메타게놈을분석하기위한새로운기술적접근법및표준화된일련의분석파이프라인을구축하였다. 2단계 HMP는 Integrative HMP (ihmp) 라는프로젝트명으로 2013년에시작되었다. 1단계 HMP가 Who is there? 즉, 우리몸에어떤미생물들이있는지를밝히는것에목표를두었다면, 2단계 ihmp는 What are they doing? 즉, 인체에존재하는미생물들이어떤역할을하는가를밝히는것에목표를두었다. 3년간 275억원규모의이프로젝트는총 15개연구기관을중심으로세가지중점질병 ( 조산, IBD, 당뇨병 ) 을분담하여연구중이다. 3. 유럽 1) META genomics of the Human Intestinal Tract 유럽연합 8개국, 15개기관이참여하는 인체장내메타지노믹스 (META genomics of the Human Intestinal Tract) 프로젝트는 2008년에시작되었다. 유럽연합의행정부역할을담당하는유럽연합진행위원회 (European Commission) 에서 4년간총약 250억원을투입한본프로젝트는 IBD 및비만과연관된장내마이크로바 http://dx.doi.org/10.4168/aard.2016.4.5.311 315
Allergy Asthma Respir Dis Choi S, et al. Korean microbiome studies 이옴의역할파악및상관관계분석을목표로하였다. 그결과 2010 년, 인간게놈의 150배에달하는숫자인 330만개의유전자를발견하여폭넓은장내마이크로바이옴유전자카탈로그를구축하였으며, 2014년에는사상최대규모의데이터분석논문을발표하였다. 2) International Human Microbiome Standards 12개국에서 8개의파트너회사와 15명의참여자가인체마이크로바이옴분석을위한최적의프로토콜을정립하기위하여 유럽국제인체마이크로바이옴스탠다드 (International Human Microbiome Standards) 를진행하였다. 15명의자원봉사자는자신의인체내에서분석에용이한샘플을제공하였으며각파트너회사는해당샘플에서부터최상의결과를도출하기위한프로토콜을생성및정리하였다. 그결과 (1) 시료수집및핵산추출, (2) 시퀀싱, (3) 데이터분석의 3단계총 14개의표준운영절차를정리하여공개함으로써마이크로바이옴연구를위한최적화된분석법을제공하는지원역할을하였다. 3) 프랑스 MetaGenoPolis 프랑스정부는비감염성질환과장내미생물간의영향을입증하고장내미생물간상호작용의핵심요소를결정하며모델링하는새로운방법개발하고자 MetaGenoPolis 를발족하였다. 인간장내마이크로바이옴의참조균의분석및유전체분석을수행하였으며, 이과정에서샘플의생물정보학적분석도구를개발하고분석결과의데이터베이스를구축하였다. 4. 아시아 1) 일본 Japanese Consortium for Human Microbiome 도쿄공업대학이주축이되어진행하는 Japanese Consortium for Human Microbiome은일본인고유의장내마이크로바이옴의특성을찾고, 일본인만의데이터베이스의구축을하는것을목표로하는프로젝트다. 이를활용하여가장적절한질병의치료법을찾고이를위한신약과보조식품등을개발하기위해여러제약회사및사업파트너들과연구과제를수행중이다. 국내연구동향 1. 국내마이크로바이옴연구과제 2011년 5월, 한국은국제인체마이크로바이옴컨소시엄 (IHMC) 에 8번째회원국으로가입하여국제적인수준의마이크로바이옴연구를수행중에있다. IHMC 국내연구팀인서울대보건대학원고광표교수팀이미국연구팀과함께흑인, 백인, 한국인쌍둥이를대상으로마이크로바이옴을분석하여한국인의마이크로바이옴구성이미국인과는다르다는사실을발표하면서본격적인마이크 Number of projects 로바이옴연구가국내에서도시작되었다. 2016 년 1 월까지국내에서 진행되고있는국가연구과제는 42 건으로, 특히 2014 년이후부터 과제의수가급격히증가하였으며추후에도지속적으로증가할것 으로예측된다 (Fig. 2). 국제마이크로바이옴연구추세와마찬가지 로국내에서도장내마이크로바이옴연구가연구주제의대부분을 차지하고있다. 호흡기관련마이크로바이옴연구는총 5 건으로호 흡기및알레르기질환과관련된주요과제로는숙명여자대학교성 미경교수연구팀의 만성호흡기알레르기질환관련미생물군집 분석및상호작용규명, 서울대학교유현주교수연구팀의 마이크 로바이옴조절을통한천연물유래만성호흡기질환치료제개발 및고려대학교윤원석교수연구팀의 마이크로바이옴조절을통한 천연물유래만성호흡기질환치료제개발 그리고서울대천종식 교수연구팀의 호흡기감염에서세균및바이러스메타지놈분석 등이있다. 그러나그연구의수와기간이장내마이크로바이옴에 비해많이부족하기때문에많은지원및추가연구가필요한실정 이다. 25 20 15 10 5 0 No. of projects No. of publications 2008 2009 2010 2011 2012 2013 2014 2015 Fig. 2. Count of microbiome research funding and publications in Korea. 2. 마이크로바이옴연구논문 2016 년 1 월현재까지국내연구진이출간한마이크로바이옴관 련연구논문 (SCI 급, 리뷰논문제외 ) 은총 29 편으로그중호흡기 관련논문은 4 편이다 (Fig. 2) ( 부록 : Supplementary Table 1). 2014 년건강인과바이러스감염환자의상기도호흡기마이크로바이옴 구조를보고한논문과 59 2014 년천식, 만성폐쇄성폐질환, 건강인의 구인두의마이크로바이옴구성차이를보고한논문이있으며 60 2014 년만성부비동염환자의비강마이크로바이옴감소를보고한 논문 61 그리고 2015 년코의곰팡이군이알레르기비염에미치는영 향을보고한논문이있다. 62 Calendar year 결론 지금까지많은연구를통해인체마이크로바이옴과질병간의 상관성이밝혀졌으며, 마이크로바이옴의구성변화를추적하여질 316 http://dx.doi.org/10.4168/aard.2016.4.5.311
최성미외 마이크로바이옴연구현황 Allergy Asthma Respir Dis 환유발가능성을예측하는진단마커개발가능성이열렸다. 비만 과심혈관계질환의예측지표가될수있음은물론, 63,64 임산부장 내마이크로바이옴을사용하여조산가능성을예측하고 65 법의학 적인관점에서범죄수사에활용할수있다. 66 그중에서도가장활발히연구가진행되고있는분야는장내마 이크로바이옴변화를이용한치료법들이다. Clostridium difficile 감염증의경우항생제만투여한환자보다건강인의장내마이크로 바이옴을환자에이식하는분변이식법을시행한결과더높은완 치율과낮은재발률을보이는것이알려져있고 67-69 프로바이오틱스 (probiotics) 섭취는체중과신체질량지수 (body mass index) 를감소 시키고인슐린감수성을높인다는연구결과가보고되어이를활용 한비만및당뇨병보조치료제들의연구도활발히진행중이다. 70 최근에는대부분장내마이크로바이옴에한정되어있던기존의 마이크로바이옴과질병상관성연구가호흡기를비롯한여러인체 기관으로확장되고있다. 그러나아직까지천식, 만성폐쇄성폐질환 등만성호흡기질환의예방및관리를위한마이크로바이옴연구 는장내마이크로바이옴에비교하면절대적으로부족하다. 본문 에서살펴보았듯이국내호흡기마이크로바이옴연구는현재까지 다섯건정도에그치고있으며, 대부분환자와건강인사이의마이 크로바이옴구성차이를통해상관성을찾으려고하는초기단계 의연구들이주를이루고있다. 또한질병의진단및치료를위하여 건강인과환자를구별할수있는마커세균과유전자를발굴하는 것매우중요하며, 더나아가인체의면역및대사체계와마이크로 바이옴의상호작용을밝히기위해서인체와마이크로바이옴의통 합분석이필수적이다. 그러나이러한유전체 - 전사체 - 발현체 - 대사 체통합분석을수행하기위해서는상당한재원및분석기술이뒷 받침되어야하며, 이는개별연구자들이감당하기어려운수준이 다. 더욱이호흡기는국제표준프로토콜이정립되어있는장내마 이크로바이옴과달리아직시료채취위치, 채취방법등에서국제 적인표준화가이루어지지않은상태이기때문에, 임상적용을위 한대규모연구를수행하는것에한계가있다. 이러한이유로호흡기마이크로바이옴을분석하기위한연구방 법론및중장기연구전략을국가적인차원에서수립할필요가있 다. 많은만성호흡기질환중연구의시급성이요구되는질병우선 순위를정하고, 여러연구자간의결과들을통합하여비교할수있 도록범용적인연구방법론을정립하고, 유기적으로조직된연구팀 내에서통합분석의역할을분담함으로써만성호흡기질환의예방 및관리를위한근원적인연구결과를도출할수있을것이다. REFERENCES 1. Collins FS, Morgan M, Patrinos A. The Human Genome Project: lessons from large-scale biology. Science 2003;300:286-90. 2. Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI. The human microbiome project. Nature 2007;449:804-10. 3. Davies J. In a map for human life, count the microbes, too. Science 2001; 291:2316. 4. Relman DA, Falkow S. The meaning and impact of the human genome sequence for microbiology. Trends Microbiol 2001;9:206-8. 5. Lederberg J, McCray AT. 'Ome sweet 'omics: a genealogical treasury of words. Scientist 2001;15:8-10. 6. Ursell LK, Metcalf JL, Parfrey LW, Knight R. Defining the human microbiome. Nutr Rev 2012;70 Suppl 1:S38-44. 7. Savage DC. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 1977;31:107-33. 8. Grice EA, Segre JA. The human microbiome: our second genome. Annu Rev Genomics Hum Genet 2012;13:151-70. 9. O'Hara AM, Shanahan F. The gut flora as a forgotten organ. EMBO Rep 2006;7:688-93. 10. Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 1985;39:321-46. 11. Ward DM, Weller R, Bateson MM. 16S rrna sequences reveal numerous uncultured microorganisms in a natural community. Nature 1990; 345:63-5. 12. Pace NR. A molecular view of microbial diversity and the biosphere. Science 1997;276:734-40. 13. Torsvik V, Goksøyr J, Daae FL. High diversity in DNA of soil bacteria. Appl Environ Microbiol 1990;56:782-7. 14. NIH HMP Working Group, Peterson J, Garges S, Giovanni M, McInnes P, Wang L, et al. The NIH Human Microbiome Project. Genome Res 2009; 19:2317-23. 15. Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 1998;5:R245-9. 16. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 2010;7:335-6. 17. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 2009;75:7537-41. 18. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. Ribosomal Database Project: data and tools for high throughput rrna analysis. Nucleic Acids Res 2014;42(Database issue):d633-42. 19. Abarenkov K, Tedersoo L, Nilsson RH, Vellak K, Saar I, Veldre V, et al. PlutoF: a web based workbench for ecological and taxonomic research, with an online implementation for fungal ITS sequences. Evol Bioinform 2010;6:189-96. 20. Bocci V. The neglected organ: bacterial flora has a crucial immunostimulatory role. Perspect Biol Med 1992;35:251-60. 21. Wold AE. The hygiene hypothesis revised: is the rising frequency of allergy due to changes in the intestinal flora? Allergy 1998;53(46 Suppl):20-5. 22. Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A 2004;101:15718-23. 23. Carmody RN, Turnbaugh PJ. Host-microbial interactions in the metabolism of therapeutic and diet-derived xenobiotics. J Clin Invest 2014;124: 4173-81. 24. Johnson CH, Patterson AD, Idle JR, Gonzalez FJ. Xenobiotic metabolomics: major impact on the metabolome. Annu Rev Pharmacol Toxicol 2012;52:37-56. http://dx.doi.org/10.4168/aard.2016.4.5.311 317
Allergy Asthma Respir Dis Choi S, et al. Korean microbiome studies 25. Saad R, Rizkallah MR, Aziz RK. Gut Pharmacomicrobiomics: the tip of an iceberg of complex interactions between drugs and gut-associated microbes. Gut Pathog 2012;4:16. 26. Sousa T, Paterson R, Moore V, Carlsson A, Abrahamsson B, Basit AW. The gastrointestinal microbiota as a site for the biotransformation of drugs. Int J Pharm 2008;363:1-25. 27. Miller AW, Dearing D. The metabolic and ecological interactions of oxalate-degrading bacteria in the Mammalian gut. Pathogens 2013;2:636-52. 28. Siva S, Barrack ER, Reddy GP, Thamilselvan V, Thamilselvan S, Menon M, et al. A critical analysis of the role of gut Oxalobacter formigenes in oxalate stone disease. BJU Int 2009;103:18-21. 29. Jones ML, Tomaro-Duchesneau C, Prakash S. The gut microbiome, probiotics, bile acids axis, and human health. Trends Microbiol 2014;22:306-8. 30. Ridlon JM, Kang DJ, Hylemon PB, Bajaj JS. Bile acids and the gut microbiome. Curr Opin Gastroenterol 2014;30:332-8. 31. Sagar NM, Cree IA, Covington JA, Arasaradnam RP. The interplay of the gut microbiome, bile acids, and volatile organic compounds. Gastroenterol Res Pract 2015;2015:398585. 32. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009;9:313-23. 33. Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science 2012;336:1268-73. 34. Hanski I, von Hertzen L, Fyhrquist N, Koskinen K, Torppa K, Laatikainen T, et al. Environmental biodiversity, human microbiota, and allergy are interrelated. Proc Natl Acad Sci U S A 2012;109:8334-9. 35. Sampson TR, Mazmanian SK. Control of brain development, function, and behavior by the microbiome. Cell Host Microbe 2015;17:565-76. 36. Diaz Heijtz R, Wang S, Anuar F, Qian Y, Björkholm B, Samuelsson A, et al. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci U S A 2011;108:3047-52. 37. Messaoudi M, Lalonde R, Violle N, Javelot H, Desor D, Nejdi A, et al. Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects. Br J Nutr 2011;105:755-64. 38. Steenbergen L, Sellaro R, van Hemert S, Bosch JA, Colzato LS. A randomized controlled trial to test the effect of multispecies probiotics on cognitive reactivity to sad mood. Brain Behav Immun 2015;48:258-64. 39. Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI. Human nutrition, the gut microbiome and the immune system. Nature 2011;474:327-36. 40. Newburg DS, Walker WA. Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatr Res 2007;61: 2-8. 41. Pop M, Walker AW, Paulson J, Lindsay B, Antonio M, Hossain MA, et al. Diarrhea in young children from low-income countries leads to largescale alterations in intestinal microbiota composition. Genome Biol 2014; 15:R76. 42. Youmans BP, Ajami NJ, Jiang ZD, Campbell F, Wadsworth WD, Petrosino JF, et al. Characterization of the human gut microbiome during travelers' diarrhea. Gut Microbes 2015;6:110-9. 43. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, et al. Enterotypes of the human gut microbiome. Nature 2011;473:174-80. 44. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022-3. 45. Turnbaugh PJ, Bäckhed F, Fulton L, Gordon JI. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host Microbe 2008;3:213-23. 46. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, et al. A core gut microbiome in obese and lean twins. Nature 2009; 457:480-4. 47. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027-31. 48. Everard A, Belzer C, Geurts L, Ouwerkerk JP, Druart C, Bindels LB, et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 2013;110:9066-71. 49. Shin NR, Lee JC, Lee HY, Kim MS, Whon TW, Lee MS, et al. An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. Gut 2014;63: 727-35. 50. Qin J, Li Y, Cai Z, Li S, Zhu J, Zhang F, et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 2012;490:55-60. 51. Wlodarska M, Kostic AD, Xavier RJ. An integrative view of microbiomehost interactions in inflammatory bowel diseases. Cell Host Microbe 2015;17:577-91. 52. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 2003;3:521-33. 53. Willing BP, Dicksved J, Halfvarson J, Andersson AF, Lucio M, Zheng Z, et al. A pyrosequencing study in twins shows that gastrointestinal microbial profiles vary with inflammatory bowel disease phenotypes. Gastroenterology 2010;139:1844-54.e1. 54. Bhattacharjee S, Lukiw WJ. Alzheimer's disease and the microbiome. Front Cell Neurosci 2013;7:153. 55. Gatz M, Pedersen NL. Study of dementia in Swedish twins. Twin Res Hum Genet 2013;16:313-6. 56. Miklossy J. Alzheimer's disease: a neurospirochetosis. Analysis of the evidence following Koch's and Hill's criteria. J Neuroinflammation 2011;8: 90. 57. Shoemark DK, Allen SJ. The microbiome and disease: reviewing the links between the oral microbiome, aging, and Alzheimer's disease. J Alzheimers Dis 2015;43:725-38. 58. Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med 2013;19:576-85. 59. Yi H, Yong D, Lee K, Cho YJ, Chun J. Profiling bacterial community in upper respiratory tracts. BMC Infect Dis 2014;14:583. 60. Park H, Shin JW, Park SG, Kim W. Microbial communities in the upper respiratory tract of patients with asthma and chronic obstructive pulmonary disease. PLoS One 2014;9:e109710. 61. Choi EB, Hong SW, Kim DK, Jeon SG, Kim KR, Cho SH, et al. Decreased diversity of nasal microbiota and their secreted extracellular vesicles in patients with chronic rhinosinusitis based on a metagenomic analysis. Allergy 2014;69:517-26. 62. Jung WH, Croll D, Cho JH, Kim YR, Lee YW. Analysis of the nasal vestibule mycobiome in patients with allergic rhinitis. Mycoses 2015;58:167-72. 63. Griffin JL, Wang X, Stanley E. Does our gut microbiome predict cardiovascular risk? A review of the evidence from metabolomics. Circ Cardiovasc Genet 2015;8:187-91. 64. Tang WH, Hazen SL. The contributory role of gut microbiota in cardiovascular disease. J Clin Invest 2014;124:4204-11. 65. DiGiulio DB, Callahan BJ, McMurdie PJ, Costello EK, Lyell DJ, Robaczewska A, et al. Temporal and spatial variation of the human microbiota during pregnancy. Proc Natl Acad Sci U S A 2015;112:11060-5. 66. Metcalf JL, Xu ZZ, Weiss S, Lax S, Van Treuren W, Hyde ER, et al. Microbial community assembly and metabolic function during mammalian corpse decomposition. Science 2016;351:158-62. 318 http://dx.doi.org/10.4168/aard.2016.4.5.311
최성미외 마이크로바이옴연구현황 Allergy Asthma Respir Dis 67. Emanuelsson F, Claesson BE, Ljungström L, Tvede M, Ung KA. Faecal microbiota transplantation and bacteriotherapy for recurrent Clostridium difficile infection: a retrospective evaluation of 31 patients. Scand J Infect Dis 2014;46:89-97. 68. Gough E, Shaikh H, Manges AR. Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clin Infect Dis 2011;53:994-1002. 69. Suwantarat N, Bobak DA. Fecal bacteriotherapy for recurrent Clostridium difficile infection: What's Old Is New Again? Curr Infect Dis Rep 2013;15: 101-3. 70. Delzenne NM, Neyrinck AM, Bäckhed F, Cani PD. Targeting gut microbiota in obesity: effects of prebiotics and probiotics. Nat Rev Endocrinol 2011;7:639-46. http://dx.doi.org/10.4168/aard.2016.4.5.311 319
Allergy Asthma Respir Dis Choi S, et al. Korean microbiome studies < 부록 > Supplementary Table 1. Publications of Korean microbiome research Title Journal Year Pyrosequencing-based molecular monitoring of the intestinal bacterial colonization in preterm infants J Pediatr Gastroenterol Nutr 2011 Diversity and abundance of single-stranded DNA viruses in human feces Appl Environ Microbiol 2011 Comparison of the gut microbiotas of healthy adult twins living in South Korea and the United States Appl Environ Microbiol 2011 Comparative analysis of Korean human gut microbiota by barcoded pyrosequencing PLoS One 2011 Characterization of the fungal microbiota (mycobiome) in healthy and dandruff-afflicted human scalps PLoS One 2012 Comparison of gut microbiota between sasang constitutions Evid Based Complement Alternat Med 2013 Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort PLoS One 2013 Impact of pelvic radiotherapy on gut microbiota of gynecological cancer patients revealed by massive pyrosequencing PLoS One 2013 Gene-targeted metagenomic analysis of glucan-branching enzyme gene profiles among human and animal fecal microbiota ISME J 2013 Decreased diversity of nasal microbiota and their secreted extracellular vesicles in patients with chronic rhinosinusitis based on a metagenomic analysis Differences in gastric mucosal microbiota profiling in patients with chronic gastritis, intestinal metaplasia, and gastric cancer using pyrosequencing methods Allergy 2014 Helicobacter 2014 The anti-obesity effect of Ephedra sinica through modulation of gut microbiota in obese Korean women J Ethnopharmaco 2014 Effect of metformin on metabolic improvement and gut microbiota Appl Environ Microbiol 2014 Stability of gut enterotypes in Korean monozygotic twins and their association with biomarkers and diet Sci Rep 2014 Profiling bacterial community in upper respiratory tracts BMC Infect Dis 2014 Microbial communities in the upper respiratory tract of patients with asthma and chronic obstructive pulmonary disease PLoS One 2014 An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in dietinduced obese mice Gut 2014 Influence of Panax ginseng on obesity and gut microbiota in obese middle-aged Korean women J Ginseng Res 2014 Profiling of the bacteria responsible for pyogenic liver abscess by 16S rrna gene pyrosequencing J Microbiol 2014 Refractory Clostridium difficile infection cured with fecal microbiota transplantation in vancomycin-resistant enterococcus colonized patient Intest Res 2015 Analysis of the nasal vestibule mycobiome in patients with allergic rhinitis Mycoses 2015 Comparison of the gut microbiota profile in breast-fed and formula-fed Korean infants using pyrosequencing Nutr Res Pract 2015 Subgingival microbiome in smokers and non-smokers in Korean chronic periodontitis patients Mol Oral Microbiol 2015 The effect of probiotics on gut microbiota during the Helicobacter pylori eradication: randomized controlled trial Helicobacter 2015 The association of uterine cervical microbiota with an increased risk for cervical intraepithelial neoplasia in Korea Clin Microbiol Infect 2015 Pyrosequencing analysis of subgingival microbiota in distinct periodontal conditions J Dent Res 2015 Comparative analysis of gut microbiota in elderly people of urbanized towns and longevity villages BMC Microbiol 2015 Genetic associations and shared environmental effects on the skin microbiome of Korean twins BMC Genomics 2015 Faecalibacterium prausnitzii subspecies-level dysbiosis in the human gut microbiome underlying atopic dermatitis J Allergy Clin Immunol 2015 320 http://dx.doi.org/10.4168/aard.2016.4.5.311