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수의학 박사 학위논문

Development of alternative control methods using bacteriophages against antibiotic-resistant Aeromonas salmonicida

infections in Korean salmonid fish

한국 연어과 어류에 감염하는 항생제 내성 Aeromonas salmonicida 에

대한 박테리오파아지 구제법 개발

2012 년 8 월

서울대학교 대학원

수의학과 수의공중보건학 전공

김 지 형

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A Dissertation for the Degree of Doctor of Philosophy

infections in Korean salmonid fish

By

Ji Hyung Kim

August, 2012

Department of Veterinary Public Health College of Veterinary Medicine

The Graduate school of Seoul National University

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infections in Korean salmonid fish

By

Ji Hyung Kim

Supervisor: Professor Se Chang Park, D.V.M., Ph.D.

A dissertation submitted to the faculty of the Graduate School of Seoul National University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in

Veterinary Public Health

August, 2012

Department of Veterinary Public Health College of Veterinary Medicine

The Graduate school of Seoul National University

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Development of alternative control methods using bacteriophages against antibiotic- resistant Aeromonas salmonicida infections

in Korean salmonid fish

한국 연어과 어류에 감염하는 항생제 내성 Aeromonas salmonicida에

대한 박테리오파아지 구제법 개발

지도교수: 박 세 창

이 논문을 수의학박사 학위논문으로 제출함 2012 년 5 월

서울대학교 대학원

수의학과 수의공중보건학 전공

김 지 형

김지형의 박사학위 논문을 인준함 2012 년 6 월

위 원 장 이 병 천

₍인)

부위원장 박 세 창

_(인)

위 원 조 성 준

_(인)

위 원 신 기 욱

(인)

위 원 Mahanama De Zoysa

(인)

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Abstract

Development of alternative control methods using bacteriophages against antibiotic- resistant Aeromonas salmonicida infections

in Korean salmonid fish

Ji Hyung Kim Department of Veterinary Public Health College of Veterinary Medicine The Graduate School of Seoul National University

Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis in salmonid fish and bacterial septicemia in a broad variety of fish, and is thus responsible for significant economic losses in the global aquaculture industry. Recently, the acquisitions of antibiotic resistance in A.

salmonicida subsp. salmonicida have been recognized as a serious concern, owing to their potential health risks to humans and animals. However, the acquisition and prevalence of antibiotic resistance in A. salmonicida subsp.

salmonicida have not yet been investigated in Korean aquaculture industry.

Therefore, in the first step towards, we collected a total of 16 A.

salmonicida (14 of A. salmonicida subsp. salmonicida and 1 each of A.

salmonicida subsp. achromogenes and subsp. flounderacida) strains from diseased fish and environmental samples in Korea from 2006 to 2009, and evaluated its antibiotic resistance against tetracycline and quinolones.

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Tetracycline and quinolone resistances were observed in 8 and 16 of the isolates, respectively, based on the measurement of minimal inhibitory concentrations. Among the tetracycline-resistant strains, 7 of the isolates harbored tetA and one isolate harbored tetE. Additionally, quinolone- resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A.

salmonicida strains, 15 strains showed point mutations in the gyrA codon 83, which were responsible for the corresponding amino acid substitutions of Ser⁸³→Arg⁸³ or Ser⁸³→Asn⁸³. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84.

Genetic similarity was assessed via pulsed field gel electrophoresis (PFGE), and the results indicated high clonality among the Korean antibiotic- resistant strains of A. salmonicida subsp. salmonicida.

In order to develop alternative control methods against this fish pathogen, in the second step towards, we isolated several bacteriophages (phages) infecting A. salmonicida subsp. salmonicida from various environmental waters or fish in Korea. Among those phages, we fully sequenced the two T4-like Myoviridae phages (named as phiAS4 and phiAS5) isolated from environmental waters in Korea. The two phages showed broad host ranges to other Aeromonadaceae as well as A.

salmonicida, and their biological properties were simultaneously investigated. Furthermore, the complete genomes of phiAS4 and phiAS5 were sequenced, and final assembly yielded linear double-stranded DNA genomes of 163,875-bp and 225,268-bp with G+C content of 41.3 and

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43.0%, respectively. Genomic analysis uncovered 271 and 343 putative ORFs, 67 and 69 putative promoters, 25 and 33 terminator regions, and 16 and 24 tRNA-encoding genes, respectively. A high degree of similarity to the Aeromonas phages 25 and Aeh1 were found in most ORFs of phiAS4 and phiAS5, respectively. The phages were further compared with their relatives including enterobacter phage T4, and the results demonstrated that they could be classified as new members of the T4-like group. Moreover, the functional activity of the putative lysozyme murein hydrolase (orf117) in phiAS5, which had no holin or holin-like gene, was investigated, and the result revealed that it may use a dual lysis system during host cell lysis.

Based on these results, we confirmed that the two phages will have the potential for controlling A. salmonicida subsp. salmonicida in Korean aquaculture and may also advance our understanding of the biodiversity of T4-like phages.

To search for candidate control agents and to evaluate its therapeutic potential against A. salmonicida subsp. salmonicida in aquaculture, we selected one novel lytic phage among those isolated Aeromonas phages in the third step towards. The novel Aeromonas phage (designated as PAS-1) was isolated from the environmental water and its several biological properties were preliminarily investigated. The phage showed broad host ranges to other subspecies of A. salmonicida as well as A. salmonicida subsp. salmonicida including antibiotic-resistant strains. The PAS-1 was morphologically classified as Myoviridae and possessed approximately 48 kb of double-strand genomic DNA. Moreover, partial genomic and

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structural proteomic analysis of PAS-1 revealed that the phage was closely related to other Myoviridae phages infecting enterobacteria or Aeromonas species. For the therapeutic applications of PAS-1, the phage was preferentially co-cultured with one virulent A. salmonicida subsp.

salmonicida strain that possesses the ascV gene, and strong bacteriolytic activity was observed against the bacteria. The administration of PAS-1 in rainbow trout (Oncorhynchus mykiss) demonstrated that it was cleared within 200 h post-administration, and temporal neutralizing activity against the phage was detected in the phage-administrated fish serums. The protective effects of the phage were verified in experimental rainbow trout furunculosis model therapy, showing increased survival rates and mean time to death.

Based on these results, it can be concluded that the isolated Aeromonas phages could be considered as altervative control agents against antibiotic- resistant A. salmonicida subsp. salmonicida as well as typical A.

salmonicida subsp. salmonicida, and will also have potential therapeutic or prophylactic candidate against salmonid furunculosis in Korean aquaculture.

Key words: A. salmonicida subsp. salmonicida, Aeromoans phages, salmonid, furunculosis, altervative control agents, Korea

Student number: 2008-30469

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B. Bacteriophage (phage) ... 16

B.1. General description ... 16

B.2. Phages infecting Aeromonadaceae ... 20

B.3. Therapeutic applications of phages... 22

C. References ... 26

Chapter I

Isolation and molecular characterization of tetracycline- and quinolone-

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resistant Aeromonas salmonicida strains from cultured fish in Korea

Abstract ... 41

1.1. Introduction ... 42

1.2. Materials and Methods ... 43

1.3. Results ... 46

1.4. Discussion ... 49

1.5. References ... 51

Chapter II Isolation, characterization and genomic analysis of the two T4-like

Aeromonas phages (phiAS4 and phiAS5) infecting A. salmonicida subsp. salmonicida as potential candidates for furunculosis control Abstract ... 61

2.2. Materials and methods ... 64

2.3. Results ... 72

Chapter III Isolation and characterization of a novel Aeromonas phage PAS-1 infecting A.

salmonicida subsp. salmonicida and its applications in rainbow trout

(Oncorhynchus mykiss) furunculosis model therapy

Abstract ... 133

3.2. Materials and Methods ... 136

3.3. Results ... 143

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General conclusion ... 166

Abstracts in Korean ... 168

List of published articles ... 173

List of conference attendances ... 181

Acknowledgements ... 189

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List of Figures

Literature review

Figure I. Schematic representation of major phage groups.

Chapter I

Figure 1.1. Multiplex PCR assay of tetracycline resistance genes (tetA of 211bp and tetE of 744bp) in two reference strains and 16 isolates of Aeromonas salmonicida.

Figure 1.2. PFGE profiles of 18 Aeromonas salmonicida strains and UPGMA dendrogram.

Chapter II

Figure 2.1. Electron microscopy of the two T4-like Myoviridae phages infecting A.

salmonicida subsp. salmonicida: phiAS4 (A) and phiAS5 (B).

Figure 2.2. One step growth curves of Aeromonas phage phiAS4 and phiAS5 in A.

salmonicida subsp. salmonicida strain AS01.

Figure 2.3. Genome map of Aeromonas phage phiAS4.

Figure 2.4. Genome map of Aeromonas phage phiAS5.

Figure 2.5. Genome comparison of Aeromonas phage phiAS4 (A) and phiAS5 (B) to related phages using the Artemis Comparison Tool (ACT).

Figure 2.6. SDS-PAGE analysis (A) and zymogram assay (B) of recombinant phiASL5.

Chapter III

Figure 3.1. Electron micrographs of negatively stained Aeromonas phage PAS-1 virions.

Figure 3.2. One step growth of Aeromonas phage PAS-1 in A. salmonicida subsp.

salmonicida AS01 strain.

Figure 3.3. Time course of lytic activity against the host cell by Aeromonas phage PAS-1.

Figure 3.4. Fate of the Aeromonas phage PAS-1 in the rainbow trout kidney (PFU/g) and its aquarium water (PFU/ml).

Figure 3.5. The neutralizing activities against Aeromonas phage PAS-1 in rainbow trout serum after administration of phage.

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List of Tables

Literature review

Table I. Classification and its biological properties of phage.

Table II. Phages that carries toxin genes and their gene products.

Table III. Sources and properties of the sequenced Aeromonas phages up to 2012.

Table IV. The representative use of phages to control bacterial pathogens in aquaculture.

Chapter I

Table 1.1. Aeromonas salmonicida strains used in this study.

Table 1.2. PCR primers used in this study.

Table 1.3. Minimal inhibitory concentrations (MICs), tetracycline resistance (tet) genes, mutations in QRDRs in A. salmonicida strains.

Chapter II

Table 2.1. Host ranges and EOPs of Aeromonas phage phiAS4 and phiAS5 against all the bacterial strains used in this study.

Table 2.2. Predicted ORFs and its products of Aeromonas phage phiAS4.

Table 2.2. Predicted ORFs and its putative functions of Aeromonas phage phiAS5.

Chapter III

Table 3.1. Bacterial strains used in this study and infectivity of Aeromonas phage PAS-1.

Table 3.2. Partial and complete ORFs of Aeromonas phage PAS-1.

Table 3.3. SDS-PAGE profile of the PAS-1 virion and their protein profiles by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis

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Abbreviations

MIC Minimum Inhibitory Concentration QRDR Quinolone Resistant Determining Region PFGE Pulsed Field Gel Electrophoresis

CLSI Clinical and Laboratory Standards Institute

MDR Multi Drug Resistant

PCR Polymerase Chain Reaction

UPGMA Unweighted Pair Group Method with Arithmetic mean CAMHB Cation Adjusted Muller Hinton Broth

NCBI National Center for Biotechnology Information TEM Transmission Electron Microscopy

EOP Efficiency Of Plating

CFU Colony Forming Unit

PFU Plaque Forming Unit

MOI Multiplicity Of Infection

IACUC Institutional Animal Care and Use Committee

ORF Open Reading Frame

SDS-PAGE Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis LC-MS/MS Liquid Chromatography-tandem Mass Spectrometry

IM Intra Muscular

OD Optical Density

SPSS Statistical Package for the Social Sciences

TSA Tryptic Soy Agar

TSB Tryptic Soy Broth

PEG PolyEthylene Glycol

EDTA Ethylene Diamine Tetraacetic Acid IPTG IsoPropyl-β-d-Thio-Galactoside

PAS Phage of Aeromonas Salmonicida

TE Tris-EDTA

TBE Tris-Borate-EDTA

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General introduction

Salmonid fish (salmonids) are the most important biological and political fish resources in the pacific oceans due to the characteristics of transboundary distributions and economical importances (5). In the recent decades, the scientific interests in 3 species of salmonids such as chum salmon (Oncorhynchus keta), masou salmon (O. masou) and rainbow trout (O. mykiss) were much increased in Korea due to the involvement to the North Pacific Anadromous Fisheries Commission (NPAFC). Chum salmon is the most important anadromous salmonid species in Korea, because it is the only indigenous fish species that migrate from Korea to north pacific oceans (3). Another salmonid fish, masou salmon, is distributed in natural freshwaters in Korea, but its natural population is very small (5). In the middle of 1980s, the enhancement program of anadromous salmonids has been established in Korea since the foundation of the Cold-water Fish Research Center (formerly Yangyang inland hatchery) of National Fisheries Research and Development Institute, and the biological and political interests of salmonid preservations were also increased (7). However, those two anadromous salmonid species recently showed rapid decrease in the late return and are now in danger of extinction in Korea, due to environmental pollutions, global warming, overfishing and diseases. In additions, rainbow trout, which is not indigenous salmonid species and was transplanted from Japan and USA approximately 40 years ago, is now artificially propagated and cultured on a large scale in Korean aquaculture (1), but it also suffered from several diseases caused by bacterial or viral pathogens (6).

Aeromonas salmonicida subsp. salmonicida is the causative agent of

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furunculosis in salmonid fish, and is thus responsible for significant economic losses in the global aquaculture industry as well as salmonid cultures (4). Recently, the acquisions of antibiotic resistance in A. salmonicida subsp. salmonicida have been recognized as a serious world-wide concern, owing to their potential health risks to animals and human (4). Therefore, alternative control methods against this fish pathogen are urgently needed. From the 20th century, phages have received attention due to their potential as alternative antimicrobial agents for a variety of bacterial pathogens. In aquaculture, phages have been used as control agents against several fish and shellfish pathogens, and its applications showed promising results (8).

In Korea, A. salmonicida subsp. salmonicida was first isolated from cultured masu salmon in 1986 (2), and more recently detected from rainbow trout farm (6).

However, the prevalence of A. salmonicida subsp. salmonicida has not been investigated in Korean aquaculture industry, and the potential acquisition or spreadness of antibiotic-resistance in this bacterium was not also studied until yet.

Therefore, this study was planed to preferentially provide recent prevalence and antibiotic resistance of A. salmonicida subsp. salmonicida isolated from Korean aquaculture, and ultimately focused on development of alternative control methods of this fish pathogen using its infectious phages to adopt it in Korean aquaculture industry.

References

1. Baik, K. K., et al. 2007. Studies on seed production of rainbow trout, Oncorhynchus mykiss. J. Aquaculture 20:85-89.

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2. Fryer, J., R. Hedrick, J. Park, and Y. Hah. 1988. Isolation of Aeromonas salmonicida from masu salmon in the Republic of Korea. J. Wildl. Dis. 24:364-365.

3. Jeon, C. H., et al. 2011. Mornitoring of viruses in chum salmon (Oncorhynchus keta) migrating to Korea. Arch. Virol. 156:1025-1030.

4. Kim, J. H., et al. 2011. Molecular characterization of tetracycline- and quinolone- resistant Aeromonas salmonicida isolated in Korea. J. Vet. Sci. 12:41-48.

5. Kim, S., C. S. Lee, and S. Kang. 2007. Present status and future prospect in salmon research in Korea. J. Korean Soc. Oceanogr. 12:57-60.

6. Lee, C., J. C. Cho, S. H. Lee, D. G. Lee, and S. J. Kim. 2002. Distribution of Aeromonas spp. as identified by 16S rDNA restriction fragment length polymorphism analysis in a trout farm. J. Appl. Microbiol. 93:976-985.

7. Lee, C. S., K. B. Seong, and C. H. Lee. 2007. History and status of the chum salmon enhancement program in Korea. J. Korean Soc. Oceanogr. 12:73-80.

8. Park, S. C., and T. Nakai. 2003. Bacteriophage control of Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis. Dis. Aquat. Org. 53:33-39.

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Literature Review

A. Aeromonas salmonicida

A.1. Taxonomy of Aeromonadaceae

The genus Aeromonas (Superkingdom, Bacteria; Phylum, Proteobacteria; Class, γ-proteobacteria; Order, Aeromonadales; Family, Aeromonadaceae) comprise a collection of gram-negative bacteria that are widespread in aquatic environments , and have been implicated as causative agents of a number of human and animal diseases (117). The taxonomy of this genus is in a continual state of flux as new species are identified by its phenotypic and genotypic classifications, and the descriptions of the existing taxa are refined (117). In a broad point of view, Aeromonas spp. could be devided as motile and non-motile species. Up to recent, approximately 30 motile Aeromonas spp. were identified (such as A.

allosaccharophila, A. aquariorum, A. bestiarum, A. bivalvium, A. cavernicola, A.

caviae, A. diversa, A. encheleia, A. enteropelogenes, A. eucrenophila, A. fluvialis, A.

hydrophila, A. jandaei, A. media, A. molluscorum, A. piscicola, A. popoffii, A. rivuli, A. sanarellii, A. sharmana, A. schubertii, A. simiae, A. taiwanensis, A. tecta, A.

trota, A. veronii biovar sobria and A. veronii biovar veronii), and those species have been associated with various human infections including gastro-enteritis, wound infections and septicaemia (43), and have also been implicated as the causative agents of various fish diseases (77). A. hydrophila is also associated with red leg disease in amphibians and infections in turtles (107) and birds (125). In addition to their role as disease agents, Aeromonas species can be found in non-

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pathogenic association with a variety of animals (82, 122, 142). Most Aeromonas species are opportunistic pathogens, entering through wounds or affecting only stressed or immune-compromised hosts (43). On the other hand, A. salmonicida is known as the only a non-motile Aeromonas sp. and is the specific aetiological agent of a bacterial septicaemia in fish, named as furunculosis (12, 61, 62, 148).

A.2. A. salmonicida and its classification

Furunculosis caused by A. salmonicida subsp. salmonicida is an important bacterial disease in wild and cultured salmonids and other fish species, and can have significant economical losses on worldwide aquaculture operations (62). In the early of 20^th century, this bacterium was initially referred as Bacterium or Bacillus salmonicida (93), but it was later designated as ‘Aeromonas salmonicida’

by Griffin et al. (52). Isolates of the bacterium initially appeared to be homogeneous, but an increasing number of studies reported several isolates with different biological or biochemical properties from those of the typical ones from the 1960s (127). Since then, the bacteria were classified into two groups as typical and atypical ones (91), and divided into three subspecies: subsp. salmonicida, subsp. achromogenes and subsp. masoucida (116). Afterwards, the fourth and fifth subspecies, subsp. smithia and subsp. pectinolytica were proposed by Austin et al.

(8) and Pavan et al. (111), respectively. In the recent years, the Bergey's manual of systematic bacteriology (64) recognizes five subspecies of A. salmonicida: subsp.

salmonicida, achromogenes, masoucida, smithia, and pectinolytica, and currently classify A. salmonicida subsp. salmonicida as "typical" and any isolate deviating phenotypically as "atypical".

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The typical isolates form a homogeneous group (7, 36, 47, 102), while the taxonomy of atypical strains is still ambiguous, regardless of attempts to classify them into several subspecies (148). In general, typical strains grow well on blood agar with large colonies, produce a brown diffusible pigment, are β-haemolytic and do not ferment sucrose (89). Therefore, morphological and biochemical differences (7, 35, 89, 148), such as pigment production, colony size and growth rate, haemolysis, and sucrose fermentation, are used to distinguish typical and atypical isolates. Recently, phylogenetic analyses based on gene sequences (90, 98) or biochemical analyses based on carbohydrates (143) appear to be better able to sort out the complex taxonomy and classification of several subspecies in this bacterium and its related species.

With the recent technical advances in genome sequencing, the complete genome sequence of A. salmonicida subsp. salmonicida strain A449 was determined (117), and the chromosome was 4,702,402 bp and encodeed 4388 genes, while the two large plasmids were 166,749 and 155,098 bp with 178 and 164 genes, respectively.

Notable features were a large inversion in the chromosome and the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integronen coding genes for resistance to streptomycin-spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. Additionally, another draft genome sequence of A. salmonicida subsp. salmonicida strain 01-B526 which isolated from a brook trout (27), is also available in GenBank database.

A.3. A. salmonicida subsp. salmonicida and furunculosis

Furunculosis was first reported in Germany at 1894 (41). The name

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‘furunculosis’ was given due to its symptom showed furuncle-like swellings, which were ulcerative at a later stage of the disease. However, the discrepancy in the taxonomy of A. salmonicida has also affected the nomenclature used for the diseases caused by this pathogen. In pioneer days, the term ‘furunculosis’ was used principally to cover all fish diseases caused by A. salmonicida species, even though it was specifically used for those infections of salmonids which showed the furuncle-like swellings (92). However, Ljungberg and Johansson (87) suggested that it was essential from an epizootiological point of view to identify typical and atypical A. salmonicida infections as two separate diseases. Subsequently, the diseases caused by atypical isolates in non-salmonid fish have been variously referred; such as carp erythrodermatitis, goldfish ulcer disease, skin ulcer disease of flounder, A.S.A. infection in salmonid fish, ulcerative furunculosis, infectious dermatitis, atypical A. salmonicida infection or atypical furunculosis (62).

Therefore, only infections caused by A. salmonicida subsp. salmonicida should be called as furunculosis (109, 148). However, the taxonomy of atypical isolates is still ambiguous and the terms used for the diseases caused by A. salmonicida vary between geographical regions (62). Therefore in this thesis, the term furunculosis is used for infections caused by A. salmonicida subsp. salmonicida. Other infections caused by atypical strains are referred to as related diseases, atypical infections or atypical A. salmonicida infections.

A.4. Host range and distribution

A. salmonicida have an extensively broad host ranges in wild and farmed fish of all ages, and its infections occurr in fresh water, brackish and marine environment

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(148). Furthermore, it has been indicated that almost all the fish species can serve as a reservoir of infections caused by A. salmonicida (60), and salmonids are considered to be the most susceptible to furunculosis, especially Atlantic salmon (Salmo salar L.), brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta L.), whereas, rainbow trout (O. mykiss) is considered being relatively resistant to this bacterium (92).

Typical and atypical infections have been reported in worldwide aquaculture (148). However, atypical infections mostly occur in the temperate regions of Canada, USA, Japan and Europe (148). In Korea, A. salmonicida subsp.

salmonicida was was first isolated from cultured masu salmon (O. masou) in 1986 (46), and more recently detected from rearing water on a rainbow trout farm (84).

The history of atypical infections in Korea is relatively not well documented as compared to typical ones, and only one case of its infections in the black rockfish (Sebastes schlegeli) were reported (58).

A.5. Clinical signs

In general, furunculosis is considered as a septicaemic disease which can be considered as a peracute, acute, subacute or chronic form (62, 92). And its clinical features were previously reviewed by McCarthy and Roberts (92) as follows:

i) The acute form cause high mortality and is common in growing and adult fish, which show signs that are typical of an acute bacterial septicemia: darkening in colour, lack of appetite, lethargy, tachy branchia and small hemorrhages at the base of the fins. Furuncles may develop, but not continuously, and they may rupture to release highly infective material. The fish usually die within two or three days.

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ii) The peracute form of furunculosis usually occurs in fingerling fish with the following clinical signs: darkening in colour, tachy branchia, exophthalmosis, haemorrhages at the pectoral fin base and high mortality.

iii) Subacute and chronic forms are more common in older fish, which are lethargic and have one or more furuncles on the flank or dorsum. There may be congestion of the blood vessels at the base of fins, injection of the sclera, slight exophthalmia and paleness of the gills. The furuncles may be large and when ruptured the fluid is more viscous and contains more formed, necrotic elements than the furuncles found in acute cases. The onset of disease is more gradual and mortality is relatively low as compared to acute form. In addition, a latent form of infection, in which there is no mortality and no symptoms but the bacterium still isolated, was reported (60). The latent form of infection was further suggested to be changed to

“clinically inapparent” or “covert” infection by Hiney et al. (61).

In contrast, atypical infections in farmed salmonids also have a septicemic disease and the bacterium is usually isolated from both skin ulcers and internal organs (103, 108, 146). The mortalities caused by atypical infections have been extremely varied from 10% (103) to more than 90% (87). Additionally, Wichardt et al. (146) found differences in susceptibility among the salmonid species: Atlantic salmon or rainbow trout was somehow resistant under normal farming conditions, whereas brown trout or arctic char (Salvelinus alpinus L.) were highly suspectible.

Rintamäki and Valtonen (118) also reported higher mortalities among sea trout (Salmo trutta m. trutta L.) than in other salmonids at the same farm. The clinical signs of atypical infections in salmonids were varied; such as emaciation and paleness of the gills (103), black discoloration, surface ulcers and lesions, ranging

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in appearance from a small superficial wound with little necrosis to deep jagged lesions surrounded by necrotic muscle (108), pronounced dermatitis (146), haemorrhages and erosion at the base of the fins, skin ulcers and small furuncles in the musculature (118), lethargy, aimless swimming, respiratory distress, fin erosion and haemorrhagic cutaneous and muscular ulcers (53).

The clinical signs caused by atypical infection in non-salmonids have been reviewed by Wiklund and Dalsgaard (148). In most signs of disease outbreaks among wild or farmed non-salmonid fish, the symptoms manifested ulcerations and lesions in a variety of locations in fish skin (62). For example, ulcerations or lesions were found in all over the body surface except on the head (in carp), those appeared on any part of the body and vary in size and depth (in goldfish), while the skin ulcers were superficial (in flounder). In case of eels, atypical A. salmonicida have been reported to cause severe necrosis, lesions in the skin and tissue swelling on the head (62). In additions, haemorrhage and erosion of the fins, necrosis of the tail and haemorrhage or lesions in the eyes have also been associated with atypical infections in non-salmonid fish (62). Septicemic infections have been reported in naturally infected cod (Gadus morhua L.) (88), and in experimentally infected turbot (Scophthalmus maximus L.) (14). However, the mortality rates for atypical A.

salmonicida outbreaks in wild fish are not well presented (62).

A.6. Transmission

The mechanism of horizontal or vertical transmission in A. salmonicida subsp.

salmonicida is still uncertain and controversial. However, contact with infected fish or contaminated water and fish farm materials, and trans-ovarian transmission have

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all been speculated as possible routes of disease transmission, and carrier fish with latent infection were also suggested as another possible route for horizontal transmission of the disease (91). Moreover, the bacteria-contaminated water can be a possible way of disease transmission to susceptible fish: fish species such as brown trout easily become infected via bath challenges, but more resistant salmonids such as rainbow trout need to be abraded before the onset of disease (91).

During infection, gills, skins and wounds were suspected as the main routes of entry for A. salmonicida subsp. salmonicida (63, 133). Additionally, bacterial transmissions through the gastro-intestinal tract via oral (91) or intra-gastric challenge (120) have been investigated, but those results were contradictory.

Vertical transmission through infected ova has been investigated by several authors. McCarthy (91) indicated that vertical transmission is not a significant route for furunculosis, and routine disinfection of eyed eggs is unnecessary.

Bullock and Stuckey (18) investigated vertical transmission following the health status of the progeny of carrier and artificially infected broodstock, and concluded that it does not occur. In contrast, Wichardt et al. (146) stated that the spread of furunculosis between Swedish fish farms occurred through the transportation of infected fish or contaminated equipment, and also through infected ova.

A.7. Virulence factors

Many fundamental aspects of the host-pathogen relationship between A.

salmonicida subsp. salmonicida and its fish hosts were poorly understood.

Therefore in recent decades, the mechanims of bacterial virulence were extensively investigated. To date, several proteins and systems in A. salmonicida subsp.

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salmonicida have been implicated in its virulence including the S-layer (136) [vapA], siderophores and their receptors (38) [fstC, fstB and hupA], superoxide dismutase (9, 33) [soda and sodB] and extracellular toxins (121) [glycerol phospholipid: cholesterol acetyl-transferase and the serine protease AspA].

However, the nature of its virulence is indeed complex and apparently varies between strains, and despite the presence of multiple virulence systems, no single system appeared to significantly contribute to its virulence as shown by the retention of virulence by strains deficient in any given system (40, 104, 140).

A type III secretion system (TTSS) in A. salmonicida subsp. salmonicida has been recently described (21, 23, 131). Gram-negative bacteria utilize TTSSs as a transmembrane injection apparatus composed of integral membrane proteins and a needle-like structure to translocate a range of effect or proteins from the cytosol directly into host cells (34), and it is known as a major virulence factor for several pathogenic bacteria (66) including Pseudomonas aeruginosa, Shigella flexneri, Salmonella enterica serovar typhimurium, entero-pathogenic E. coli, as well as A.

hydrophila AH-1 (153) which belongs to Aeromonadaceae. Likewise, the TTSS in A. salmonicida subsp. salmonicida consists of inner- and outer-membrane secretory pores, a host-cell translocation pore and a number of effector molecules. In addition, the various genes of the TTSS of A. salmonicida subsp. salmonicida are carried both on plasmids and chromosome (22, 131). Moreover, the 2 laboratory- derived TTSS-deficient strains have been described as avirulent in a rainbow trout challenge model; One strain was deficient in the 140 kbp plasmid that carries the TTSS system, and the other was a knockout mutant strain in ascV which forms part of the inner bacterial membrane pore (20, 22). And ultimately, it was proved that

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the TTSS gene in A. salmonicida subsp. salmonicida is responsible for secretion of the ADP-ribosylating toxin, AexT, and encoded on a thermolabile plasmid, and the absence of the TTSS gene ascV disabled the bacteria to secret AexT, even though the strain contained the aexT gene (131). Based on these results, the TTSS is now considered as a major virulence factors in A. salmonicida subsp. salmonicida, but things still remains to be proved.

A.8. Disease control

Furunculosis was the first bacterial disease in fish which was treated with antibiotics such as sulfonamides and nitrofurans (55), and the oubreaks caused by A. salmonicida subsp. salmonicida were usually controlled with chemotherapy (29, 92). Although other antibiotics effectively control this disease (60), the U.S. Food and Drug Administration imposes stringent requirements for the antibiotics used on aquaculture industry, and only the use of sulfamerazine, oxytetracycline and the potentiated sulfonamide Ro5-0037 or ROMET^®(19) is approved in USA. In other country, several antimicrobial agents have been used to control furunculosis, including chloramphenicol, thiophenicol, furazolidone and oxytetracycline, sulphamerazine, tetracycline and a combination of trimethoprim and sulphonamide, flumequine, oxolinic acid, florfenicol, amoxicillin and enrofloxacin (59, 60, 70, 71, 92, 95, 130).

On the other hands, several vaccines against typical strains were recently developed with providing long-lasting protection, and their use is promoted in commercial aquaculture (29, 39, 86). However, the vaccines were not guaranted further expression of furunculosis within or transmission of furunculosis from

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covertly infected vaccinated carriers (61). Furthermore, the serologic relatedness among A. salmonicida subsp. salmonicida strains (109) suggests that immunization of fish against atypical infection is also a realistic possibility. However, Atlantic salmon inoculated with a commercial vaccine against typical strain or with this vaccine and another prepared against atypical strain of A. salmonicida subsp.

achromogenes were equally protected against A. salmonicida subsp. salmonicida by cohabitation challenge. However, the salmonids vaccinated with A. salmonicida subsp. achromogenes only were not protected against furunculosis (54).

A.9. Emergence of antibiotic resistance

As early as in 1967, the increased frequency of antimicrobial resistance among A. salmonicida subsp. salmonicida was first reported in USA (151). Resistance has also been reported to sulphonamides (60), oxytetracycline (51, 69, 137), and combinations of sulphonamide and trimethoprim (51, 137), oxolinic acid (51, 59, 69, 137) and to amoxicillin (51). Furthermore, several typical strains showing multi-drug resistance have been isolated in recent years (51, 69, 72, 100).

Among the antibiotics utilized in the treatment of furunculosis, both tetracycline and quinolone resistance have been most widely documented (37, 99). Those studies indicated that tetracycline resistance in A. salmonicida subsp. salmonicida was plasmid-encoded, and tetA was predominant among the different classes of tetracycline-resistant genes. Schmidt et al. (123) reported the isolation and characterization of oxytetracycline-sulfonamide/trimethoprim-resistant Aeromonas spp. from Danish rainbow trout farms, and the results indicate that tetE was the predominant determinant, followed by tetA and tetD. Whereas, DePaola et al. (37)

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had determined that 86% of Aeromonas spp. isolated from catfish contained tetA and the rest harbored tetE. Quinolones are mainly used as drugs of choice for the treatment of human Aeromonas infections (4, 76), and are also used for the treatment of other bacterial fish disease as well as furunculosis (48). These drugs can persist for a long time in the environment (57), which could cause the emergence of resistant strains in environmental samples. Quinolone resistance in gram-negative bacteria is mainly due to DNA mutations in the quinolone resistance determining regions (QRDRs) which consist of DNA gyrase and topoisomerase IV that alter the target enzymes for these drugs (3, 4). DNA gyrase and topoisomerase IV are hetero-tetramers formed by two types of subunits: GyrA, GyrB and ParC, ParE, respectively (114). Mutations in the gyrA and parC genes in QRDRs also proved to be related to quinolone resistance in the motile and non-motile Aeromonas spp. (49). Moreover, an active efflux pump belonging to the resistance nodulation cell division family that could contribute to its quinolone resistance in A.

salmonicida subsp. salmonicida also have been presented (48). Additionally, the plasmid-mediated qnr gene was also known to be associated with low level quinolone resistance (128).

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B. Bacteriophage (phage)

B.1. Genaral description

Phages are bacterial viruses that infect bacterial cells, disrupt bacterial metabolism and cause the bacterium to lyse. Phages are the most abundant living entities on earth, and play major roles in bacterial ecology, adaptation, evolution and pathogenesis (1). Phages are common in soils (approximately 10⁷ to 10⁹ virions/g), and highly abundant in fresh water and marine waters (approximately 10⁷ virions/ml), and its total amount on earth was estimated as 10³¹ virions (132).

The phages were discovered twice at the beginning of the 20^th century in a short time (25). Frederick W. Twort, an English medical bacteriologist, described a marked antibacterial activity in Micrococcus by an unknown agent in 1915 (25).

And 2 years later, phages were “officially” discovered by Felix H. d’Herelle, a French-Canadian microbiologist at the Institut Pasteur. He discovered the destruction of Shigella in broth culture, and recognized the viral nature of this phenomenon and suggested the term ‘bacteriophage’ (32). The viral nature of phages was recognized in 1940 with the development of electron microscope, and the basis of the present phage classification was proposed by Bradley in 1967 (17) as six types: such as tailed phage, filamentous phages, and icosahedral phages with single-stranded (ss) DNA or ssRNA. In 1971, the International Committee on Taxonomy of Viruses (ICTV) classified phages into 6 genera (T4, λ, φX174, MS2, fd and PM2) (145). From that time, new phage groups were added over time, and the ICTV presently recognize one order, 13 families and 31 genera of phages (25).

Most phages contain dsDNA, but there are other groups with ssDNA, ssRNA and

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dsRNA. A few phage types which have lipid-containing envelop or contain lipid as part of its molecule were also found.

Up to recent, a total of 5500 tailed phages (96% of phages) are now classified into the order Caudovirales and 3 large phylogenetically related families (Myoviridae, Siphoviridae and Podoviridae). In contrast, filamentous or pleomorphic phages comprise less than 190 viruses only (3.6% of phages), and classified into 10 small families. These results indicated that phages are extremely diversified by their basic properties and morphology. Therefore, there are no available universal criteria for its genus and species delineation up to date (25). In Figure I and Table I, the recently morphologically presented phage and its classifications are summarized.

Figure I. Schematic representation of major phage groups (24)

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Table I. Classification and its biological properties of phages (24)

C, circular; L, linear; S, segmented; T, superhelical; ss, single-stranded; ds, double-stranded.

Briefly, phages are known to have two possible life cycles; the ‘lytic’ (or virulent) and ‘lysogenic’ (or temperate) cycle (152). Lytic phages rapidly multiply and kill the host cell at the end of the replication cycle. On the other hands, temperate phages which undergo the lysogenic cycle persist in a lysogenic state, whereby the phage genome can exist indefinitely by being inserted in the bacterial chromosome (known as the prophage state). The lysogenic life cycle of λ phage, for example, ensures the replication of the integrated prophage along with the bacterial genome for many generations. When induction occurs through damage of the DNA (UV irradiation or exposure to mutagens), which signifies the imminent death of the host, the phage switches to the lytic cycle which results in the release of new phage particles. Interestingly, some prophages can change non-pathogenic bacteria to pathogenic one by lysogenic conversion mechanism (94). Several examples of toxin genes or pathogenic islands insertion of temperate phage to host

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bacterium were reported and summarized in Table II.

Table II. Phages that carries toxin genes and their gene products (94).

Phage Gene Gene product/phenotype Bacterial host 933, H-19B stx Shiga toxins

Escherichia coli O157:H7 ΦFC3208 hly2 Enterohaemolysin

Λ lom Serum resistance

Λ bor Host-cell envelope protein Sfi6 oac O-antigen acetylase

Shigella flexneri Sfll, sfV, sfX gtrll Glucosyltransferase

SopEΦ sopE Type III effector

Salmonella enterica Gifsy-2 sodC-1 Superoxide dismutase

Gifsy-2 nanH Neuraminidase Gifsy-1 gipA Insertion element

ε³⁴ rfb Glucosylation

CTXΦ ctxAB Cholera toxin

Vibrio cholera K139 glo G-protein like

VPIΦ tcp Toxin co-regulated pilus

ΦCTX ctx Cytotoxin Pseudomonas aeruginosa

C1 C1 Neurotoxin Clostridium botulinum

NA see, sel Enterotoxin

Staphylococcus aureus

Φ13 entA,

sak

Enterotoxin A, Staphylokinase

TSST-1 tst Toxic shock syndrome-1

T12 speA Erythrogenic toxin Streptococcus pyogenes β-phage tox Diptheria toxin Corynebacterium diptheriae

The prevalence of phage-mediated lytic and lysogenic infections in the aquatic environment is still controversial; Freifelder stated that more than 90% of known phages are temperate (6), but Cochran et al. suggested that only around 50% of

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bacterial strains contained inducible temperate (or lysogenic) phages (30).

Although a large percentage of phages are lysogenic, they are not suitable candidates for phage therapy since they may not immediately kill the host bacteria.

Therefore, we will only focus on lytic phages in the further section ‘Therapeutic applications of phages’ in this review.

B.2. Phages infecting Aeromonadaceae

The first phages infecting Aeromonadaceae (Hereinafter referred as Aeromonas phages) was studied in the electron microscope in 1965 (16). Its host, which was identified as an Acelobacter sp., was later reclassified as Aeromonas sp. (124).

Subsequently, Paterson isolated nine Aeromonas phages infecting A. salmonicida from trout ponds and fish hatcheries, and described the characteristics of 4 selected isolates (110). A halophilic and psychrophilic phage, specific for a marine Aeromonas spp., was isolated from sea water collected at a depth of 825 m (147).

In 1971, 35 Aeromonas phages infecting A. salmonicida, which isolated from sewage, surface water, fish hatcheries and lysogenic bacteria, were characterized by serology and various biological criteria. Sixteen of these phages were studied by electron microscopy and were divided into three morphological groups (115). At least 8 additional phages infecting A. salmonicida were described since 1980 (73, 74, 119), and two phages infecting A. hydrophila were recently isolated from sewage (28). However, those isolated Aeromonas phages have not been classified in that time. Among the phages of known morphology, all but one had contractile tails and isometric or elongated heads. The exception is Bradley's phage, which had a short tail and resembles Salmonella phage P22 (16). Moreover, since physico-

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chemical properties of Aeromonas phages were almost completely unknown, their classification depended largely on morphology and serological data (2). In addition, many Aeromonas phages were described without accurate morphological micrographs, until the first morphological characteristics of about 35 Aeromonas phages, mostly infecting A. salmonicida, were thoroughly investigated by Ackermann in 1985 (2).

In a recent review of Ackermann in 2007 (1), a total of 43 phages infecting Aeromonadaceae (especially in A. hydrophila and A. salmonicida) have been reported, and all of those were morphologically classified as tailed phages belonging to Caudovirales (33 of Myoviridae, 7 of Siphoviridae and 3 of Podoviridae). And among the Aeromonas phages belong to family Myoviridae, most of them were classified into P1-, P2- and T4-like viruses in the VIIIth ICTV Report (http://www.ictvdb.org/Ictv/index.htm) (42). With the technological advances in phage research, the morphology and genetic functions of T4 phage and T4-like phages infecting Escherichia coli or other bacteria were thoroughly investigated (31, 112), and provided an attractive model for the study of comparative genomics and evolution of phages. In this respect, recent studies of Aeromonas phages have also focused on virulent (or lytic) T4-like phages and have included extensive genomic investigations (31, 83, 101, 110, 113, 134); the complete genome sequences of 4 T4-like phages (Aeromonas phage 25, 31, 44RR2.8t and Aeh1), and only one exception of the complete genome of P2-like temperate Aeromonas phage (designated as φO18P) infecting A. media have already been published in GenBank. And in 2012, we fully sequenced the two T4- like Aeromonas phages (phiAS4 and phiAS5) infecting A. salmonicida subsp.

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salmonicida, and one T7-like Aeromonas phage (phiAS7) which belongs to Podoviridae and infects A. salmonicida subsp. salmonicida. The characteristics of those previously sequenced 8 Aeromonas phages were summarized in Table III.

Table III. Sources and properties of the sequenced Aeromonas phages up to 2012

Phage Family Host Isolation

source/contry

Genome

size (bp) Reference 25 Myoviridae

(T4-like)

A. salmonicida

subsp. salmonicida Fish farm/France 161,475 (112)

31 Myoviridae (T4-like)

A. salmonicida

subsp. salmonicida Fish farm/France 172,963 (112)

44RR2.8t Myoviridae (T4-like)

A. salmonicida

subsp. salmonicida Fish farm/Canada 173,591 (87)

Aeh1 Myoviridae

(T4-like) A. hydrophila Sewage/USA 233,234 (28)

φO18P Myoviridae

(P2-like) A. media A. media O18

strain/Germany 33,985 (11)

phiAS4 Myoviridae (T4-like)

A. salmonicida

subsp. salmonicida River/Korea 163,875 (79)

phiAS5 Myoviridae (T4-like)

A. salmonicida

subsp. salmonicida River/Korea 225,268 (80)

phiAS7 Podoviridae (T7-like)

A. salmonicida

subsp. salmonicida Fish farm/Korea 41,572 (81)

B.3. Therapeutic application of phages

Even though phages were discovered in the early of 20^th century, the research of the past half-century is almost rare on the possible therapeutic applications against infectious bacterial diseases (6). The poor understanding of bacterial pathogenesis and phage-host interactions led to a succession of badly designed and executed

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experiments. Furthermore, with the advent of antibiotic therapy, the use of phages became underestimated after the World War II. The discovery of antibiotics diverted research attention from phage therapy, mainly in the USA and Western Europe in 1940s. However, the use of the phage therapy has persisted without interruption in Eastern Europe and Soviet Union, and phages were commercialized by a number of companies (65). With regards to human health, in the past, phage was commercialized and administered in Poland and the Soviet Union orally, tropically or systemically to treat a wide variety of human infections (suppurative wound, gastro-enteritis, sepsis, osteomyelitis, dermatitis, emphysemas and pneumonia) in both adults and children with showing promising results (5). And in the 1970s, previous enthusiasm on the application of phages to prevent and treat bacterial infections in human was recovered (5, 10); studies of Smith et al. using E.

coli models with mice and farm animals, showed that phages could be used for both treatment and prophylaxis against bacterial infections (129). From then, several other Polish and Soviet Union study groups presented successful clinical applications of phages against drug-resistant bacterial infections in humans as well as animal models (5). The therapeutic efficacy of phage against infectious diseases caused by Pseudomonas aeruginosa (56, 144), Staphylococcus aureus (including MRSA) (149), E. coli (10), Enterococcus faecium (including VRE) (13), Streptococcus pneumoniae (75), Helicobacter pylori (26), Klebsiella pneumoniae (6) and Salmonella enteritidis (44, 135) has been shown in experimental animal models. However in recent decades, the emergence of antibiotic-resistant bacteria has substancially enhanced the interesting phage therapy even by USA and Western Europe. And nowadays, more than a dozen of companies and universities are

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working on phage therapy for human, using current standards of clinical and microbiological research (15).

Recent studies evaluated phages as biocontrol agents in food (50, 67, 85, 135), in plants (45), to control cyanobacterial blooms and for wastewater treatment (150).

Additionally, bacterial diseases are a major problem in the expanding aquaculture (6, 126, 141). The increasing problems related to worldwide emergence of antibiotic resistance in common pathogenic bacteria, and the concerns about its spreadings in the aquaculture environments demanded alternative methods to control bacterial pathogens in fish and shellfish. Phage therapy has been showed a potentially viable alternative to antibiotics used in aquaculture to control indigenous and non-indigenous bacterial disease in farmed fish (6). In addition, some studies of phages were concerned with identifying those phages for use in bacterial typing schemes or for the characterization of its properties, including their potential role in virulence. Remarkably, there have been several attempts of phages to prevent bacterial infections in aquaculture (Table IV), and these previous experimental applications proved that phage could be useful for controlling bacterial infections of fish or shellfish. In the same manner, the experimental applications of phages to control A. salmonicida subsp. salmonicida have been attempted (68, 138), but those studies faced several difficulties with failures regarding fish protection. Therefore, our goal of this study was to find novel Aeromonas phages infecting A. salmonicida subsp. salmonicida, and to verify its therapeutic efficacy in Korean salmonids against furunculosis.

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Table IV. The representative use of phages to control bacterial pathogens in aquaculture (6).

Bacteria Phage Treated

fish/shellfish Effects References Aeromonas

salmonicida HER1107

HER 110

Brook trout (Salvelinus fontinalis)

The onset of furunculosis in brook trout was delayed by 7 days

(68)

Vibrio harveyi

Siphoviridae phage isolated from oyster tissue

and from shrimp hatchery water

Shrimp larvae

(Penaeus monodon) Improved larval survival (78)

Lactococcus garvieae

Siphoviridae phage isolated from diseased fish

and sea water in fish culture cages.

Yellow tail (Seliora quinqueradiata) and Ayu (Plecoglossus

altivelis)

Protective/curative effect (increase in the survival

rate)

(96)

Lactococcus garvieae

Siphoviridae phage isolated from diseased fish

and sea water in fish culture cages.

Yellowtail (Seliora quinqueradiata)

Protective/curative effect (increase in the survival

rate)

(97)

Pseudomonas plecoglossicida

PPp-W4 (Podoviridae)

and PPpW-3 (Myoviridae)

Ayu (Plecoglossus

altivelis)

Reduced infection and

increased fish survival (105)

Pseudomonas plecoglossicida

Myoviridae and Podoviridae isolated from diseased ayu and the rearing pond

water

Ayu (Plecoglossus

altivelis)

Protection against

experimental infection (106)

Aeromonas salmonicida subsp.

salmonicida

Aeromonas salmonicida phages O, R and B

Atlantic salmon (Salmo salar L.)

Lower rate mortality but similar absolute mortality. No protection was offered by any of the

phage treatments.

(138)

Vibrio harveyi Siphoviridae phage

Shrimp larvae

(Penaeus monodon) Improved larval survival (139)

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C. References

1. Ackermann, H. W. 2007. 5500 Phages examined in the electron microscope. Arch. Virol.

152:227-243.

2. Ackermann, H. W., et al. 1985. Aeromonas bacteriophages: Reexamination and classification. Ann. Inst. Pasteur Virol. 136:175-199.

3. Akasaka, T., M. Tanaka, A. Yamaguchi, and K. Sato. 2001. Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: Role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob. Agents Chemother. 45:2263-2268.

4. Alcaide, E., M. D. Blasco, and C. Esteve. 2010. Mechanisms of quinolone resistance in Aeromonas species isolated from humans, water and eels. Res. Microbiol. 161:40-45.

5. Alisky, J., K. Iczkowski, A. Rapoport, and N. Troitsky. 1998. Bacteriophages show promise as antimicrobial agents. J. Infect. 36:5-15.

6. Almeida, A., et al. 2009. Phage therapy and photodynamic therapy: Low environmental impact approaches to inactivate microorganisms in fish farming plants. Mar. Drugs 7:268-313.

7. Austin, B., et al. 1998. Characterization of atypical Aeromonas salmonicida by different methods. Syst. Appl. Microbiol. 21:50-64.

8. Austin, D. A., D. McIntosh, and B. Austin. 1989. Taxonomy of fish associated Aeromonas spp., with the description of Aeromonas salmonicida subsp. smithia subsp.

nov. Syst. Appl. Microbiol. 11:277-290.

9. Barnes, A. C., M. T. Horne, and A. E. Ellis. 1996. Effect of iron on expression of superoxide dismutase by Aeromonas salmonicida and associated resistance to superoxide anion. FEMS Microbiol. Lett. 142:19-26.

10. Barrow, P., M. Lovell, and A. Berchieri. 1998. Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves. Clin.

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11. Beilstein, F., and B. Dreiseikelmann. 2008. Temperate bacteriophage φO18P from an Aeromonas media isolate: Characterization and complete genome sequence. Virology 373:25-29.

12. Bernoth, E., A. E. Ellis, P. J. Midtlyng, G. Olivier, and P. Smith. 1997. Furunculosis - Multidisciplinary Fish Disease Research. Academic Press, London.

13. Biswas, B., et al. 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect. Immun. 70:204-210.

14. Björnsdóttir, B., S. Gudmundsdóttir, S. H. Bambir, and B. K. Gudmundsdóttir. 2005.

Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine. J. Fish Dis. 28:181-188.

15. Brüssow, H. 2005. Phage therapy: the Escherichia coli experience. Microbiology 151:2133-2140.

16. Bradley, D. E. 1965. The isolation and morphology of some new bacteriophages specific for Bacillus and Acetobacter species. J. Gen. Microbiol. 41:233-241.

17. Bradley, D. E. 1967. Ultrastruc