ABSTRACT
The complete genome of Kalamiella piersonii clinical isolate URMC-2103A041 from human bacteremia was determined using the hybrid assembly of short- and long-read sequencing chemistry. The genome contains a 3.93 Mb chromosome, three circular plasmids, and one linear plasmid.
KEYWORDS: Kalamiella, Pantoea, Enterobacteriaceae, blood culture, bloodstream infections
ANNOUNCEMENT
Kalamiella piersonii is a Gram-negative Enterobacterales in the Erwiniaceae family. First described from the International Space Station environment, a recent phylogenomic analysis of Erwiniaceae has proposed reclassification as Pantoea piersonii comb. nov (1, 2).
From humans, K. piersonii has been isolated from saliva and urine, the latter involving a patient with kidney stone disease, suggesting potential roles in human infection (3, 4). URMC-2103A041 was recovered from a patient whose bacteremia was previously described (5). The organism was detected after 9 hours of incubation at 37°C using a BACT/ALERT SA blood culture bottle on a Virtuo automated blood culture instrument (bioMérieux). Colony growth was isolated on tryptic soy agar with 5% sheep blood agar plates (BAP) after 24 hours at 35°C.
MALDI-TOF (MALDI Biotyper, Bruker) identified the organism as Pantoea septica [log score of 1.8 (low confidence)]. Biochemical identification (API 20E, bioMérieux) produced Pantoea spp. (98.2% identification). The BD Phoenix NID panel identification was Shigella flexneri. Identification was resolved by 16S rRNA V1-V3 sequencing (MicroSeq 500 16S rDNA Sequening Kit) (6) showing 100% identity with K. piersonii (Pathogenomix database; Genbank: OR250797.1). Susceptibility was determined using zone diameter breakpoints for Enterobacterales (CLSI M100, 30th Ed.) (7).
The bacilli were non-hemolytic and a late lactose fermenter (Fig. 1A). Like other Pantoea spp., URMC-2103A041 was Voges-Proskauer- and ONPG-positive, fermented mannitol, arabinose, and rhamnose, but was arginine dihydrolase positive (8, 9).
Fig 1.
Gram stain and colonial appearance of K. piersonii URMC-2103A041.
For DNA extraction of both short- and long-read sequencing library preparation, colonies were purified on a BAP, then inoculated into tryptic soy broth, and incubated for 18 hours (30°C shaking at 200 rpm). DNA was extracted following the protocol for “Pretreatment for Gram-Negative Bacteria” (DNeasy Blood and Tissue kit; Qiagen). DNA shearing and size selection were not performed. DNA was quantified with the QuantiFluor dsDNA system (Promega).
For short reads, paired-end sequencing following library preparation (DNA Prep Kit, Illumina) was performed on a MiSeq instrument (MiSeq Reagent v2 500-cycle kit, Illumina). Unless otherwise specified, sequences were analyzed with tools available on the Galaxy server (v22.05, usegalaxy.eu), using default parameters (10). Reads were trimmed with Trimmomatic v0.36 (11) and quality checked with FastQC v0.11.5 (12) to generate 645,637 paired-end reads (average read length: 224 bp).
For long-read sequencing, 40 ng DNA was used to prepare libraries with the Rapid Barcoding Sequencing Kit [Oxford Nanopore Technologies (ONT), SQK-RBK004]. Long-read libraries were sequenced (MinION flow cell, FLO-MIN106), and base calling was performed with Guppy v6.1.5 (MinKNOW v22.05.5). Filtering (quality score of >8) generated 93,023 reads (read N50: 11,125 bp) for hybrid assembly (Unicycler v0.5.0, with rotation to default start gene) and polishing (Quast v5.2.0) (13, 14).
The hybrid assembly genome was 4,990,348 bp with 57.02% GC content; it consisted of one circular chromosome (3.93 Mb, GenBank: CP115895.1), three circular plasmids (527.8 kb, CP115896.1; 277.8 kb, CP115897.1; and 174.2 kb, CP115898.1), and one linear plasmid (81.1 kb, CP115899.1). Annotation was added by the NCBI Prokaryotic Genome Annotation Pipeline (15 - 17).
ACKNOWLEDGMENTS
The authors would like to thank Stepan Volhejn for sequencing expertise and the staff of UR Medicine Labs.
Funding from the URMC Department of Pathology and Laboratory Medicine supported this study.
Contributor Information
Andrew Cameron, Email: andrew_cameron@urmc.rochester.edu.
David Rasko, University of Maryland School of Medicine, Baltimore, Maryland, USA .
DATA AVAILABILITY
The genome sequence and raw sequencing reads for URMC-2103A041 were deposited under BioProject accession number PRJNA916888 (BioSample accession number SAMN32512754) and SRA accession numbers SRX18896138 and SRX18895383. The partial 16S sequence was deposited under Genbank accession number OR250797.1.
ETHICS APPROVAL
This study was performed under University of Rochester Medical Center (URMC) institutional review board-approved protocol RSRB00007269.
REFERENCES
- 1. Singh NK, Wood JM, Mhatre SS, Venkateswaran K. 2019. Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the international space station. Appl Microbiol Biotechnol 103:4483–4497. doi: 10.1007/s00253-019-09813-z [DOI] [PubMed] [Google Scholar]
- 2. Soutar CD, Stavrinides J. 2022. Phylogenomic analysis of the Erwiniaceae supports reclassification of Kalamiella piersonii to Pantoea piersonii comb. Nov. and Erwinia gerundensis to the new genus Duffyella gen. nov. as Duffyella gerundensis comb. nov. Mol Genet Genomics 297:213–225. doi: 10.1007/s00438-021-01829-3 [DOI] [PubMed] [Google Scholar]
- 3. McDonagh F, Singh NK, Venkateswaran K, Lonappan AM, Hallahan B, Tuohy A, Burke L, Kovarova A, Miliotis G. 2023. First complete genome of a multidrug resistant strain of the novel human pathogen Kalamiella piersonii (GABEKP28) identified in human saliva. J Glob Antimicrob Resist 32:31–34. doi: 10.1016/j.jgar.2022.12.003 [DOI] [PubMed] [Google Scholar]
- 4. Rekha PD, Hameed A, Manzoor MAP, Suryavanshi MV, Ghate SD, Arun AB, Rao SS, Bajire SK, Mujeeburahiman M, Young C-C. 2020. First report of pathogenic bacterium Kalamiella piersonii isolated from urine of a kidney stone patient: draft genome and evidence for role in struvite crystallization. Pathogens 9:711. doi: 10.3390/pathogens9090711 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Rodolfo Alpizar-Rivas AC, Hay CM. 2022. Human disease caused by Kalamiella piersonii. AIMCC 1. doi: 10.7326/aimcc.2022.0220 [DOI] [Google Scholar]
- 6. Arosio M, Nozza F, Rizzi M, Ruggeri M, Casella P, Beretta G, Raglio A, Goglio A. 2008. Evalutaion of the MICROSEQ 500 16S rDNA-based gene sequencing for the diagnosis of culture-negative bacterial meningitis. New Microbiol. 31:343–349. [PubMed] [Google Scholar]
- 7. CLSI. 2022. Performance standands for antimicrobial susceptibiltiy testing, 32nd ed. CLSI supplement M100. [Google Scholar]
- 8. De Baere T, Verhelst R, Labit C, Verschraegen G, Wauters G, Claeys G, Vaneechoutte M. 2004. Bacteremic infection with Pantoea ananatis. J Clin Microbiol 42:4393–4395. doi: 10.1128/JCM.42.9.4393-4395.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Siwakoti S, Sah R, Rajbhandari RS, Khanal B. 2018. Pantoea agglomerans infections in children: report of two cases. Case Rep Pediatr 2018:4158734. doi: 10.1155/2018/4158734 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Grüning BA, Guerler A, Hillman-Jackson J, Hiltemann S, Jalili V, Rasche H, Soranzo N, Goecks J, Taylor J, Nekrutenko A, Blankenberg D. 2018. The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46:W537–W544. doi: 10.1093/nar/gky379 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Andrews S. 2017. FastQC: a quality control tool for high throughput sequence data. 2010.
- 13. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi: 10.1371/journal.pcbi.1005595 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V, O’Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR, Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A, Pruitt KD. 2018. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res 46:D851–D860. doi: 10.1093/nar/gkx1068 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Li W, O’Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ, Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Research 49:D1020–D1028. doi: 10.1093/nar/gkaa1105 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569 [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
The genome sequence and raw sequencing reads for URMC-2103A041 were deposited under BioProject accession number PRJNA916888 (BioSample accession number SAMN32512754) and SRA accession numbers SRX18896138 and SRX18895383. The partial 16S sequence was deposited under Genbank accession number OR250797.1.