PostScript LETTER a functional and potentially pathogenic 5.8S-Fun and ITS4-Fun on an Illumina link between the intestinal bacteriota and MiSeq machine. Sequencing data were Gut: first published as 10.1136/gutjnl-2019-320008 on 25 October 2019. Downloaded from 2 3 Gut mycobiome of primary liver inflammation in PSC, the aetiology subjected to quality control by using the of the disease remains largely unknown. open source package DADA2 (V.1.10)5 sclerosing cholangitis patients We here report on the fungal myco- in R (V.3.5.1; https:// github. com/ mrue- is characterised by an increase biome results of our cohort from hlemann/ ikmb_ amplicon_ processing). of Trichocladium griseum and Northern Germany approved by the Amplicon sequence variants were taxo- local ethics committees (A148/14 and nomically annotated using the UNITE ITS Candida species MC-111/15) comprising stool samples database (V.7.2).6 of 66 healthy control (HC) subjects, 65 In disagreement with the findings in 1 LETTER TO THE EDITOR patients with well-characterised PSC the French cohort, overall fungal alpha We read with interest the recent Gut article (including a subgroup with concomitant diversity in the German cohort was by Lemoinne et al describing a dysbiosis colitis (PSC-IBD), n=32) and 38 subjects neither altered in PSC nor in UC versus of the fungal gut community in faeces of with UC.3 PCR and sequencing of the HC as calculated by Shannon species patients suffering from primary sclerosing fungus-specific internal transcribed spacer equivalent (figure 1A). None of the cholangitis (PSC).1 Though several reports, 2 genomic region was performed as previ- disease groups significantly deviated in including our own previous data, support ously described4 using the primer pair community composition from healthy http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright. Figure 1 Mycobiome of individuals with primary sclerosing cholangitis (PSC) and UC as well as healthy controls (HC) (all of northern German origin). Rarefaction curves for Shannon diversity of sequence variants reached plateau between 50 and 100 sequences per samples, thus samples were normalised to 100 random reads per sample. (A) Alpha diversity as presented by Shannon species equivalents (all p>0.05). (B) Beta diversity ordination of the Bray-Curtis dissimilarity based on genus-level fungal abundances (all padonis >0.05). (C) Phylum-level and (D) genus-level mean abundances of all taxa with >1% mean abundance and present in at least 10 samples. (E) Group-wise box-and-whisker plots for significant genus level annotations tested for differential abundances with individual values represented as data points. (F) Differences in group-mean abundances of patients with PSC and UC, as compared with HC. *q<0.05, **q<0.01. Gut Month 2019 Vol 0 No 0 1 PostScript individuals (all p >0.05; figure 1B). Malte Christoph Rühlemann ,1 different terms, provided the original work is properly adonis 1 2 Fungal composition on phylum level was Miriam Emmy Leni Solovjeva, Roman Zenouzi, cited, appropriate credit is given, any changes made Gut: first published as 10.1136/gutjnl-2019-320008 on 25 October 2019. Downloaded from 2 3,4 found to be mainly dominated by Ascomy- Timur Liwinski , Martin Kummen , indicated, and the use is non-commercial. See: http:// Wolfgang Lieb,5 Johannes Roksund Hov ,3,4 creativecommons.org/ licenses/ by- nc/ 4. 0/. cota (figure 1C), particularly by the genera 2,6 1 Saccharomyces, Candida and Dipodascus Christoph Schramm, Andre Franke , © Author(s) (or their employer(s)) 2019. Re-use Corinna Bang 1 permitted under CC BY-NC. No commercial re-use. See (figure 1D) in relatively higher abun- 1 rights and permissions. Published by BMJ. dance of reads when compared with the Institute of Clinical Molecular Biology, Christian- 1 Albrechts-Universität zu Kiel, Kiel, Germany AF and CB contributed equally. findings of Lemoinne et al. Though our 2Department of Internal Medicine, University Medical results generally validate the previously Center Hamburg-Eppendorf, Hamburg, Germany described overall fungal composition in 3Norwegian PSC Research Center, Oslo University Hospital, Rikshospitalet, Oslo, Norway To cite Rühlemann MC, Solovjeva MEL, Zenouzi R, stool, we were not able to detect the genus 4 Exophiala, which was found in five PSC Institute of Clinical Medicine, University of Oslo, Oslo, et al. Gut Epub ahead of print: [please include Day Norway Month Year]. doi:10.1136/gutjnl-2019-320008 patients from France exclusively. Whether 5Institute of Epidemiology and Biobank POPGEN, this is due to methodological differences Christian-Albrechts-University of Kiel, Kiel, Germany Received 4 October 2019 (choice of primer sets, data analysis tools 6Martin Zeitz Centre for Rare Diseases, University Revised 10 October 2019 Accepted 11 October 2019 and sampling depth) or presence of this Medical Center Hamburg-Eppendorf, Hamburg, Germany fungus in only a subset of PSC patients not Gut 2019;0:1–2. doi:10.1136/gutjnl-2019-320008 sampled in the German cohort needs to be Correspondence to Dr Corinna Bang, Institute determined. of Clinical Molecular Biology, Christian-Albrechts- ORCID iDs University Kiel, Kiel 24105, Germany; Malte Christoph Rühlemann http:// orcid. org/ 0000- Disease-associated differential abun- c. bang@ ikmb. uni- kiel. de 0002- 0685- 0052 dance of fungal taxa was investigated Timur Liwinski http:// orcid. org/ 0000- 0002- 1041- 9142 by applying Student’s t-test to the Twitter Malte Christoph Rühlemann @mruehlemann Martin Kummen http:// orcid. org/ 0000- 0001- 9660- and Johannes Roksund Hov @hov_jer arcsin-squareroot-transformed relative 6290 Johannes Roksund Hov http:// orcid. org/ 0000- 0002- abundances of all genera with mean abun- Acknowledgements We would like to thank Ms Ilona Urbach, Ms Ines Wulf and Mr Tonio Hauptmann of 5900- 8096 dance >1% and present in at least 10 indi- the IKMB microbiome laboratory for excellent technical Andre Franke http:// orcid. org/ 0000- 0003- 1530- 5811 viduals. This analysis revealed increased support. Corinna Bang http:// orcid. org/ 0000- 0001- 6814- 6151 levels of the genera Candida and Humi- Contributors AF and CB designed the study. CS and cola (species level annotation suggests H. AF obtained funding. RZ, CS and WL acquired and REFERENCES grisea) in PSC patients with and without quality-controlled patient samples and data. CB and 1 Lemoinne S, Kemgang A, Ben Belkacem K, et al. concomitant colitis compared with HC MELS supervised sample processing and sequencing. Fungi participate in the dysbiosis of gut microbiota (all q <0.05; figure 1E and F) and UC MCR performed statistical analyses. MCR, MELS, RZ, TL, in patients with primary sclerosing cholangitis. Gut BH MK, JRH, CS, AF and CB interpreted data and drafted 2019:gutjnl-2018-317791 (published Online First: (all q <0.1; figure 1E) individuals. H. BH the manuscript with input and critical revision from 2019/04/19). grisea, recently reclassified as Trichocla- all authors. All authors revised and approved the final 2 Kummen M, Holm K, Anmarkrud JA, et al. The gut dium griseum,7 belongs to the fungal class version of the manuscript. microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative http://gut.bmj.com/ Sordariomycetes, thus our results repro- Funding This study was supported by the Deutsche colitis without biliary disease and healthy controls. Gut duce the significant increase of this class Forschungsgemeinschaft (DFG) Clinical Research 2017;66:611–9. Group 306’ Primary sclerosing cholangitis’ (no: in PSC patients, as previously described by 3 Rühlemann M, Liwinski T, Heinsen F-A, et al. KFO306) as well as Research Training Group 1743 and Lemoinne and colleagues, but at increased Consistent alterations in faecal microbiomes received infrastructure support from the DFG Cluster of patients with primary sclerosing cholangitis taxonomic resolution. Previous research of Excellence ’Inflammation at Interfaces’ (http:// independent of associated colitis. Aliment Pharmacol on T. griseum showed that it is most www. inflammation- at- interfaces. de, no: EXC306 and Ther 2019;50:580–9. frequently isolated from soil and plants EXC306/2) and the German Ministry of Education and 4 Taylor DL, Walters WA, Lennon NJ, et al. Accurate on September 28, 2021 by guest. Protected copyright. Research (BMBF) program e:Med sysINFLAME (http:// but also occasionally found in patients estimation of fungal diversity and abundance through www. gesundheitsforschung- bmbf. de/ de/ 5111. php, no: suffering from peritonitis.8 In addition, improved lineage-specific primers optimized for 01ZX1306A). the validated increase of Candida species Illumina amplicon sequencing. Appl Environ Microbiol in PSC patients argues for an immuno- Competing interests None declared. 2016;82:7217–26. Patient consent for publication Not required. 5 Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: genic role of these fungi, particularly with high-resolution sample inference from Illumina respect to earlier findings that demon- Provenance and peer review Not commissioned; amplicon data. Nat Methods 2016;13:581–3. strated their high potential to induce externally peer reviewed. 6 Nilsson RH, Larsson K-H, Taylor AFS, et al. The unite Th17 response in T cells.9 Increased Th17 Data availability statement Sequencing and database for molecular identification of fungi: handling numbers have previously been reported in clinical data of the patient samples used in this study dark taxa and parallel taxonomic classifications. can be applied for via the Popgen Biobank (Institute Nucleic Acids Res 2019;47:D259–64. PSC patients and recently been shown to 7 Wang XW, Yang FY, Meijer M, et al. Redefining 10 of Epidemiology, Christian-Albrechts-University of Kiel, be involved in PSC pathogenesis. Germany). Humicola sensu stricto and related genera in the In summary, both the significant increase Chaetomiaceae. Stud Mycol 2019;93:65–153. of the fungal class Sordariomycetes, likely 8 Burns N, Arthur I, Leung M, et al. Humicola sp. as a T.