Quinolone-resistant phenotype of Flavobacterium columnare isolates harbouring point mutations both in gyrA and parC but not in gyrB or parE.

OBJECTIVES: The aim of this study was to determine mutations associated with a quinolone-resistant (QR) phenotype of Flavobacterium columnare isolates. METHODS: The susceptibility of 53 F. columnare isolates to 11 antimicrobials, including 2 quinolones, was investigated by the disk diffusion method. Oxolinic acid (OXO) was subsequently chosen for minimum inhibitory concentration (MIC) assay. Sequence analysis of four genes within the quinolone resistance-determining regions (QRDRs) of OXO-resistant F. columnare compared with susceptible isolates was subsequently performed. RESULTS: The disk diffusion assay revealed that the majority of isolates were susceptible to all tested antimicrobials. However, 14 and 8 isolates were resistant to the quinolone antibiotics OXO and nalidixic acid, respectively. No multidrug resistance was observed. The MIC assay revealed five additional isolates that were resistant to OXO (≥4µg/mL), making a total of 19 OXO-resistant isolates observed in this study. DNA sequencing identified missense mutations both in parC and gyrA but not in gyrB or parE in QR F. columnare isolates. Mutation in parC resulted in the change His87→Tyr. For gyrA, 15 isolates of Thai origin exhibited a change at residue Ser83 to either Phe, Tyr or Ala, whereas 3 Vietnamese isolates contained two mutation sites (Ser83→Phe and Asp87→Tyr). CONCLUSION: This study is the first to reveal that QR phenotype F. columnare isolates harboured missense mutations both in parC and gyrA but not in gyrB or parE of the QRDRs.

Genome characterization of piscine 'Scale drop and Muscle Necrosis syndrome'-associated strain of Vibrio harveyi focusing on bacterial virulence determinants.

AIMS: Genomic characterization of Harveyi clade vibrio strain Y6 causing 'Scale drop and Muscle Necrosis syndrome' (SDMN) isolated from barramundi (Lates calcarifer) in Vietnam. METHODS AND RESULTS: A bacterial genome was sequenced using Illumina MiSeq platform. Multilocus sequence analysis confirmed that the bacterium belongs to Vibrio harveyi species. Further phylogenetic analysis inferred from core genome SNPs revealed a close relationship between our bacterium and the V. harveyi isolated from groupers in Taiwan and China. blastp results indicated that V. harveyi piscine strains carried numerous adhesin, secretion system, siderophore and toxin-related genes. Genome comparison between Y6 and 32 strains of V. harveyi from different origins showed that at least 17 potential virulence genes were present exclusively in the strain Y6. Many of these (six of 17 genes) were homologous to pyoverdine siderophore, a secreted high-affinity iron chelator, clusters originally found in Pseudomonas aeruginosa. Genome of V. harveyi Y6 was incorporated by a bacteriophage VHY6φ and replication protein of the phage was most similar to CTXφ described previously in Vibrio cholerae and Vibrio fischeri. However, the cholera toxin-encoding genes, namely ctxA and ctxB, were absent from VHY6φ, while the CTXφ-enterotoxin gene (zonula occludens toxin; zot) remained intact. CONCLUSIONS: Several putative virulence genes and a phage carrying toxin gene were identified in the genomes of SDMN-associated V. harveyi Y6. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confers genomic information of the piscine pathogenic V. harveyi which recently caused widespread mortality. Such information is of importance to gain insight into bacterial molecular pathogenesis.

Infectious spleen and kidney necrosis disease (ISKND) outbreaks in farmed barramundi (Lates calcarifer) in Vietnam.

Emergence of a disease with clinical signs resembling megalocytivirus infection seriously affected large-scale barramundi farms in Vietnam in 2012-2014 with estimated losses reaching $435,810 per year. An oil-based, inactivated vaccine against red sea bream iridovirus (RSIV) was applied in one farm for disease prevention without analysis of the causative agent, and the farmer reported inadequate protection. Here we describe histological and molecular analysis of the diseased fish. PCR targeting the major capsid protein (MCP) of megalocytiviruses yielded an amplicon with high sequence identity to infectious spleen and kidney necrosis virus (ISKNV) genotype II previously reported from other marine fish but not barramundi. Detection of the virus was confirmed by positive in situ hybridization results with fish tissue lesions of the kidney, liver, pancreas, and brain of the PCR-positive samples. Based on the complete sequence of the MCP gene, the isolate showed 95.2% nucleotide sequence identity and 98.7% amino acid sequence identity (6 residue differences) with the MCP of RSIV. Prediction of antigenic determinants for MCP antigens indicated that the 6 residue differences would result in a significant difference in antigenicity of the two proteins. This was confirmed by automated homology modeling in which structure superimpositioning revealed several unique epitopes in the barramundi isolate. This probably accounted for the low efficiency of the RSIV vaccine when tested by the farmer.

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite).

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70-90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.