Comparative genome analysis of Lactococcus garvieae  RTCLI04 with Lactococcus garvieae ATCC 49156 and Lactococcus garvieae Lg2  isolated from fish

 

Authors

Neetu Shahi1, Sumanta Kumar Mallik1*, Monalisha Sahoo2 & Debajit Sarma1

Affiliations

1ICAR-Directorate of Coldwater Fisheries Research (ICAR-DCFR)

Anusandhan Bhavan, Industrial Area, Bhimtal 263 136

Nainital, Uttarakhand, India

2ICAR-Indian Veterinary Research Institute (ICAR-IVRI), Bareilly, 243122, Uttarpradesh, India

*Correspondence should be addressed to Sumanta Kumar Mallik, Fish health Section, ICAR-DCFR, Bhimtal 263 136, Nainital, Uttarakhand, India,

email: sumanta1@rediffmail.com

PUBMED

 

Abstract

The present study reports the whole genome sequence (WGS) of Lactococcus garvieae RTCLI04, isolated from cultured rainbow trout, Oncorhynchus mykiss in India, showing clinical signs of haemorrhagic septicaemia. The genome of L. garvieae RTCLI04 was a single circular chromosome of 2,054,885 bp that encoded 1,993 proteins. Comparative analysis of L. garvieae RTCLI04 genome with two other existing reference genomes, L. garvieae ATCC 49156 (avirulent strain) and L. garvieae Lg2 (virulent strain) revealed  that the pangenome was consisted of 2,239 protein-coding genes in which 1,850 (83%) genes were core gene, 389 genes (17%) were dispensable gene and 19 genes were unique to our strain. Similar to L. garvieae ATCC 49156, L. garvieae RTCLI04 of the current study was lack of genomic island of 16.5 Kb capsule gene cluster. The study provides new insight into understanding the virulence mechanism, antimicrobial resistance and development of therapeutic measures against L. garvieae disease outbreak in aquaculture.

Keywords: Whole genome sequence; Lactococcus garvieae; rainbow trout; aquaculture

 

Supplement

Lactococcus garvieae is a deadly zoonotic pathogen that causes fatal hemorrhagic septicaemia and meningoencephalitis in humans, animals and fishes [1]. It is a Gram-positive, α-hemolytic, chain-forming and non-motile cocci. This bacterium is one of the economically important, emerging fish pathogen [2], affecting fish inhabiting freshwater to marine habitat. This bacterium causes considerable economic loss in aquaculture in Mediterranean region, Japan, Europe, South-East Asian countries and North America. Originally L. garvieae was first isolated in the United Kingdom from the clinical sample of mastitis [3]. However, due to movement of the carrier or infected fish from one location to another, lack of suitable control measures, zoonotic nature of the pathogen and rise in inland water temperature due to global warming, this bacterium has spread to naive rainbow trout populations, globally [4]. Thus, reported in farmed rainbow trout, Oncorhynchus mykiss from Spain, the disease has now spread to rainbow trout farms of Asia, Middle-East, Australia, America and Africa.

Rainbow trout mortality due to L. garvieae infection can be up to 60% of the total standing crop that may depend upon the water temperature, stress to fish and type strain of the bacterium (capsulated or non-capsulated) [4]. So far, the virulence of L. garvieae is linked to its ability to form capsule, and therefore serologically classified into two groups, KG+ (non-capsulated and non-virulent) and KG- (capsulated and virulent) [5].  Despite severe mortality caused by L. garvieae, a little is known about the genome of this bacterium.

In the current study, two available WGS of genus Lactococcus, L. garvieae ATCC 49156 (GenBank accession number: AP009332) and L. garvieae Lg2 (GenBank accession number: AP009333) were used for the comparative genome analysis with WGS of L. garvieae RTCLI04. L. garvieae RTCLI04 isolated in the year 2015 from intestine of a diseased rainbow trout, O. mykiss showing the clinical symptoms of L. garvieae infection from India. The bacterium was whole genome sequenced using HiSeq Illumina platform to generate approximately 2.5 GB data. Assembly was performed using Velvet v1.2.10, and optimised at kmer109. Assembly statistics were calculated by in-house perl scripts.  tRNAscan-SE v1.31 and RNAmmer v1.2  was used for identification of tRNAs and rRNAs genes respectively from identified scaffolds. Prodigal v2.6.1 was used for the identification of protein-coding genes (www.github.com/hyattpd/prodigal), whereas BlastP v2.2.30 was used for similarity search against NR (non-redundant) database for annotation of protein-coding genes with a cut-off E-values of 10-5. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed by Blast2GO Pro v4.1.9 and KAAS (KEGG automatic annotation server), respectively. The genome sequence of L. garvieae strain RTCLI04 has been deposited in the EMBL database under BioProject accession number PRJNA508270 and annotated assembly is available under accession number RSFD00000000.

Dot plots of complete genome sequences of all the three strains were generated by MUMmer v3.22 and the Mummerplot script. The orthologous and strain specific number of genes were identified using OrthoMCL v2.09, and a phylogenetic tree was generated from matrix file showing the relationship between the L. garvieae RTCLI04, L. garvieae ATCC 49156 and L. garvieae Lg2.

Preliminary comparison of the genome of L. garvieae RTCLI04 with genome of L. garvieae ATCC 49156 and L. garvieae Lg2 showed a difference of 1,04,750 bp and 9,092 bp in genome size respectively (Table 1).  Our L. garvieae isolate has the larger genome size (~ 2.0 Mbp) and has a slightly higher number of protein-coding genes (1,993 bp) than that of the two reference strains used for comparison in the current study.

 

Table 1. Genome characteristics of L. garvieae RTCLl04, L. garvieae ATCC 49156 and L. garvieae Lg2

 

A nucleotide based sequence alignment analysis for comparing the genome using MUMmer revealed the co-linearity of L. garvieae RTCLI04 to L. garvieae ATCC 49156 and L. garvieae Lg2 (Figure 1A and B), with 99.2% sequence (2,034,336 bp)  identity in the aligned regions. Aligned nucleotide segments were represented by dots or lines (Figure 1). Phylogenetically, our strain was more closely related to L. garvieae ATCC 49156 (Figure 2) among the two reference strains used for comparison in this study. Examination of ortholog’s via orthoMCL showed that the pangenome consisted of 2,239 protein-coding genes, in which 1,850 protein-coding genes (83%) were core gene and 389 protein-coding genes (17%) were a dispensable gene (Figure 3). Around 19 protein-coding genes were unique to L. garvieae RTCLI04.

 

 

 

Figure 1. Dot-plot alignment of 2.0 Mbp chromosome of L. garvieae RTCLI04 (x-axis) to a 1.9 Mbp scaffold of L. garvieae ATCC49156 (y-axis; A) and L. garvieae Lg2 (y-axis; B). This nucleotide based alignment is derived with Nucmer. Each dot point on the plot represents reciprocal best matches by BLASTP comparisons between the orthologs and line or dot represent the aligned nucleotide sequences.

 

Figure 2. Genome based phylogenetic analysis of L.garvieae RTCLI04, L. garvieae ATCC49156 and L. garvieae Lg2.

 

Figure 3. Venn diagram showing the overlap of gene repertoires of L.garvieae RTCLI04, L.garvieae ATCC 49156 and L.garvieae Lg2. The core gene of L.garvieae genome contains 1850 genes and 19 genes specific to L.garvieae RTCLI04 genome.

 

The WGS of microbe provides insight into the characteristics of that pathogen, intraspecific diversity, epidemiological information and functional characteristics and therefore it helps in the development of useful diagnostic tools and effective disease control strategy in aquaculture. In the current study, using Illumina HiSeq platform, WGS of zoonotic bacterial pathogen, L. garvieae type strain RTCLI04 isolated for the first time from the diseased food fish, rainbow trout, O. mykiss in India has been determined. Comparative genome analysis of L. garvieae RTCLI04 with two other reference strains, L. garvieae ATCC 49156 and L. garvieae Lg2 is also carried out to understand the functions of various genes and evolution of its genome. 

 

References

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