Jin Woo Jun

Isolation, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus in Korean Seafood

The principal objective of this study was to investigate the incidence, risk assessment, antibiotic resistance, and genotyping of Vibrio parahaemolyticus in Korean seafood. The incidence of V. parahaemolyticus in seafood obtained from several fish markets in Korea was investigated from May to December of 2009, except between July and September. Two selective mediums (TCBS [thiosulfate, citrate, bile salts, and sucrose] agar and CHROMagar™ Vibrio) were used, and the V. parahaemolyticus strains were identified via polymerase chain reaction (PCR) amplification (Vp. flaE, tl, and toxR). 16S rRNA gene sequencing and their virulence were analyzed via the detection of tdh, trh, ORF8, toxRS/old, and toxRS/new genes. We collected 24 strains of V. parahaemolyticus: 19 seafood isolates, three environmental isolates, and two clinical (human) isolates. Among these strains, two tdh+ strains, two ORF8+ strains, 16 toxRS/old+ strains, and one toxRS/new+ strain were isolated. Twenty-two commercial antibiotics were used to assess the antibiotic susceptibility of isolates, and all the strains evidenced resistance to more than four antibiotics. The strains harboring antibiotic-resistant genes such as TetA (25%) and strB (4.16%) were detected via PCR. Repetitive extragenic palindromic sequence (REP)-PCR analysis revealed differences in the V. parahaemolyticus strains from other species and intraspecific strains.

Bacteriophage Therapy of a Vibrio parahaemolyticus Infection Caused by a Multiple-Antibiotic–Resistant O3:K6 Pandemic Clinical Strain

ABSTRACT Background. Recently isolated Vibrio parahaemolyticus strains have displayed multiple antibiotic resistance. Alternatives to conventional antibiotics are needed, especially for the multiple-antibiotic–resistant V. parahaemolyticus pandemic strain. Methods. A bacteriophage, designated pVp-1, showed effective infectivity for multiple-antibiotic–resistant V. parahaemolyticus and V. vulnificus, including V. parahaemolyticus pandemic strains. The therapeutic potential of the phage was studied in a mouse model of experimental infection using a multiple-antibiotic–resistant V. parahaemolyticus pandemic strain. We monitored the survivability and histopathological changes, quantified the bacterial and phage titers during phage therapy, and observed the immune response induced by phage induction. Results. Phage-treated mice displayed protection from a V. parahaemolyticus infection and survived lethal oral and intraperitoneal bacterial challenges. Conclusions. To the best of our knowledge, this is the first report of phage therapy in a mouse model against a multiple-antibiotic–resistant V. parahaemolyticus pandemic strain infection.

Eating oysters without risk of vibriosis: application of a bacteriophage against Vibrio parahaemolyticus in oysters.

Vibrio parahaemolyticus is a major cause of foodborne illness and related with the consumption of raw contaminated seafood, especially oysters. To evaluate the effectiveness of various applications of a bacteriophage (phage), pVp-1, against a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain (CRS 09-17), we designed artificial contamination models that are most likely to be encountered during oyster processing. When live oysters were treated with bath immersion with pVp-1 after CRS 09-17 challenge, the growth of bacterial strain was significantly reduced. After 72h of phage application with bath immersion, bacterial growth reduction was observed to be 8.9×10(6)CFU/ml (control group) to 1.4×10CFU/ml (treatment group). When pVp-1 was surface-applied on the flesh of oysters after CRS 09-17 inoculation, bacterial growth was properly inhibited. After 12h of phage application on the surface of oysters, bacterial growth inhibition was revealed to be 1.44×10(6)CFU/ml (control group) to 1.94CFU/ml (treatment group). This is the first report, to the best of our knowledge, of oyster surface-application of a phage against a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain, and our successful phage application to various situations emphasizes the potential use of the phage to avoid V. parahaemolyticus infection from aquaculture to consumption.

First description of the qnrS-like (qnrS5) gene and analysis of quinolone resistance-determining regions in motile Aeromonas spp. from diseased fish and water.

Antimicrobial resistance patterns in a collection of 33 motile Aeromonas species were described in this study. Quinolone has been frequently employed for treatment of Aeromonas-related diseases, and prolonged use of antimicrobial compounds has led to development of resistant strains. In a sample of diseased fish and environmental water, we evaluated nalidixic acid (n = 19) and ciprofloxacin (n = 4) resistance via minimum inhibitory concentration (MIC) assays and the genetic basis was also investigated. Among the isolated Aeromonas spp., 17 strains encoded for chromosomal mutations of quinolone resistance-determining regions (QRDRs) in gyrA, 11 strains encoded for mutations of QRDRs in parC, 1 strain harbored plasmid-mediated quinolone resistance (PMQR) qnrS1-like gene and 4 strains harbored the PMQR qnrS2 gene. In particular, the new variant (qnrS1-like) differed from qnrS1 by 6 amino acid substitutions at positions 5 (Asn(5)→Arg(5)), 120 (Ser(120)→Thr(120)), 148 (Asn(148)→His(148)), 206 (Leu(206)→Glu(206)), 207 (Ile(207)→ Leu(207)), and 216 (Tyr(216)→Phe(216)), and the gene was designated qnrS5. These resistant strains may function as reservoirs of quinolone resistance.

Isolation and characterization of a Myoviridae bacteriophage against Staphylococcus aureus isolated from dairy cows with mastitis

A lytic bacteriophage (phage), designated SAH-1, was isolated from sewage effluent near a dairy cow farm in Gwacheon, South Korea to search for biocontrol agents against Staphylococcus aureus infections. The SAH-1 was morphologically classified as Myoviridae and possessed an approximate 144 kb double-stranded genomic DNA. The phage showed broad host ranges within S. aureus strains including methicillin-resistant strains, and its latent period and burst size were approximately 20 min and 100 PFU/cell, respectively. Moreover, morphologic and genomic analysis of SAH-1 revealed that the phage was closely related to other Myoviridae phages infecting Staphylococcus species. The bacteriolytic activity of phage SAH-1 at a multiplicity of infection (MOI) 1 and 100 indicated its efficiency for reducing bacterial growth. Based on these results, phage SAH-1 could be considered a potential therapeutic or prophylactic candidate against S. aureus infections.

Complete Genome Sequence of a Novel Marine Siphovirus, pVp-1, Infecting Vibrio parahaemolyticus

ABSTRACT Among the abundant bacteriophages that belong to the order Caudovirales in the ocean, the genome sequences of marine siphoviruses are poorly investigated in comparison to those of myo- or podoviruses. Here we report the complete genome sequence of Vibrio phage pVP-1, which belongs to the family Siphoviridae and infects Vibrio parahaemolyticus ATCC 33844.

Characterization and complete genome sequence of the Shigella bacteriophage pSf-1

Shigellosis is a global health problem, and Shigella flexneri is the major cause of this disease. In this study, we isolated a virulent Siphoviridae bacteriophage (phage), pSf-1, that infects S. flexneri. This phage was isolated from the Han River in Korea and was found to infect S. flexneri, Shigella boydii, and Shigella sonnei. One-step growth analysis revealed that this phage has a short latent period (10 min) and a large burst size (86.86 PFU/cell), indicating that pSf-1 has good host infectivity and effective lytic activity. The double-stranded DNA genome of pSf-1 is composed of 51,821 bp with a G + C content of 44.02%. The genome encodes 94 putative ORFs, 71 putative promoters, and 60 transcriptional terminator regions. Genome sequence analysis of pSf-1 and comparative analysis with the homologous Shigella phage Shfl1 revealed that there is a high degree of similarity between pSf-1 and Shfl1 in 54 of the 94 ORFs of pSf-1. The results of this investigation indicate that pSf-1 is a novel Shigella phage and that this phage might have potential uses against shigellosis.

Complete genome sequence and characterization of a broad-host range T4-like bacteriophage phiAS5 infecting Aeromonas salmonicida subsp. salmonicida.

In this study, we report one lytic Myoviridae bacteriophage (phage) infecting Aeromonas salmonicida subsp. salmonicida. The phage (named as phiAS5) was isolated from environmental river waters in Korea, and showed broad infectivity to other bacterial species in the family Aeromonadaceae as well as antibiotic-resistant A. salmonicida subsp. salmonicida strains. The biological properties and complete genome of phiAS5 were simultaneously investigated. The complete genome of phiAS5 composed of linear double-stranded DNA of 225,268 bp with G+C content of 43.0%, and encoded 343 putative ORFs, 69 putative promoters, 33 transcriptional terminator regions and 24 tRNA-encoding genes. A high degree of similarity to other T4-like Aeromonas phage was found in most ORFs of phiAS5. Therefore, the genome of phiAS5 was further compared with T4 phage and the closest relative, Aeromonas phage Aeh1, and the result demonstrated that it could be classified as a new member of the T4-like group. The bacteriolytic activity of phiAS5 against A. salmonicida subsp. salmonicida was evaluated at different doses of multiplicity of infection using one each of virulent strain that possesses the ascV gene and multi-drug resistant strain, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, phiAS5 may have the potential for reducing the impacts of virulent or antibiotic-resistant A. salmonicida subsp. salmonicida in aquaculture and may also advance our understanding of the biodiversity of T4-like Aeromonas phages.

The phylogenetic study on Thelohanellus species (Myxosporea) in relation to host specificity and infection site tropism.

Thelohanellus kitauei (Myxobolidae) infects cyprinid fish. The evolution of species derived from common ancestors results in the sharing of biological features. To reveal the origin of T. kitauei biological features, the correlation between phylogeny and biological features of Myxobolidae was investigated by Bayesian inference tree and Bayesian tip association significance testing. The results demonstrated that host specificity and infection site tropism were correlated with the phylogeny of Myxobolidae, and that the biological features of T. kitauei originated from the ancient Myxobolidae as exhibited by the non-specific infection site tropism and the ability to infect cyprinids.

Bacteriophage application to control the contaminated water with Shigella

Shigella is one of the most important waterborne and foodborne pathogens around the world. Emergence of antibiotic-resistant Shigella has made the development of alternatives to conventional antibiotics necessary. In this study, a virulent Myoviridae bacteriophage, pSs-1 was isolated from environmental water in South Korea and showed infectivity to S. flexneri as well as S. sonnei strains. One-step growth analysis showed that pSs-1 has a short latent period (25 min) and a large burst size (97 PFU/cell). According to the genomic analysis, pSs-1 contains 164,999 bp of genome with a G + C content of 35.54% and it is considered as a member of the T4-like bacteriophage group. These results showed that pSs-1 may have potential as a biocontrol agent instead of conventional antibiotics for shigellosis.