Resistance and genomic characterization of a plasmid pkh2101 harbouring erm(B) isolated from emerging fish pathogen Lactococcus garvieae serotype II in Japan.

The emergence of antibiotic-resistant pathogenic strains of Lactococcus garvieae serotype II isolated from fish in Japan has become a growing concern in recent years. The data on drug susceptibility and its associated resistance mechanism are limited. Therefore, the present study was conducted to determine the minimum inhibitory concentrations (MICs) of chemotherapeutic agents against 98 pathogenic strains of emerging Lactococcus garvieae serotype II isolated from fish from six different prefectures in Japan from 2018 to 2021. The tested strains were resistant to erythromycin, lincomycin and tiamulin. PCR amplification revealed the presence of erm(B) in all erythromycin-resistant strains, while a conjugation experiment confirmed that these strains carried erm(B) that could be transferred to recipient Enterococcus faecalis OG1RF with frequencies from 10-4 to 10-6 per donor cells. Nucleotide sequencing of the representative isolated plasmid pkh2101 from an erythromycin-resistant strain showed that it was a 26,850 bp molecule with an average GC content of 33.49%, comprising 31 CDSs, 13 of which remained without any functional annotation. Comparative genomic analysis suggested that pkh2101 shared the highest similarity (97.57% identity) with the plasmid pAMbeta1, which was previously isolated clinically from Enterococcus faecalis DS-5. This study provides potential evidence that the plasmid harbouring erm(B) could be a source of antibiotic resistance transmission in emerging L. garvieae infection in aquaculture.

Complete genome sequence of a novel lytic bacteriophage, PLG-II, specific for Lactococcus garvieae serotype II strains that are pathogenic to fish.

A novel lytic siphophage, PLG-II, which is specific for Lactococcus garvieae serotype II strains that are pathogenic to fish, was isolated from seawater samples collected from Miyazaki Prefecture, Japan. Whole-genome sequencing showed that the PLG-II genome is a 32,271-bp double-stranded DNA molecule, with an average GC content of 37.74%. It contains 69 open reading frames (ORFs), 43 of which currently have no reliable functional annotation for their product, as well as a single tRNA. Comparative genomics analysis suggests that phage PLG-II might represent a novel species in the genus Uwajimavirus.

A novel jumbo Tenacibaculum maritimum lytic phage with head-fiber-like appendages.

A novel jumbo bacteriophage (myovirus) is described. The lytic phage of Tenacibaculum maritimum, which is the etiological agent of tenacibaculosis in a variety of farmed marine fish worldwide, was plaque-isolated from seawater around a fish aquaculture field in Japan. The phage had an isometric head 110-120 nm in diameter, from which several 50- to 100-nm-long flexible fiber-like appendages emanate, and a 150-nm-long rigid contractile tail. The full genomes of the two representative phages (PTm1 and PTm5) were 224,680 and 226,876 bp long, respectively, both with 29.7% GC content, and the number of predicted open reading frames (ORFs) was 308 and 306, respectively. The average nucleotide sequence identity between PTm1 and PTm5 was 99.95%, indicating they are quite similar to each other. A genetic relationship was found in 15.0-16.6% of the predicted ORFs among the T. maritimum phages PTm1 and PTm5, the Tenacibaculum spp. phage pT24, and the Sphingomonas paucimobilis phage PAU. Phylogenetic analysis based on the terminase large subunit genes revealed that these four phages (PTm1, PTm5, pT24 and PAU) are more closely related than the other 10 jumbo myoviruses that have similar genome sizes. Transmission electron microscopy observations suggest that the head fibers of the T. maritimum phage function as tentacles to search and recognize the host cell surface to facilitate infection.

Complete genome sequence and characterization of virulence genes in Lancefield group C Streptococcus dysgalactiae isolated from farmed amberjack (Seriola dumerili).

Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.

Host range and influence of a cell capsule on the phage efficacy of three Lactococcus garvieae lytic phages.

Three lytic phages (PLgW-1, PLgY-16, and PLgY-30) were previously used for phage-typing Lactococcus garvieae, a bacterial pathogen of various marine fish species, and were demonstrated to be potential therapeutants for infections caused by L. garvieae. The morphology, host range, and efficacy of these phages have not been investigated in detail, however. The current study examined the lysis spectrum of these 3 phages against 16 different genotypes of L. garvieae and the influence of a bacterial capsule on phage efficacy, to aid in developing an effective treatment for lactococcosis in fish. Morphological analysis by transmission electron microscopy revealed that all 3 phages belonged to the family Siphoviridae and had a minor difference in morphology. These phages lysed a high proportion of their bacterial host (93.7% of the different L. garvieae genotypes). In addition, the efficacy of the plating assays was affected by both the phages and their bacterial host, in which phage efficacy was clearly affected by a bacterial capsule. The results of this study may be useful for developing appropriate strategies to use these phages to control various genotypes of L. garvieae causing disease in marine fish.

Multilocus sequence analysis of Vibrionaceae isolated from farmed amberjack and the development of a multiplex PCR assay for the detection of pathogenic species.

Since 2011, high mortality rates and symptoms consistent with vibriosis have been observed in farmed amberjack (Seriola dumerili) in Japan. To identify 41 strains isolated from diseased amberjack, a multilocus sequence analysis using nine concatenated genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) was conducted. Twenty-seven strains were identified as Vibrio harveyi, suggesting an epidemic of V. harveyi infection in amberjack farms. Other strains were identified as Vibrio anguillarum, Vibrio owensii and Photobacterium damselae subsp. damselae. To develop an efficient diagnostic method for vibriosis in amberjack, a multiplex PCR system was developed to identify V. anguillarum, V. harveyi and P. damselae subsp. damselae. The method successfully discriminated between these three bacterial species, with amplification products of 350 bp for V. anguillarum, 545 bp for V. harveyi and 887 bp for P. damselae subsp. damselae and can be used for diagnosis in aquaculture farms.

Full-genome sequence of a novel myovirus, GF-2, infecting Edwardsiella tarda: comparison with other Edwardsiella myoviral genomes.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.

Complete Genome Sequence of Lactococcus garvieae MS210922A, Isolated from Farmed Greater Amberjack (Seriola dumerili) in Japan.

Here, we report the complete genome sequence of a potentially new serotype, Lactococcus garvieae strain MS210922A, isolated from greater amberjack. It is nonreactive to serotype I or II antisera. The complete genome comprises 2,007,159 bp, including 1,929 coding, 16 rRNA, and 58 tRNA genes, with 38.9% G+C content.

Characterization of lsa(D), a Novel Gene Responsible for Resistance to Lincosamides, Streptogramins A, and Pleuromutilins in Fish Pathogenic Lactococcus garvieae Serotype II.

Aims: Fish pathogenic Lactococcus garvieae serotype II has been isolated from cultured fish species in Japan. This study aimed to investigate the molecular mechanisms of lincomycin (LCM)-resistant L. garvieae serotype II and assess the molecular basis for lincosamides-streptogramins A-pleuromutilins (LSAP)-resistant phenotype. Results: We identified a novel lsa(D)-encoded 497-aa ATP-binding cassette F (ABC-F) protein in the LSAP-resistant strains. Amino acid identities of 41.25-54.73% were obtained between the deduced amino acids from Lsa(D) and other Lsa-type ABC-F proteins. Furthermore, comparative analysis revealed that the allele of lsa(D) with single point mutation at 233 aa position (TGG → TAG; tryptophan→premature termination codon [PTC]) in LSAP-sensitive strains. The minimum inhibitory concentrations of antimicrobials against the lsa(D) complementary strain and lsa(D)-disrupted mutant confirmed that lsa(D) conferred the LSAP-resistant phenotype. The reverse transcription-polymerase chain reaction could not detect the noncoding region of lsa(D) allelic variant in the LSAP-sensitive strains. Additionally, the PTC (TAG) in LCM-sensitive strains was replaced by TGG, CAG, or TAT in the laboratory-induced revertant mutants. Conclusions: The novel lsa(D) conferred the LSAP-resistant phenotype in clinically LCM-resistant L. garvieae serotype II strains. However, the allele of lsa(D) gene containing the PTC was found in L. garvieae serotype II, resulting in the LSAP-susceptible phenotype.

A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone.

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

Author Correction: A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparative genomic analysis of three lytic Lactococcus garvieae phages, novel phages with genome architecture linking the 936 phage species of Lactococcus lactis.

To date, a number of bacteriophages that infect Lactococcus garvieae isolated from marine fish have been identified. However, the evolutionary insight between L. garvieae phages and other viral community have not yet been immersedly investigated. In this study, completed genomic sequence of phage PLgY-30 was obtained, a comparative analysis of three lytic phages, which have been using for phage typing and treatment of L. garvieae infecting marine fish, is conducted. The results revealed that the genomes of lytic phages specific for L. garvieae isolated from diseased marine fish share a high level of homology and almost all proteins are conserved. At genome level, no similarity was detected for either PLgY-30 or PLgY-16, while PLgW-1 shares only very limited homology (1%) with other sequences in Genbank database. In addition, the function of only 35% of ORFs in the PLgY-30 phage genomes could be predicted, demonstrating that it is novel phage. At protein level, lytic phage proteins shared a significant similarity to various proteins of global phage species isolated from dairy fermentation facilities that utilize L. lactis as a primary starter culture, called the 936 phage group. Genome organization and architecture of three lytic phages are also similar to that of the 936 phage group. To our knowledge, this is the first time lytic bacteriophages infecting L. garvieae from marine fish were characterized to genome level.

Improvement of the Pacific bluefin tuna (Thunnus orientalis) reference genome and development of male-specific DNA markers.

The Pacific bluefin tuna, Thunnus orientalis, is a highly migratory species that is widely distributed in the North Pacific Ocean. Like other marine species, T. orientalis has no external sexual dimorphism; thus, identifying sex-specific variants from whole genome sequence data is a useful approach to develop an effective sex identification method. Here, we report an improved draft genome of T. orientalis and male-specific DNA markers. Combining PacBio long reads and Illumina short reads sufficiently improved genome assembly, with a 38-fold increase in scaffold contiguity (to 444 scaffolds) compared to the first published draft genome. Through analysing re-sequence data of 15 males and 16 females, 250 male-specific SNPs were identified from more than 30 million polymorphisms. All male-specific variants were male-heterozygous, suggesting that T. orientalis has a male heterogametic sex-determination system. The largest linkage disequilibrium block (3,174 bp on scaffold_064) contained 51 male-specific variants. PCR primers and a PCR-based sex identification assay were developed using these male-specific variants. The sex of 115 individuals (56 males and 59 females; sex was diagnosed by visual examination of the gonads) was identified with high accuracy using the assay. This easy, accurate, and practical technique facilitates the control of sex ratios in tuna farms. Furthermore, this method could be used to estimate the sex ratio and/or the sex-specific growth rate of natural populations.

Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

A lytic bacteriophage of the newly emerging rainbow trout pathogen Weissella ceti.

This study was conducted to isolate and characterize a bacteriophage of a newly emerging pathogen, Weissella ceti, which causes weissellosis outbreaks of intensively farmed rainbow trout worldwide. The phage appeared together with the cultured Weissella ceti during isolation of pathogen from kidney of diseased rainbow trout. The morphological, physiological, proteomic and lytic spectrum were characterized. This phage, named PWc, belonged to the family Siphoviridae and possessed an isometric head (approximately 65 nm in diameter) and a flexible, non-contractile tail of 170-180 nm in length. The latent time and burst size of PWc were approximately 25 min and 16 PFU/infected cells, respectively. The PWc was relatively stable over a wide range of temperatures and pH values and possessed a broad lytic spectrum, lysing all 36 tested W. ceti strains isolated from diseased rainbow trout in Japan. The protein profile of the phage was obtained using SDS-PAGE analysis, and the potential packaging strategy was determined based on terminase large subunit sequence analysis. This is the first study to investigate a lytic bacteriophage of a newly emerging pathogen W. ceti that causes infectious disease in rainbow trout.

Identification of genetic linkage group 1-linked sequences in Japanese eel (Anguilla japonica) by single chromosome sorting and sequencing.

Japanese eel (Anguilla japonica) constitutes one of the most important food fish in Japan; accordingly, genome sequencing and linkage mapping have been conducted for the purpose of artificial cultivation. In the next stage, integration of genomic sequences within linkage groups (LG) is required to construct higher-resolution genetic markers for quantitative trait loci mapping and selective breeding of beneficial traits in farming. In order to identify LG1-linked scaffolds from the draft genome assembly (323,776 scaffolds) reported previously, we attempted to isolate chromosomes corresponding to LG1 by flow sorting and subsequent analyses. Initially, single chromosomes were randomly collected by chromosome sorting and subjected to whole-genome amplification (WGA). A total of 60 WGA samples were screened by PCR with primers for a known LG1-linked scaffold, and five positive WGA samples were sequenced by next-generation sequencing (NGS). Following reference mapping analysis of the NGS reads, four of the five WGA samples were found to be enriched by LG1-linked sequences. These samples were cytogenetically assigned to chromosome 5 by fluorescence in situ hybridization. Using blastn searches with 82,081 contigs constructed from the NGS reads of the four WGA samples as queries, 2323 scaffolds were identified as putative LG1-linked scaffolds from the draft genome assembly. The total length of the putative LG1-linked scaffolds was 99.0 Mb, comparable to the estimated DNA amounts of chromosome 5 (91.1 Mb). These results suggest that the methodology developed herein is applicable to isolate specific chromosome DNAs and integrate unanchored scaffold sequences onto a particular LG and chromosome even in teleost fishes, in which isolation of specific chromosomes by flow sorting is generally difficult owing to similar morphologies, sizes, and GC-contents among chromosomes in the genome. The putative LG1-linked scaffolds of Japanese eel contain a total of 6833 short tandem repeats which will be available for higher-resolution linkage mapping.

Genetic parameters and quantitative trait loci analysis associated with body size and timing at metamorphosis into glass eels in captive-bred Japanese eels (Anguilla japonica).

The Japanese eel (Anguilla japonica) is among the most important aquaculture fish species in Eastern Asia. The present study aimed to identify the genetic parameters underlying body size and the timing at metamorphosis from leptocephali to glass eels in captive-bred Japanese eels, with the intent to foster sustainable development. Larvae from a partly factorial cross (14 sires × 11 dams) were reared until the point of metamorphosis into glass eels. In these organisms, we observed moderate heritability and mild genetic correlations among traits related to body size (h2 = 0.16-0.33) and timing at metamorphosis (h2 = 0.36-0.41). In an F1 full-sib family, quantitative trait loci (QTL) mapping for these traits identified one significant (genome-wide P < 0.05) and five suggestive QTLs (chromosome-wide P < 0.05). These results suggest that in the Japanese eel, metamorphic traits exhibit a polygenic genetic structure comprising many QTLs with small effects. In addition, we updated the genetic linkage map for the Japanese eel and integrated it with our newly constructed de novo genome assembly. The information and tools generated from this study will contribute to the development of freshwater eel genetics and genomics.

The yellowtail (Seriola quinqueradiata) genome and transcriptome atlas of the digestive tract.

Seriola quinqueradiata (yellowtail) is the most widely farmed and economically important fish in aquaculture in Japan. In this study, we used the genome of haploid yellowtail fish larvae for de novo assembly of whole-genome sequences, and built a high-quality draft genome for the yellowtail. The total length of the assembled sequences was 627.3 Mb, consisting of 1,394 scaffold sequences (>2 kb) with an N50 length of 1.43 Mb. A total of 27,693 protein-coding genes were predicted for the draft genome, and among these, 25,832 predicted genes (93.3%) were functionally annotated. Given our lack of knowledge of the yellowtail digestive system, and using the annotated draft genome as a reference, we conducted an RNA-Seq analysis of its three digestive organs (stomach, intestine and rectum). The RNA-Seq results highlighted the importance of certain genes in encoding proteolytic enzymes necessary for digestion and absorption in the yellowtail gastrointestinal tract, and this finding will accelerate development of formulated feeds for this species. Since this study offers comprehensive annotation of predicted protein-coding genes, it has potential broad application to our understanding of yellowtail biology and aquaculture.

Analysis of the complete genome sequence of Nocardia seriolae UTF1, the causative agent of fish nocardiosis: The first reference genome sequence of the fish pathogenic Nocardia species.

Nocardiosis caused by Nocardia seriolae is one of the major threats in the aquaculture of Seriola species (yellowtail; S. quinqueradiata, amberjack; S. dumerili and kingfish; S. lalandi) in Japan. Here, we report the complete nucleotide genome sequence of N. seriolae UTF1, isolated from a cultured yellowtail. The genome is a circular chromosome of 8,121,733 bp with a G+C content of 68.1% that encodes 7,697 predicted proteins. In the N. seriolae UTF1 predicted genes, we found orthologs of virulence factors of pathogenic mycobacteria and human clinical Nocardia isolates involved in host cell invasion, modulation of phagocyte function and survival inside the macrophages. The virulence factor candidates provide an essential basis for understanding their pathogenic mechanisms at the molecular level by the fish nocardiosis research community in future studies. We also found many potential antibiotic resistance genes on the N. seriolae UTF1 chromosome. Comparative analysis with the four existing complete genomes, N. farcinica IFM 10152, N. brasiliensis HUJEG-1 and N. cyriacigeorgica GUH-2 and N. nova SH22a, revealed that 2,745 orthologous genes were present in all five Nocardia genomes (core genes) and 1,982 genes were unique to N. seriolae UTF1. In particular, the N. seriolae UTF1 genome contains a greater number of mobile elements and genes of unknown function that comprise the differences in structure and gene content from the other Nocardia genomes. In addition, a lot of the N. seriolae UTF1-specific genes were assigned to the ABC transport system. Because of limited resources in ocean environments, these N. seriolae UTF1 specific ABC transporters might facilitate adaptation strategies essential for marine environment survival. Thus, the availability of the complete N. seriolae UTF1 genome sequence will provide a valuable resource for comparative genomic studies of N. seriolae isolates, as well as provide new insights into the ecological and functional diversity of the genus Nocardia.