Crohn’s disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that Parabacteroides distasonis could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.
ABSTRACT
Crohn’s disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that Parabacteroides distasonis could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.
ANNOUNCEMENT
Crohn’s disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans that affects all layers of the gut wall. Although CD is not primarily caused by bacteria, there is evidence that gut microbiome alterations are associated with CD (1, 2). However, it remains unclear which, and how, microbes worsen CD. We recently isolated Parabacteroides distasonis (NCBI taxid 823, confirmed by 16S rRNA gene Sanger sequencing and matrix-assisted laser desorption ionization–time of flight [MALDI-TOF] mass spectrometry) from an intramural cavernous fistulous tract (IM-CavFT) (3) microlesion from the gut wall of a patient who underwent surgical removal of the ileum as treatment for CD. The surgical specimen was obtained as a deidentified sample via the Biorepository Core of the NIH Silvio O. Conte Cleveland Digestive Disease Research Core Center (Cleveland, OH, USA), which obtained the consent and approval from surgical patients following protocols approved by the Case Western Reserve University and University Hospitals Cleveland Medical Center institutional review boards.
Parabacteroides distasonis (NCBI lineage: bacteria; FCB group; Bacteroidetes/Chlorobi group; Bacteroidetes; Bacteroidia; Bacteroidales; Tannerellaceae; Parabacteroides) has been reported to be a beneficial commensal gut microorganism (4–6); however, evidence indicates that P. distasonis could contribute to IBD (7, 8). Herein, we announce the complete genome sequence of a strain we designated CavFT-hAR46 (pronounced “cavftar46”) since its human origin in a gut wall lesion could help identify potential pathogenic mechanisms of bacterial invasion, intramural lesions, and complications in CD.
This strain was isolated anaerobically from IM-CavFT debris which was obtained by microdissection of a fresh specimen, as we previously reported (3). The microscopically dissected debris was then plated onto and purified using prereduced tryptic soy 5% defibrinated sheep blood agar (80% N–10% H–10% CO2, at 37°C; Thermo Fisher Scientific) using a variable-atmosphere anaerobic Whitley workstation A85 (540 plate capacity; Microbiology International, Inc.). Individual colonies were subcultured and purified in the same agar and then banked in prereduced brain heart infusion (BHI) broth with 7% dimethyl sulfoxide (DMSO). DNA was extracted from the original isolate using the MagAttract high-molecular-weight (HMW) DNA kit (Qiagen).
Genome sequencing was conducted using Pacific Biosciences (PacBio) reagents (DNA Link, Inc., South Korea). The SMRTbell library was constructed with the SMRTbell template prep kit 1.0 (product number 100-259-100; PacBio), with removal of small fragments (<20 kb) using the BluePippin size selection system. The constructed library was validated using an Agilent 2100 bioanalyzer and sequenced using one single-molecule real-time (SMRT) cell, P6-C4 chemistry (DNA sequencing reagent 4.0), and PacBio RS II 240-minute movies (9). Default parameters were used for all software, unless otherwise specified. We produced 128,581 long reads and 1,132,591,489 bp after subread filtering. De novo assembly was conducted using the Hierarchical Genome Assembly Process (HGAP, version 2.3) (10), including consensus polishing with Quiver. Since the average sequencing coverage was 168× for a total of 5,079,886 bp distributed across 5 contigs, we performed error correction based on the longest (150,079,410 bp) seed bases (~30× coverage) and then assembled them with error-corrected reads (contig N50, 4,988,677 bp for a 5 total contigs with a length of 5,079,886 bp). To examine the circularity form of the contigs, we used MUMmer 3.5 and trimmed one of the self-similar ends for manual genome closure using previously described protocols (10, 11). Combined sequencing produced one polished single circular contig with no gaps or plasmids.
For contextualization, we used the Pathosystems Resource Integration Center (PATRIC v.3.5.38) genome database for automated annotation and overall comparison to 49 other P. distasonis sequenced strains (1 May 2019; https://patricbrc.org/; 99% DNA purity, free of other bacterial contaminants). Because most strains (n = 47) were whole-genome shotgun (WGS) sequenced and/or had multiple contigs (range, 3 to 1,920), we used the 1-contig P. distasonis reference genome/strain (ATCC 8503) and three other strains to conduct comparative single-nucleotide polymorphism (SNP) analysis of our contig using EDGE Bioinformatics (12, 13). Collectively, our strain matched the ATCC 8503 reference genome at ∼85% identity. The other strains matched at 80% (Parabacteroides sp. strain CT06) or <10% (Table 1).
TABLE 1.
Genomic features of Parabacteroides distasonis strain CavFT-hAR046 and single nucleotide polymorphism differences compared to other complete Parabacteroides genomes
| Featurea | Data for strain: |
|||
|---|---|---|---|---|
| CavFT-hAR46 | ATCC 8503 | CT06 | 82G9 | |
| Location of origin (yr) | Ohio, USA (2017) | USA | South Korea | Japan |
| Accession no. | CP040468 | NC_009615 | NZ_CP022754 | NZ_LR215978 |
| Genome size (bp) | 4,952,323 | 4,811,379 | 5,372,666 | 5,212,259 |
| No. of CDSb | 4,263 | 4,216 | 4,686 | 4,365 |
| G+C content (%) | 45.2 | 45.1 | 45.2 | 45.2 |
| No. of rRNAs | 21 | 21 | 21 | 21 |
| No. of proteins with functional assignments | 2,694 | 2,639 | 2,875 | NDc |
| No. of hypothetical proteins | 1,569 | 1,487 | 1,811 | ND |
| No. of antibiotic resistance genes | 24 | 22 | 28 | ND |
| No. of genomic polymorphisms of CavFT-hAR046 strain compared to ATCC 8503, CT06, 82G9 strains | ||||
| Synonymous SNPs | NAd | 10,600 | 14,748 | ND |
| Nonsynonymous SNPs | NA | 3,443 | 4,986 | ND |
| Indels | NA | 478 | 689 | ND |
Basic genome features in this table were derived from pairwise analysis using PATRIC annotation definitions.
CDS, coding sequences.
ND, not determined.
NA, not applicable.
In contrast to two previously available complete genomes of P. distasonis (ATCC 8503 and 82G9 in Table 1) and strain NBRC 113806, which seemed to have been isolated from feces, herein, we report the complete genome sequence of a potential pathogenic strain with relevance to chronic forms of intestinal inflammation, especially to severe and incurable surgical forms of CD. Since this strain was isolated from chronic inflammatory lesions present in the gut wall of CD patients, we expect that future functional microbiological and genetic analyses with this strain could help determine the role that intestinal and invasive bacteria play in IBD. This is the lead genome of a series of potential pathogenic bacteria we have identified in Crohn’s disease.
Data availability.
This genome sequence has been deposited in GenBank under the accession number CP040468. Its associated BioProject, BioSample, and Sequence Read Archive (SRA) numbers are PRJNA542869, SAMN11642307, and SRR9221602, respectively. For public availability as gold standard, this genome has been annotated by the National Center of Biotechnology Information using the latest Prokaryotic Genome Annotation Pipeline which uses new hidden Markov models for antimicrobial resistance proteins, and curated complex domain architectures for functional annotation of proteins (assembly name ASM614918v1, RefSeq sequence NZ_CP040468.1; www.ncbi.nlm.nih.gov/genome/annotation_prok/).
ACKNOWLEDGMENTS
This project was primarily supported by NIH grant R21 DK118373 to A.R.-P., entitled “Identification of pathogenic bacteria in Crohn’s disease.” We acknowledge the Biorepository Core of the NIH Silvio O. Conte Cleveland Digestive Disease Research Core Center (P30DK097948) for clinical advice and provision of deidentified surgical specimens for the above-mentioned R21 project. Partial support also originated for A.R.-P. from a career development award from the Crohn’s and Colitis Foundation and National Institutes of Health award 2P01DK091222-06 (Germ-Free and Gut Microbiome Core to A.R.-P. and to F.C., Case Western Reserve University). Further support came from the Los Alamos National Laboratory (LANL) internal grants, the Laboratory Directed Research and Development grant 20160340ER, and the LANL Director’s Postdoctoral Fellowship, grant 20170671PRD2. F.Y. was awarded a scholarship from the China Scholarship Council to join the laboratory of A.R.-P. as visiting professor. J.C.E. was supported by an internship summer award from The Ohio State University Second-Year Transformational Experience (STEP) program.
REFERENCES
- 1.Pascal V, Pozuelo M, Borruel N, Casellas F, Campos D, Santiago A, Martinez X, Varela E, Sarrabayrouse G, Machiels K, Vermeire S, Sokol H, Guarner F, Manichanh C. 2017. A microbial signature for Crohn's disease. Gut 66:813–822. doi: 10.1136/gutjnl-2016-313235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Halfvarson J, Brislawn CJ, Lamendella R, Vázquez-Baeza Y, Walters WA, Bramer LM, D'Amato M, Bonfiglio F, McDonald D, Gonzalez A, McClure EE, Dunklebarger MF, Knight R, Jansson JK. 2017. Dynamics of the human gut microbiome in inflammatory bowel disease. Nat Microbiol 2:17004. doi: 10.1038/nmicrobiol.2017.4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Rodriguez-Palacios A, Kodani T, Kaydo L, Pietropaoli D, Corridoni D, Howell S, Katz J, Xin W, Pizarro TT, Cominelli F. 2015. Stereomicroscopic 3D-pattern profiling of murine and human intestinal inflammation reveals unique structural phenotypes. Nat Commun 6:7577. doi: 10.1038/ncomms8577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Kverka M, Zakostelska Z, Klimesova K, Sokol D, Hudcovic T, Hrncir T, Rossmann P, Mrazek J, Kopecny J, Verdu EF, Tlaskalova-Hogenova H. 2011. Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition. Clin Exp Immunol 163:250–259. doi: 10.1111/j.1365-2249.2010.04286.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Koh GY, Kane A, Lee K, Xu Q, Wu X, Roper J, Mason JB, Crott JW. 2018. Parabacteroides distasonis attenuates toll-like receptor 4 signaling and Akt activation and blocks colon tumor formation in high-fat diet-fed azoxymethane-treated mice. Int J Cancer, in press. [DOI] [PubMed] [Google Scholar]
- 6.Wang K, Liao M, Zhou N, Bao L, Ma K, Zheng Z, Wang Y, Liu C, Wang W, Wang J, Liu SJ, Liu H. 2019. Parabacteroides distasonis alleviates obesity and metabolic dysfunctions via production of succinate and secondary bile acids. Cell Rep 26:222–235.e5. doi: 10.1016/j.celrep.2018.12.028. [DOI] [PubMed] [Google Scholar]
- 7.Dziarski R, Park SY, Kashyap DR, Dowd SE, Gupta D. 2016. Pglyrp-regulated gut microflora Prevotella falsenii, Parabacteroides distasonis and Bacteroides eggerthii enhance and Alistipes finegoldii attenuates colitis in mice. PLoS One 11:e0146162. doi: 10.1371/journal.pone.0146162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Lopetuso LR, Petito V, Graziani C, Schiavoni E, Paroni Sterbini F, Poscia A, Gaetani E, Franceschi F, Cammarota G, Sanguinetti M, Masucci L, Scaldaferri F, Gasbarrini A. 2018. Gut microbiota in health, diverticular disease, irritable bowel syndrome, and inflammatory bowel diseases: time for microbial marker of gastrointestinal disorders. Dig Dis 36:56–65. doi: 10.1159/000477205. [DOI] [PubMed] [Google Scholar]
- 9.Varela-Álvarez E, Andreakis N, Lago-Leston A, Pearson GA, Serrao EA, Procaccini G, Duarte C, Marba N. 2006. Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction. J Phycol 42:741–745. doi: 10.1111/j.1529-8817.2006.00218.x. [DOI] [Google Scholar]
- 10.Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. doi: 10.1038/nmeth.2474. [DOI] [PubMed] [Google Scholar]
- 11.Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. 2004. Versatile and open software for comparing large genomes. Genome Biol 5:R12. doi: 10.1186/gb-2004-5-2-r12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Li PE, Lo CC, Anderson JJ, Davenport KW, Bishop-Lilly KA, Xu Y, Ahmed S, Feng S, Vishwesh P, Mokashi VP, Chain P. 2017. Enabling the democratization of the genomics revolution with a fully integrated Web-based bioinformatics platform. Nucleic Acids Res 45:67–80. doi: 10.1093/nar/gkw1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kumar A, Davenport KW, Vuyisich G, Kunde YA, Johnson SL, Chain PSG, Dichosa AEK, Rodriguez-Palacios A. 2018. Complete genome sequences of historic Clostridioides difficile food-dwelling ribotype 078 strains in Canada identical to that of the historic human clinical strain M120 in the United Kingdom. Microbiol Resour Announc 7:e00853-18. doi: 10.1128/MRA.00853-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
This genome sequence has been deposited in GenBank under the accession number CP040468. Its associated BioProject, BioSample, and Sequence Read Archive (SRA) numbers are PRJNA542869, SAMN11642307, and SRR9221602, respectively. For public availability as gold standard, this genome has been annotated by the National Center of Biotechnology Information using the latest Prokaryotic Genome Annotation Pipeline which uses new hidden Markov models for antimicrobial resistance proteins, and curated complex domain architectures for functional annotation of proteins (assembly name ASM614918v1, RefSeq sequence NZ_CP040468.1; www.ncbi.nlm.nih.gov/genome/annotation_prok/).