ABSTRACT
We report the complete genome sequence of Lactococcus garviae strain 20-1E, isolated from a Danish freshwater moat. The genome was generated using hybrid Oxford Nanopore and Illumina sequencing. Taxonomic identity was confirmed by GTDB-Tk and average nucleotide identity analysis, highlighting virulence and its potential as a free-living species in freshwater.
KEYWORDS: Lactococcus garvieae, complete genome sequence, hybrid genome assembly, freshwater isolate, virulence genes
ANNOUNCEMENT
Lactococcus garviae is a fish pathogen and may cause human infections (1–3). Strain 20-1E was isolated from a freshwater stream (03 February 2021, Table 1) and sequenced to assess its pathogenic potential. We collected 10 mL water sample with a sterile plastic pipette from approximately 5 cm depth and within 1 m from shore and transferred it into a sterile 13 mL propylene centrifuge tube (TRP, Switzerland) and stored it for less than 48 h below 10°C before arrival at the laboratory.
TABLE 1.
Sampling location description and genome characteristics for Lactococcus garviae 20-1E
| Description of location | |
|---|---|
| Location | E Jutland, Moat, Stagsevold, Stagsrode Forest (4) |
| Time | 3 February 2021 |
| Type | Freshwater stream, pH 6.73, forming a part of a medieval castle moat within a beech forest |
| Geographic coordinates | 55°40′45.7″N 9°50′48.4″E |
| Genome statistics | |
| Assembly size (bp) | 2,140,176 |
| Number of contigs | 1 |
| Contig N50 (bp) | 2,140,176 |
| Contig L50 | 1 |
| GC (%) | 38.5% |
| Number of 5S ribosomal RNA | 6 |
| Number of 16S ribosomal RNA | 5 |
| Number of 23S ribosomal RNA | 5 |
| Number of transfer RNAs | 61 |
| Total number of genes | 2,111 |
| Total number of CDSa | 2,049 |
| GTDB taxonomy | Lactococcus garvieae |
| Read characteristics | |
| Illumina reads 1 | Total reads: 805,066; total bases: 110,891,409 |
| Illumina reads 2 | Total reads: 805,066; total bases: 110,904,335 |
| Genome Coverage Illumina | 102× |
| Nanopore reads | Total reads: 228,521; total bases: 500,778,065; raw reads N50: 7,196 |
| Genome Coverage Nanopore | 230× |
| Read data accession | |
| BioSample | SAMN54194371 |
| Illumina Reads Accession | SRR36501241 |
| ONT Reads Accession | SRR36502162 |
| Assembly Accession | JBUEDY010000001 |
CDS, coding sequences.
One milliliter sample was transferred to APT broth (Difco, Sparks, MD, USA), incubated at 15°C under microaerophilic conditions for 3 days, streaked on nitrite-pyridoxal (NP) agar (APT agar [Merck, Darmstadt] with 1 mL 12% sodium nitrite [Ampliqon A/S, Odense, Denmark] and 0.2 mL polymyxin supplement [Oxoid SR 099] per 200 mL agar) and incubated at 15°C for 3 days. Gram-positive, catalase-negative colonies were selected for sequencing.
DNA was extracted using the Bead-Beat Micro AX Gravity kit (cat#106-100-M1; A&A Biotechnology, Gdynia, Poland). Long-read libraries were prepared using the Oxford Nanopore Rapid Barcoding Kit (SQK-RBK114.24) without prior size selection and sequenced on a MinION MK1D device and basecalled using Dorado v0.8.1. Paired-end 151-bp read libraries were sequenced on Illumina NovaSeq X and filtered using Trimmomatic v.0.40 (5), while Nanopore reads were filtered by minimum length of 1,000 bp using Filtlong 0.3.1. Hybrid genome assembly was performed using Trycycler v0.5.6 following the full developer-recommended pipeline (6) (step-by-step guide here: https://github.com/rrwick/Trycycler/wiki/How-to-run-Trycycler), where assembly polishing was conducted using the short reads on the long-read-assembled genomes.
Genome quality and completeness were assessed using CheckM2v1.1.0 and QUASTv5.3.0. Genome coverage was calculated using Qualimapv2.3 based on Minimap2v2.30 read alignments. Taxonomic classification was performed using GTDB-Tkv2.3.2 against the Genome Taxonomy Database (release 214) (7). Genome annotation was carried out using prokaryotic genome annotation pipeline (PGAP v2.0) (8), and ribosomal RNA genes were identified with Barrnapv0.9.
Virulence genes were identified using ABRicatev.1.4.0 with the VFDB v2025 core database, as well as a custom L. garviae virulence protein database (https://figshare.com/s/4c5eb3d394b85051380e). Genome assemblies were screened using TBLASTN (BLAST+ v2.17.0), retaining hits with ≥50% amino acid identity, ≥90% query coverage, and an e-value 1 × 10⁻²⁰. For each query, the best hit was selected based on bitscore and results summarized as a presence/absence matrix. Antimicrobial resistance and bacteriocin encoding genes were identified using ABRicate with the ResFinder v4.7.2 database and AntiSMASH 6.0 (9) and BAGEL 4 (10).
Average nucleotide identity (ANI) was calculated using FastANI v.1.34 (11) by comparing strain 20-1E against all publicly available L. garviae genomes in National Center for Biotechnology Information (NCBI) as of 12 December 2025. Public genomes were retrieved using ncbi-genome-download and dereplicated using dRep v.3.6.2 (11), and a pairwise ANI was visualized as a heatmap in RStudio v4.3.1.
Pairwise ANI analysis (Fig. 1) revealed only four closely related (≥99% ANI) strains: GCA_006782925 (from fish, India, 2019), GCA_030219685 (cattle, Spain, 2023), GCA_044988645.1 (fish, China, 2024), and GCA_900114555.1 (unknown source, 2016).
Fig 1.
Heatmap of pairwise average nucleotide identity (ANI) of our isolate 20-1E vs public genomes that are within at least 98% ANI to it.
The complete genome of L. garviae strain 20-1E is 2,140,176 bp in length, with a GC content of 38.5%, and encodes 2,103 predicted genes. The genome did not harbor bacteriocin-encoding genes but contained the mdt(A) gene, associated with resistance to erythromycin, azithromycin, and tetracycline, and the full constellation of known L. garviae virulence genes, including capsule biosynthesis genes, hemolysins (hly1-hly3), and multiple LPxTG-anchored adhesins, underscoring pathogenic potential.
ACKNOWLEDGMENTS
This research was supported by the United Kingdom’s Foreign, Commonwealth and De-velopment Office (FCDO) and Canada’s International Development Research Centre (IDRC) through the Climate Adaptation and Resilience (CLARE) programme, under grant number 110150–001 (to Y.M.G.H.). Use of the HPC compute nodes and resources for complete genome reconstruction was covered by the I-CRECT project, which received funding from Innovation Fund Denmark, Swedish Research Council, Federal Ministry of Education and Research, Germany, The French National Research Agency, and the International Centre for Antimicrobial Resistance Solutions (www.icars-global.org) under the umbrella of the JPIAMR-Joint Programming Initiative on Antimicrobial Resistance (to Y.M.G.H.).
Contributor Information
Yaovi Mahuton Gildas Hounmanou, Email: gil@sund.ku.dk.
Elinne Becket, California State University San Marcos, San Marcos, California, USA.
DATA AVAILABILITY
Paired-end Illumina data are submtted to sequence reads archive (SRA) under accession SRR36501241 and Oxford Nanopore Technologies (ONT) sequencing reads have been deposited under SRA accession number SRR36502162. Accession number of the complete genome assembly is available in the Nucleotide’s database in NCBI as JBUEDY000000000 with the full circular chormosome available at https://www.ncbi.nlm.nih.gov/nuccore/CM155296. The ABRicate output for resistance gene and BLAST virulence gene searches are deposited in FigShare with the DOI 10.6084/m9.figshare.31385272.
REFERENCES
- 1. Meyburgh CM, Bragg RR, Boucher CE. 2017. Lactococcus garvieae: an emerging bacterial pathogen of fish. Dis Aquat Org 123:67–79. doi: 10.3354/dao03083 [DOI] [PubMed] [Google Scholar]
- 2. Francés-Cuesta C, Ansari I, Fernández-Garayzábal JF, Gibello A, González-Candelas F. 2022. Comparative genomics and evolutionary analysis of Lactococcus garvieae isolated from human endocarditis. Microb Genom 8:000771. doi: 10.1099/mgen.0.000771 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Sirakov I, Strateva TV, Boyanov VS, Orozova P, Yordanov D, Rusenova N, Gergova R, Dimov SG, Sirakova B, Radosavljević V, Boyanova L, Mitov I. 2025. Identification, characterization, and epidemiological analysis of Lactococcus garvieae fish isolates obtained in a period of eighteen years. Microorganisms 13:436. doi: 10.3390/microorganisms13020436 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Tang T, Martinenghi LD, Hounmanou YMG, Leisner JJ. 2023. Distribution and ecology of the generalist lactic acid bacterium Carnobacterium maltaromaticum in different freshwater habitats: metabolic and antagonistic abilities. Environ Microbiol 25:3556–3576. doi: 10.1111/1462-2920.16508 [DOI] [PubMed] [Google Scholar]
- 5. 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]
- 6. Wick RR, Judd LM, Cerdeira LT, Hawkey J, Méric G, Vezina B, Wyres KL, Holt KE. 2021. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol 22:266. doi: 10.1186/s13059-021-02483-z [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. 2020. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics 36:1925–1927. doi: 10.1093/bioinformatics/btz848 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. 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]
- 9. Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP, Medema MH, Weber T. 2021. antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res 49:W29–W35. doi: 10.1093/nar/gkab335 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. van Heel AJ, de Jong A, Song C, Viel JH, Kok J, Kuipers OP. 2018. BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins. Nucleic Acids Res 46:W278–W281. doi: 10.1093/nar/gky383 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114. doi: 10.1038/s41467-018-07641-9 [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
Paired-end Illumina data are submtted to sequence reads archive (SRA) under accession SRR36501241 and Oxford Nanopore Technologies (ONT) sequencing reads have been deposited under SRA accession number SRR36502162. Accession number of the complete genome assembly is available in the Nucleotide’s database in NCBI as JBUEDY000000000 with the full circular chormosome available at https://www.ncbi.nlm.nih.gov/nuccore/CM155296. The ABRicate output for resistance gene and BLAST virulence gene searches are deposited in FigShare with the DOI 10.6084/m9.figshare.31385272.