Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei.

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.

Complete genomic sequence of a T4-like bacteriophage, phiAS4, infecting Aeromonas salmonicida subsp. salmonicida.

A newly identified virulent phage (named phiAS4) infecting Aeromonas salmonicida subsp. salmonicida was isolated from river water in Korea. Morphological analysis of phiAS4 by transmission electron microscopy revealed that it belonged to the family Myoviridae. The genome of phiAS4 comprised a linear double-stranded DNA of 163,875 bp with a G + C content of 41.3%, and genomic analysis revealed 271 putative ORFs, 67 putative promoters, 25 putative terminator regions, and 16 tRNA-encoding genes. Most of the ORFs of phiAS4 showed a high degree of similarity to those of Aeromonas phage 25, which belongs to the T4-like group. Moreover, the comparison of the genome of phiAS4 with those of its relatives demonstrated that phage phiAS4 is closely related to members of the T4-like group and can be classified as a new member of the T4-like phages infecting bacteria of the family Aeromonadaceae.

Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from Aeromonas species in South Korea.

AIMS: To investigate the tetracycline resistance related to tet genes in Aeromonas isolates collected from water and diseased fish in South Korea. METHODS AND RESULTS: A total of 34 Aeromonas strains were examined for their susceptibility to tetracycline using the minimum inhibitory concentration (MIC) assay, and the genetic determinants (tetA to E) were analysed. Among these strains, the tetA and tetE genes were predominant (tetA was found in six strains, and tetE was found in nine strains), and 15 strains were tetracycline-resistant by the MIC assay. Additionally, the 8979-bp plasmid that contains the tetE gene was fully sequenced. CONCLUSIONS: These data may be important with regard to the spread and persistence of tetracycline resistance genes in the bacterial populations that are present in aquaculture systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Interestingly, no isolate has previously been shown to harbour three tet genes that are mediated by efflux systems, but the tetA, tetD and tetE genes were all isolated from one strain, which had the highest MIC value for tetracycline among the strains analysed in this study. We also investigated the full-length plasmid that encoded the tetE gene from a tetracycline-resistant strain.

Isolation and characterization of a lytic Myoviridae bacteriophage PAS-1 with broad infectivity in Aeromonas salmonicida.

To search for candidate control agents against Aeromonas salmonicida subsp. salmonicida infections in aquaculture, one bacteriophage (phage), designated as PAS-1, was isolated from the sediment samples of the rainbow trout (Oncorhynchus mykiss) culture farm in Korea. The PAS-1 was morphologically classified as Myoviridae and possessed approximately 48 kb of double-strand genomic DNA. The phage showed broad host ranges to other subspecies of A. salmonicida as well as A. salmonicida subsp. salmonicida including antibiotic-resistant strains. Its latent period and burst size were estimated to be approximately 40 min and 116.7 PFU/cell, respectively. Furthermore, genomic and structural proteomic analysis of PAS-1 revealed that the phage was closely related to other Myoviridae phages infecting enterobacteria or Aeromonas species. The bacteriolytic activity of phage PAS-1 was evaluated using three subspecies of A. salmonicida strain at different doses of multiplicity of infection, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, PAS-1 could be considered as a novel Aeromonas phage and might have potentiality to reduce the impacts of A. salmonicida infections in aquaculture.

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene.

AIMS: Isolation and full sequence analysis of ColE-type plasmid, which carries the qnrS2 gene. METHODS AND RESULTS: Quinolone resistance (qnrS2) gene-carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer-walking approach. The total sizes of these plasmids (pAQ2-1 and pAQ2-2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2-type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1-type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. CONCLUSIONS: Two plasmids were assumed to be the same plasmid, and this identification of a plasmid-mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first finding of the ColE-type plasmid carrying the qnrS2 gene.

A small IncQ-type plasmid carrying the quinolone resistance (qnrS2) gene from Aeromonas hydrophila.

AIMS: To investigate the qnrS2 gene encoded by a plasmid obtained from Aeromonas hydrophila. METHODS AND RESULTS: To investigate the full-length sequence of the plasmid carrying qnrS2 (plasmid designated pAHH04) from the strain SNUFPC-A10, the full-length coding sequence of the qnrS region was first amplified. The remaining part of the plasmid was read outwards from this region. The plasmid pAHH04 contained the repC, repA, mobA and mobC genes, and its total size was 7191 bp with a G+C content of 60%. CONCLUSIONS: This study describes the full-length sequence of a plasmid carrying the qnrS2 gene from Aer. hydrophila. The plasmid pAHH04 carried plasmid replication and mobilization genes from IncQ-type plasmids. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated qnrS2 gene encoded by a plasmid from an Aer. hydrophila strain is of significant importance because it emphasizes the problem of antibiotic resistance as well as the ability of the determinants to spread among the different bacterial species that impact human health.

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea.

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S-23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype-specific biochemical characteristics and genetic characteristics of 16S-23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.

Effect of dimethyl sulfoxide on cell cycle synchronization of goldfish caudal fin derived fibroblasts cells.

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin-derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.

Biological control of Aeromonas salmonicida subsp. salmonicida infection in rainbow trout (Oncorhynchus mykiss) using Aeromonas phage PAS-1.

The potential control efficacy of Aeromonas phage PAS-1 was evaluated against Aeromonas salmonicida subsp. salmonicida infection in rainbow trout (Oncorhynchus mykiss) model in this study. The phage was co-cultured with the virulent A. salmonicida subsp. salmonicida strain AS05 that possesses the type III secretion system (TTSS) ascV gene, and efficient bacteriolytic activity was observed against the bacteria. The administration of PAS-1 in rainbow trout demonstrated that the phage was cleared from the fish within 200 h post-administration, and a temporal neutralizing activity against the phage was detected in the sera of phage-administrated fish. The administration of PAS-1 (multiplicity of infection: 10 000) in A. salmonicida subsp. salmonicida infected rainbow trout model showed notable protective effects, with increased survival rates and mean times to death. These results demonstrated that Aeromonas phage PAS-1 could be considered as an alternative biological control agent against A. salmonicida subsp. salmonicida infections in rainbow trout culture.