ABSTRACT
We report here an update to the draft genome sequence of Kluyvera intestini sp. nov. strain GT-16, generated using MinION long-read sequencing technology. The complete genome sequence of the human-derived strain GT-16 measured 5,768,848 bp. An improved high-quality complete genome sequence provides insights into the mobility potential of resistance genes in this species.
GENOME ANNOUNCEMENT
Kluyvera intestini sp. nov. strain GT-16 is a newly described species that was isolated, using an original genomic workflow, from the stomach of a patient with gastric cancer (1–3). This strain is characterized by numerous virulence factors and antibiotic resistance genes (ARGs) that correspond to those in other clinically significant Kluyvera sp. pathogens (4–6). To update the draft genome sequence, we used a combination of Illumina short-read and Oxford Nanopore Technologies (ONT) MinION long-read sequencing technologies (7).
Whole-genome shotgun libraries for Illumina sequencing were prepared as follows: 500 ng of genomic DNA were sheared to 500-bp fragments using a Covaris E220 ultrasonicator, and a library was prepared using the Kapa low-throughput “with bead” preparation kit (Kapa Biosystems, catalog no. KK8231) without PCR amplification. The DNA was sequenced as 300-bp paired-end reads on the Illumina MiSeq platform. DNA was also sequenced using a flow cell system on the ONT MinION sequencer.
Base-calling of the initial ONT MinION FAST5 files was performed with the Metrichor service. The base-called FAST5 files were converted to FASTQ format using poretools (8). Assembly was performed using Canu (9), and nanopolish (10) was used to compute an improved consensus sequence.
De novo assembly, combined with manual integration of Nanopore data via LASTZ alignments, yielded two scaffolds of 5,520,555 bp and 248,293 bp, with an average coverage of 150× and a total of 6,098 coding sequences. The DNA-DNA hybridization value showed 99.5% similarity with K. intestini sp. nov. GT-16 (GenBank accession no. MKZW00000000), meaning that these genomes belong to the same species (11).
Numerous virulence-related genes, such as those coding hemolysin D, peptidases, permeases, chemotaxis regulators, and exonucleases, were found in K. intestini sp. nov. GT-16. High-confidence ARGs (containing over 75% amino acid identity) were identified using the NCBI and Comprehensive Antibiotic Resistance databases (12, 13).
Using complete genome sequencing, we evaluated the mobility potential of the identified ARGs. We identified all mobile genetic elements (MGEs), such as insertion and/or transposable elements, or phages, using the ISFinder, PHAST, and GypsyGenes algorithms (14, 15). The genome was shown to harbor 24 insertion sequence (IS) elements, 2 transposases, 8 resolvases, 11 integrases, and 5 Tn3 transposons. Next, we merged the outputs to identify all composite elements that were suggested as ARGs and colocalized within the MGEs. The composite elements were determined to be an ARG flanked by two ISs belonging to the same family within a 10-kb region, a transposon with an ARG within a 5-kb region, or a transposable phage.
Overall, the genome of K. intestini strain GT-16 harbored 18 ARGs associated with MGEs, including those encoding resistance to tellurium, quaternary ammonium compounds, arsenic, beta-lactams, copper, chloramphenicol, polymyxin, and multidrug resistance (MDR) transporters. This indicates that these ARGs can be involved in horizontal gene transfer (16). Follow-up studies of K. intestini and its harbored bacteriophages would enable us to understand its possible pathogenicity and role in cancer (17).
Accession number(s).
This complete genome sequencing project has been deposited in GenBank under the accession no. MKZW00000000. The version described in this paper is the second version, MKZW02000000.
ACKNOWLEDGMENTS
We thank members of the Genome Technology Center for expert technical assistance, as well as the high-performance computing group at New York University Langone Medical Center.
The genome technology center is partially supported by grant P30CA016087 from the Laura and Isaac Perlmutter Cancer Center.
Footnotes
Citation Tetz G, Vecherkovskaya M, Zappile P, Dolgalev I, Tsirigos A, Heguy A, Tetz V. 2017. Complete genome sequence of Kluyvera intestini sp. nov., isolated from the stomach of a patient with gastric cancer. Genome Announc 5:e01184-17. https://doi.org/10.1128/genomeA.01184-17.
REFERENCES
- 1.Tetz G, Tetz V. 2016. Draft genome sequence of Kluyvera intestini strain GT-16 isolated from the stomach of a patient with gastric cancer. Genome Announc 4(6):e01432-16. doi: 10.1128/genomeA.01432-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Tetz G, Tetz V. 2017. Introducing the sporobiota and sporobiome. Gut Pathog 9:38. doi: 10.1186/s13099-017-0187-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Tetz G, Tetz V, Vecherkovskaya M. 2016. Genomic characterization and assessment of the virulence and antibiotic resistance of the novel species Paenibacillus sp. strain VT-400, a potentially pathogenic bacterium in the oral cavity of patients with hematological malignancies. Gut Pathog 8:6. doi: 10.1186/s13099-016-0089-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Carter JE, Evans TN. 2005. Clinically significant Kluyvera infections: a report of seven cases. Am J Clin Pathol 123:334–338. doi: 10.1309/61XP-4KTL-JYWM-5H35. [DOI] [PubMed] [Google Scholar]
- 5.Saul SR, Chen R, Jiang P, Schade M, Jaker M. 2014. Kluyvera cryocrescens presenting as a complicated urinary tract infection: case report and literature review. Infect Dis Clin Pract 22:e50–e51. doi: 10.1097/IPC.0b013e3182a01f35. [DOI] [Google Scholar]
- 6.Sarria JC, Vidal AM, Kimbrough RC. 2001. Infections caused by Kluyvera species in humans. Clin Infect Dis 33:E69–E74. doi: 10.1086/322686. [DOI] [PubMed] [Google Scholar]
- 7.Mikheyev AS, Tin MM. 2014. A first look at the Oxford Nanopore MinION sequencer. Mol Ecol Resour 14:1097–1102. doi: 10.1111/1755-0998.12324. [DOI] [PubMed] [Google Scholar]
- 8.Loman NJ, Quinlan AR. 2014. Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics 30:3399–3401. doi: 10.1093/bioinformatics/btu555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. doi: 10.1101/gr.215087.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Loman NJ, Quick J, Simpson JT. 2015. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods 12:733–735. doi: 10.1038/nmeth.3444. [DOI] [PubMed] [Google Scholar]
- 11.Auch AF, von Jan M, Klenk HP, Göker M. 2010. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2:117–134. doi: 10.4056/sigs.531120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FS, Wright GD, McArthur AG. 2017. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 45:D566–D573. doi: 10.1093/nar/gkw1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Ciufo S, Li W. 2013. Prokaryotic genome annotation pipeline. In Beck J, Benson D, Coleman J, Hoeppner M, Johnson M, Maglott D, Mizrachi I, Morris R, Ostell J, Pruitt K, Rubinstein W, Sayers E, Sirotkin K, Tatusova T (ed), The NCBI handbook, 2nd ed. NCBI, Bethesda, MD. [Google Scholar]
- 14.Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. 2006. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 34:D32–D36. doi: 10.1093/nar/gkj014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. Nucleic Acids Res 39:W347–W352. doi: 10.1093/nar/gkr485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Stokes HW, Gillings MR. 2011. Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens. FEMS Microbiol Rev 35:790–819. doi: 10.1111/j.1574-6976.2011.00273.x. [DOI] [PubMed] [Google Scholar]
- 17.Tetz GV, Ruggles KV, Zhou H, Heguy A, Tsirigos A, Tetz V. 2017. Bacteriophages as potential new mammalian pathogens. Sci Rep 7:7043. doi: 10.1038/s41598-017-07278-6. [DOI] [PMC free article] [PubMed] [Google Scholar]