Gut Online First, published on October 7, 2017 as 10.1136/gutjnl-2017-314814 Gut microbiota ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2017-314814 on 7 October 2017. Downloaded from The oral microbiota in colorectal cancer is distinctive and predictive Burkhardt Flemer,1,2 Ryan D Warren,1 Maurice P Barrett,1,2 Katryna Cisek,3 Anubhav Das,3 Ian B Jeffery,1,2 Eimear Hurley,2,4 Micheal O’Riordain,5 Fergus Shanahan,1,5 Paul W O’Toole1,2 ► Additional material is ABSTRACT published online only. To view Background and aims Microbiota alterations are Key messages please visit the journal online linked with colorectal cancer (CRC) and notably higher (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2017- 314814). abundance of putative oral bacteria on colonic tumours. The gut microbiota is associated with colorectal 1 However, it is not known if colonic mucosa-associated ► APC Microbiome Institue, cancer (CRC) development. University College Cork, taxa are indeed orally derived, if such cases are a distinct National University of Ireland, subset of patients or if the oral microbiome is generally ► Faecal microbiota has potential as a biomarker Cork, Ireland suitable for screening for CRC. for CRC. 2 Schools of Microbiology, Methods We profiled the microbiota in oral swabs, ► Putatively oral bacteria are more abundant on University College Cork, colonic mucosae and stool from individuals with CRC (99 CRC biopsies and Fusobacterium nucleatum has National University of Ireland, been reported to be enriched in IBD. Cork, Ireland subjects), colorectal polyps (32) or controls (103). 34D Pharma Cork Ltd, Cork, Results Several oral taxa were differentially ► A ‘Western diet’ contributes to CRC Ireland abundant in CRC compared with controls, for example, development. 4 Department of Dentistry, Streptococcus and Prevotellas pp. A classification model University College Cork, What are the new findings? of oral swab microbiota distinguished individuals with National University of Ireland, We developed an oral and faecal microbiota- CRC or polyps from controls (sensitivity: 53% (CRC)/67% ► Cork, Ireland based classifier that distinguished individuals 5Department of Medicine, (polyps); specificity: 96%). Combining the data from with CRC and adenomas from healthy controls. University College Cork, faecal microbiota and oral swab microbiota increased the The discriminatory power particularly for National University of Ireland, sensitivity of this model to 76% (CRC)/88% (polyps). We Cork, Ireland adenomas was higher than for currently used detected similar bacterial networks in colonic microbiota tests. and oral microbiota datasets comprising putative oral http://gut.bmj.com/ Correspondence to We detected similar networks of oral bacteria biofilm forming bacteria. While these taxa were more ► Dr Paul W O’Toole, School of at both oral and colonic mucosal surfaces, Microbiology & APC Microbiome abundant in CRC, core networks between pathogenic, including in individuals with colonic lesions (on Institute, University College CRC-associated oral bacteria such as Peptostreptococcus, and off the tumour), healthy controls and Cork, 17 T12 YN60 Cork, Parvimonas and Fusobacterium were also detected in Ireland; pwotoole@ ucc. ie children with and without Crohn’s disease. healthy controls. High abundance of Lachnospiraceae A microbiota rich in Lachnospiraceae was was negatively associated with the colonisation ► Received 12 July 2017 negatively correlated with ‘Western diet’ Revised 20 September 2017 of colonic tissue with oral-like bacterial networks on September 27, 2021 by guest. Protected copyright. and colonic colonisation with oral bacteria, Accepted 21 September 2017 suggesting a protective role for certain microbiota including oral pathogens associated with CRC, types against CRC, possibly by conferring colonisation suggesting a protective role, possibly mediated resistance to CRC-associated oral taxa and possibly through habitual diet. mediated through habitual diet. Conclusion The heterogeneity of CRC may relate How might it impact on clinical practice in the to microbiota types that either predispose or provide foreseeable future? resistance to the disease, and profiling the oral ► If the suitability of oral microbiota screening for microbiome may offer an alternative screen for detecting the detection of CRC and polyps can be verified CRC. in larger study groups, this could significantly improve current screening programmes. INTRODUCTION Microbes have been implicated in the pathogenesis reported changes in the faecal or colonic mucosal of several human cancers, most strikingly in the microbiota in patients with colorectal cancer case of Helicobacter pylori and gastric carcinoma (CRC),3–8 and data from several animal models and some gastric lymphomas.1 2 H. pylori is now have implicated the microbiota in the pathogenesis designated a gastric carcinogen and a preclinical risk of CRC.9–13 Our finding of a microbiota configu- To cite: Flemer B, Warren RD, factor. Current non-invasive screening approaches ration associated with benign colonic polyps that Barrett MP, et al. Gut Published Online First: [please for colon cancer such as faecal immune test (FIT) is intermediate between that of controls and those include Day Month Year]. and faecal occult blood test (FOBT) have very low with cancer suggests that the microbiota might doi:10.1136/ sensitivity for detecting early lesions, and more reli- provide a potential biomarker predictive of the risk gutjnl-2017-314814 able biomarkers are required. We and others have of later development of cancer. It also suggests that Flemer B, et al. Gut 2017;0:1–10. doi:10.1136/gutjnl-2017-314814 1 Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Gut microbiota an intervention could theoretically be applicable years before the obtained by rubbing the inside of both cheeks with a swab. development of the disease. The additional finding by us and Exclusion criteria were a personal history of CRC, IBD and irri- Gut: first published as 10.1136/gutjnl-2017-314814 on 7 October 2017. Downloaded from others of microbes that are normally associated with the oral table bowel syndrome. A breakdown of the analysed samples cavity3–8 13 being present in the faecal and mucosal microbiota is given in table 1. Detailed demographic information for each linked with CRC prompted us to investigate the oral microbiota individual is given in (online supplementary table 1). in colon cancer as a first step in determining if it might serve as Genomic DNA was extracted using the AllPrep DNA/RNA a more accessible sampling site for convenient and widespread kit from Qiagen (Hilden, Germany). 16S rRNA gene ampl- screening. Previously, several groups reported the applicability of icon sequencing libraries of the V3-V4 region were prepared, faecal microbiota profiling as a tool for detection of CRCs,4 5 14 and pools of amplicons were sequenced at GATC (Konstanz, particularly in conjunction with the FOBT5 or FIT.4 Moreover, Germany) on a MiSeq sequencing instrument (Illumina, San distinct bacterial profiles in the oral cavity have been associ- Diego, California, USA) using 2×250 bp chemistry. ated with oral cancers15 16 and with esophageal17 and pancreatic 16S amplicon sequences from our Irish cohort were processed cancers.18 19 A single study identified significant differences in as previously described.3 We also conducted a meta-analysis the bacteria present in oral rinse samples from individuals with with amplicon sequencing data pertaining to Gevers et al21 and CRC compared with healthy controls.20 processed data associated with this study similarly. In order to Here, we present the findings of an extended CRC study compare bacterial operational taxonomic units (OTUs) obtained population and include an assessment of the oral microbiota. We in the Irish CRC cohort (sequenced region: V3-V4) with OTUs developed a classifier using oral and faecal microbiota profiles obtained in the Crohn’s disease cohort (V4), we shortened the with high specificity and sensitivity particularly for the detection sequences of the CRC cohort to the sequenced region of the CD of colorectal polyps. Furthermore, we found similar bacterial cohort using cutadapt,22 and then processed the sequences of the networks at both oral and colonic mucosal surfaces that were two studies together. enriched in CRC and also detectable in healthy tissue. However, Statistical analysis was carried out in R.23 we could not find a direct link between oral and colonic micro- A more detailed description of the employed protocols is biota such that elevated abundance of bacteria in the oral cavity available as (online supplementary information). was predictive of colonic colonisation by the same taxa. Rather, colonic presence and abundance of oral pathogens was nega- CRC classifier tively associated with the colonic abundance of Lachnospiraceae. The Random forest (RF) classifier to determine OTUs suitable as We also detected weak negative correlations of Lachnospiraceae biomarkers of colonic lesions was described elsewhere.4 In brief, with dietary habits reminiscent of a ‘Western diet’. Lastly, in a we used log-ratio transformed values of OTUs present in at least meta analysis, we found similar networks of oral bacteria to be 5% of individuals as input to the function AUCRF of the AUCRF enriched in colonic tissue of children in a recent Crohn’s disease package.24 Significance of difference