Exploration of Phage-Host Interactions in the Fish Pathogen Vibrio

UNIVERSITY OF COPENH AGEN FACULTY OF SCIENCE DEPARTMENT OF BIOLOG Y MARINE BIOLOGICAL SE CTION

Exploration of phage-host interactions in the

fish pathogen Vibrio anguillarum and anti-

phage defense strategies

PhD thesis Demeng Tan

Academic advisor: Mathias Middelboe

Submitted: 16/03/2015 Academic Supervisor:

Professor Mathias Middelboe

Marine Biological Section

Department of Biology

University of Copenhagen

Denmark

Assessment committee:

Reader Martha Clokie

Department of Infection, Immunity & Inflammation

University of Leicester

United Kingdom

Associate Professor Niels-Ulrik Frigaard (Chair)

Marine Biological Section

Department of Biology

University of Copenhagen

Denmark

Associate Professor Lars Jelsbak

Infection Microbiology

Department of Systems Biology

Technical University of Denmark

Denmark

Submitted:

March 16, 2015 Contents

Contents ...... 1 Preface ...... 3 Acknowledgements ...... 4 Abstract ...... 5 Resumé ...... 7 List of abbreviations ...... 9 Introduction ...... 10 1. Aquaculture ...... 10 1.1. Current status of aquaculture ...... 10 1.2. Bacterial diseases in aquaculture ...... 10 1.3. Vibriosis and Vibrio anguillarum ...... 11 2. Bacterial communication ...... 13 2.1. Quorum sensing in Gram-negative bacteria...... 13 2.2. Quorum sensing in V. anguillarum ...... 14 3. Bacterial biofilm formation and its role in infections ...... 17 3.1. Biofilm structure ...... 17 3.2. Biofilm development and variation in biofilm formation ...... 18 3.3. Heterogeneity in biofilms ...... 20 3.4. Diagnosis of biofilms ...... 21 3.5. Treatment of vibriosis in aquaculture ...... 22 4. Alternative disease control in aquaculture ...... 24 4.1. Bacteriophages ...... 24 4.2. Phage classification ...... 24 4.3. Bacteriophage life cycle ...... 25 4.4. Vibriophages ...... 26 4.5. Application of phages to control fish pathogens: Phage therapy ...... 28 5. Bacteriophage resistance mechanisms ...... 30 5.1. Phage receptor modification ...... 30 5.2. Cutting phage nucleic acids ...... 32

5.3. CRISPR/Cas bacterial immune system cleaves bacteriophage DNA ...... 32 5.4. Abortive infection (ABi) system ...... 32 5.5. Regulation of phage-host interactions by extracellular signaling molecules ...... 33 5.6. Implications of phage protection mechanisms: Phage-host coexistence and co-evolution ...... 33 Aims of this Ph.D. project ...... 35 A summary of this Ph.D. project ...... 36 Paper I ...... 36 Paper II ...... 37 Paper III ...... 38 Future perspectives ...... 39 Paper I ...... 39 Paper II ...... 41 Paper III ...... 43 References ...... 44 Accompanying papers ...... 54

Preface The project presented in this PhD was carried out under the supervision of Prof. Mathias Middelboe primarily at the Marine Biological Section, Department of Biology, University of Copenhagen, Denmark.

The work was done in collaboration with Prof. Lone Gram (Department of Systems Biology, Technical University of Denmark) and Dr. Sine Lo Svenningsen (Department of Biology, University of Copenhagen).

List of publication and manuscripts

1. Demeng Tan, Lone Gram, and Mathias Middelboe. "Vibriophages and their interactions with the fish pathogen Vibrio anguillarum." Applied and Environmental Microbiology 80.10 (2014): 3128- 3140. 2. Demeng Tan, Amalie Dahl, and Mathias Middelboe. "Vibriophages differentially influence biofilm formation by Vibrio anguillarum strains." Applied and Environmental Microbiology. (accept, AEM00518-15) 3. Demeng Tan, Sine Lo Svenningsen, and Mathias Middelboe. "Quorum sensing determines the choice of anti-phage defense strategy in Vibrio anguillarum." mBio. (in revision, mBio00627-15)

3 Acknowledgements Studying abroad is probably one of the best things that I have ever done in my life so far. This experience has not just involved learning scientific skills, but it has also taught me a great deal about independence, taking initiative, and self-motivation, and has helped me develop a wide range of social skills. I am indebted to many people for supporting me through the numerous stages of the past three years. Unfortunately, I will not mention everybody’s name, but there are a few people who deserve a special mention here. To those who deserve but did not receive mention here, and realized my omission, I thoroughly apologize.

First of all, I want to thank my supervisor, Prof. Mathias Middelboe, for offering me such a valuable opportunity to let me do my Ph.D. studies with you. You have taught me so many invaluable things, and I have learned so many important lessons.

I would also like to thank Dr. Sine Lo Svenningsen, for letting me stay in your lab. It has been a tremendously wonderful time, and I have learned a lot about Molecular Biology and Quorum Sensing. Thanks again for your patience and enthusiasm. Hopefully, we will have more collaboration in the future about Quorum Sensing studies.

I am also grateful to Prof. Lone Gram for making our first paper possible.

Hannah Eva Blossom, thank you for proofreading my Introduction Chapter.

I would not have finished my Ph.D. without the love and support of my family, friends and relatives in China. I wish I could have spent more time with you during the past three years.

And last but not least, my host family in Snekkersten. I cannot express to you how much I appreciated you over the past three years, with so many nice memories, and your effort to make Sprydet 83 feel like home.

Abstract The disease vibriosis is caused by the bacterial pathogen Vibrio anguillarum and results in large losses in aquaculture both in Denmark and around the world. Antibiotics have been widely used in antimicrobial prophylaxis and treatment of vibriosis. Recently, numerous multidrug-resistant strains of V. anguillarum have been isolated, indicating that antibiotic use has to be restricted and alternatives have to be developed. Lytic phages have been demonstrated to play an essential role in preventing bacterial infection. However, phages are also known to play a critical role in the evolution of bacterial pathogenicity development. Therefore, successful application of phage therapy in the treatment of vibriosis requires a detailed understanding of phage-host interactions, especially with regards to anti-phage defense mechanisms in the host.

Part I. As a first approach, 24 V. anguillarum and 13 Vibrio sp. strains, representing considerable temporal (20 years) and geographic (9 countries) variation in regards to their origins of isolation, and 11 vibriophages representing three different families (Myoviridae, Siphoviridae, and Podoviridae), were characterized with respect host range, morphology, genome size and lytic properties. Together the host range of the 11 vibriophages covered all the 37 Vibrio strains in the collection. In addition, the occurrence of unique susceptibility patterns of the individual host isolates, as well as key phenotypic properties related to phage susceptibility that were common to the isolated strains were studied.

Part II. In vitro phage-host interactions in two V. anguillarum strains (BA35 and PF430-3) and their corresponding phages (ΦH20 (Siphoviridae) and KVP40 (Myoviridae)), during growth in the settings of micro-colonies, biofilms, and the free-living phase were investigated, in order to explore the resistance/tolerance mechanisms responsible for the different outcomes of these two phage-host interactions. The study demonstrated large intraspecific differences in phage-protection mechanisms, as strain BA35 obtained genetic resistance through mutational changes, whereas strain PF430-3 was protected from phage infection by formation of a biofilm. These different protection mechanisms have important implications for the phage-host dynamics and efficiency of phage infection.

Part III. To gain an insight into whether bacteria of the Vibrio genus use quorum sensing (QS) to control their phage-susceptibility, a study of the QS-mediated interaction between phage KVP40 and V. anguillarum PF430-3 was carried out. The data show that QS does indeed regulate phage-host interactions in V. anguillarum PF430-3, potentially by using QS transcription factor VanT to repress ompK expression. It was demonstrated that QS controls the choice of anti-phage defense strategies in the V. anguillarum strain

PF430-3, suggesting the presence of dynamic, temporary adaptations to phage infection pressure, while still securing the ability to produce a functional OmpK receptor.

In conclusion, this thesis provides a first insight into the dynamic vibriophage-host interactions, indicating the complexity of phage therapy in the treatment of vibriosis, regarding the evolution of anti-phage defense mechanisms, gene regulation, quorum sensing, biofilm formation, as well as pathogenesis. Together, these discoveries will enable us to evaluate these potential factors which regulate phage-host interactions, and on that background, to optimize phage therapeutic strategies against vibriosis with regards to the composition of applied phage cocktails, the timing of treatment, and the likely outcome of phage therapy and QS inhibiting molecules.

Resumé Vibrio anguillarum er en vigtig patogen bakterie i akvakultur, hvor den forårsager sygdommen vibriose, som resulterer i fiskedød og store økonomiske tab i akvakulturindustrien i både Danmark og hele resten af verden. Typisk bruges antibiotika i behandlingen af vibriose, men forøget forekomst af antibiotika- resistente V. anguillarum har medført øget fokus på behovet for alternative behandlingsmetoder. Anvendelse af lytiske bakteriofager har de senere år vist sig som et potentielt alternativ til antibiotika ifm forebyggelse og behandling af fiskeinfektioner. Denne tilgang er dog også forbundet med udfordringer og begræsninger, dels fordi fagerne kan spille en rolle i bakteriers patogene egenskaber og dels fordi bakterie også udvikler resistens overfor fager. Succesful anvendelse af bakteriofager i sygdomsbehandling kræver derfor et grundigt kendskab til fag-vært interaktioner og specielt en forståelse af anti-fag forsvarsmekanismer i værtsbakterien.

Del 1. Som en første tilgang dette undersøgtes 24 V. anguillarum og 13 Vibrio sp., stammer som repræsenterede en betydelige tidsmæssige (20 år) og geografiske (9 forskellige lande) forskellige med hensyn til stedet for deres oprindelige isolation, samt 11 vibriofager, der repræsenterede 3 forskellige moroflogiske familier (Myoviridae, Siphoviridae, and Podoviridae). Bakteriofagerne blev karakteriseret med hensyn til "host range", morfologi, genomstørrelse og lytiske egenskaber, og tilsammen kunne de 11 fag- isolater inficere alle 37 V. anguillarum stammer i stammesamlingen. Derudover blev mønstre i inficerbarhed overfore fagerne samt egenskaber associeret med resistens overfor disse undersøgt hos værtsstammerne.

Del 2. In vitro studier af fag-værtinteraktioner hos to V. anguillarum stammer (BA35 and PF430-3) og deres associerede fager (ΦH20 (Siphoviridae) og KVP40 (Myoviridae)) blev undersøgt under forskellige vækstfaser (mikrokolonier, biofilm og fritlevende) med det formål at kortlægge resistens/tolerance mekanismer i de to stammer. Studiet dokumenterede store intraspecifikke forskelle i de underliggende mekanismer for beskyttelse mod fager i de to stammer. Stamme BA35 udviklede genetisk resistens gennem mutationer mens stamme PF430-3 beskyttedes mod fag-infektioner ved dannelse af biofilm. Disse forskelle i beskyttelsesmekanismer havde store implikationer for fag-vært dynamik og infektions-effektivitet af de to fager.

Del 3. I dette studie undersøgtes i hvilket omfang Vibrio anguillarum stamme PF430-3 anvender "quorum sensing" (QS) i reguleringen af tolerance overfor faginfektion. Data påviser at QS regulerer interaktionerne mellem fag KVP40 og V. anguillarum PF430-3, idet QS medierer en aktivering af transkriptionsfaktor VanT som leder til en nedjustering af ekspressionen af ompK, som er receptor for KVP40. Arbejdet viser at QS kan

7 kontrollerer skift i anti-fag strategi hos V. anguillarum strain PF430-3, som afhænger af de givne vækstbetingelser. Disse skift i forsvarsmekanisme indikerer meget dynamiske tilpasninger til fag- infektionstryk I V. anguillarum PF430-3, som samtidigt sikrer at stammen kan opretholde en funktionel ompK receptor.

Denne afhandling bidrager således med ny viden om de dynamiske og komplekse interaktioner mellem vibriofager og deres værtsceller og understreger betydningen af en bedre forståelse af disse i relation til den løbende udforskning af nye fag-baserede behandlingsmetoder. Disse opdagelser kan således anvendes i vurderingen af de faktorer der regulerer fag-resistens under forskellige forhold og kan dermed bidrage til en optimering af fag-anvendelsen i behandling af vibriose, specielt i forhold til sammensætning af fag cocktails, timing af fag-behandling og potentielt anvendelse af QS hæmmende substrater.

List of abbreviations Abi Abortion infection system AHLs N - acyl - L - homoserine lactone AI Autoinducer CAI-1 cholerae autoinducer-1 cas CRISPR - associated CRISPR Cl ustered regularly interspaced short palindromic repeats CT Cholera toxin CVEC Conditionally viable environmental cells CWD Cold water disease eDNA Extracellular DNA EMSA Electrophoretic mobility shift assay EPS Extracellular Polymeric Substances ERIC Enterobacterial repetitiveelement intergenic consensus sequence FISH Fluorescence in situ hybridization ICTV International Committee on Taxonomy of Viruses LPS Lipopolysaccharide MLST Multi - locus sequence typing MRSA Methicillin - resistant S. aureus OA Oxolinic acid Omp Outer membrane protein PFGE Pulsed - field gel electrophoresis qrr Quorum sensing regulatory RNA QS Quorum s ensing R-M Restriction - Modification VBNC Viable but non-culturable vps Vibrio polysaccharide

Introduction

1. Aquaculture

1.1. Current status of aquaculture A daunting challenge of our time is to ensure adequate nutritional food to the expanding human population (1). Over the past 50 years, the aquaculture industry has become one of the fastest growing industries globally, with an annual growth rate of 8.3%. The aquaculture industry not only provides healthy food for human consumption, but it is extremely important for local economies which rely heavily on the sales of high valued marine fish and crustaceans (1). According to Figure 1, at the end of 2012, aquaculture production has already reached 66.6 million tons per year (1). With declining natural fish stocks and global fisheries, aquaculture plays a vital role in feeding the planet, today and in the future.

Figure 1. World capture fisheries and aquaculture production from 1950 to 2012 (1).

Aquaculture is defined as farming aquatic organisms including fish, molluscs, crustaceans and aquatic plants. In addition, aquaculture farming not only involves interventions in the rearing process to enhance production, such as stock, feeding, protection from predators, but also implies stocks cultivation (1). Despite the fact that aquaculture is a well-controlled and organized industry, problems such as disease outbreaks can still occur. Disease outbreaks are considered as one of the most significant constraints to sustainable aquaculture growth (2).

1.2. Bacterial diseases in aquaculture Fish are susceptible to a wide range of bacterial pathogens. Some of the most important opportunistic bacterial diseases are vibriosis, salmon rickettsial syndrome (SRS), furunculosis, and cold water disease (CWD), which is caused by Vibrio anguillarum, Piscirickettsia salmonis, Aeromonas salmonicida, and

Flavobacterium psychrophilum, respectively (3-5). These bacterial cells are naturally occurring in the environment but become serious pathogens when fish hosts are physiologically unbalanced, immunocompromised or exposed to other stressors, i.e., poor water quality, overstocking, and overfeeding, etc., which then allows opportunistic bacterial infections to progress (6). Once the abundance of these opportunistic pathogens reaches a threshold level, they can cause disease. Hence, farmed fish in many cases are affected by these bacterial diseases, resulting in dynamic unpredictable mortality rates, and subsequently, severe economic losses. Thus, disease management in aquaculture is becoming more complex and challenging.

1.3. Vibriosis and Vibrio anguillarum Vibriosis, a highly fatal haemorrhagic septicaemia, is one of the most prevalent fish diseases and has been found in more than 50 fresh and salt-water fish species including various species of economic importance to the larviculture and aquaculture industry, especially during the larval and juvenile stages. These species include salmon (Salmo salar), rainbow trout (Onchorhynchus mykiss), turbot (Psetta maxima), European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) and ayu (Plecoglossus altivelis) (7). The pathogen can invade the host either via contaminated food or through the skin epithelial cells, while the infection is then triggered due to environmental factors commonly found in fish farms such as high population density or poor water quality (8). Typical external clinical symptoms of the disease include weight loss, lethargy, and development of red spots on the ventral and lateral areas of the fish, with swollen, dark skin lesions causing ulceration and bleeding (Figure 2) (9). Nevertheless, in acute pandemic infections the infection spreads so rapidly that most of the infected fish die even without evidence of any clinical symptoms (10). Previous studies have reported that V. anguillarum, as one of the most prominent fish and shellfish pathogens, is associated with vibriosis infection in aquaculture (11-13).

Figure 2. Clinical symptoms of vibriosis infection a sea bass causing haemorrhagic septicaemia on the fin and around the operculum, figure adapted from Austin (10).

V. anguillarum is a Gram-negative, halophilic and facultative anaerobic bacterium, which grows rapidly in the range of temperatures between 15 and 30 °C on rich media containing 1.5-2% NaCl. According to Harrell’s study, V. anguillarum was initially divided into two distinct biotypes (1 and 2) (14). However, through advances in DNA sequence technology, V. anguillarum biotype 1 was reclassified as Listonella anguillarum, as a result of 5S ribosomal RNA gene sequence analysis (15). Nevertheless, because of its strong similarities with other Vibrio species, it is generally still identified as V. anguillarum (16). Biotype 2 was reclassified as a new species, V. ordalii (11).

V. anguillarum is composed of 2 chromosomes (3.0 and 1.2 Mbp) with a whole genome size of about 4.2 Mbp and a G+C content of 43-46%. Most of the O1 serotype strains isolated harbor a plasmid which carries many virulence factors, such as genes affecting chemotaxis and motility, an iron uptake system, lipopolysaccharides (LPSs) and extracellular products with proteolytic or haemolytic activity (11). Recently, the genome of the O1 serotype strain V. anguillarum 775 was sequenced. The genome annotation shows that the majority of the genes for essential cell functions and pathogenicity are located in chromosome 1. This chromosome also has 8 genomic islands, while chromosome 2 has only 2. It was found that some of these islands carried potential virulence genes (17).

V. anguillarum strains show close similarities according to 16S rRNA sequences irrespective of time or place of isolation, suggesting 16S rRNA is highly uniform and stable (18), indicating further genetic fingerprinting tools such as enterobacterial repetitive element intergenic consensus (ERIC) PCR (19), multi-locus sequence typing (MLST) (20), and pulsed-field gel electrophoresis (PFGE) (21) are needed for surveillance purposes, monitoring outbreaks and tracking pathogen spreading.

Currently, 23 different O serotypes (O1-O23) within V. anguillarum are discriminated, each single O serotype with its own different pathogenicity and host specificity. Among these, only serotypes O1, O2, and O3, have been reported to associate with vibriosis infection in fish (22). Within serotype O2, three host- specific sub-serotypes, O2a, O2b and O2c, have been distinguished by Rasmussen (23).

2. Bacterial communication Bacteria have adapted to thrive in diverse ecological niches. Over the long-term evolution, they have developed sophisticated mechanisms, allowing them to perceive and respond to dynamic environmental conditions (24).

Quorum sensing (QS) is a ubiquitous cell-cell communication found in bacteria, thus far (25). By using this QS system, bacteria coordinate their cell density-dependent gene expression to act in unison, via the production, secretion and subsequent detection of extracellular signaling molecules, namely, autoinducers (25). Detection of autoinducers allows bacterial cells to distinguish different behavioral modes, i.e. from low cell densities to high cell densities (25).

2.1. Quorum sensing in Gram-negative bacteria Most Gram-negative proteobacteria use acyl homoserine lactones (AHLs) as major auotoinducers for interspecies communication, with homoserine lactone (HSL) rings carrying acyl chains of a range from C4 to

C18 in length (26). The first AHL was identified in bioluminescent bacteria (V. fischeri), which have a symbiotic relationship with bobtail squid (Euprymna scolopes) (27). By using QS to activate expression of the luciferase operon to counter-illuminate itself, the squid can easily escape from predators as a cooperative symbiotic interaction between the squid and V. fischeri (25, 28, 29). The mechanism underling this phenomenon has been fully characterized. Briefly, in the V. fischeri model, LuxI synthesizes QS autoinducers, and LuxR acts as a cytoplasmic receptor for AHLs as well as transcription factor of the luciferase gene (28, 30, 31). However, without AHLs ligand, LuxR is unstable and can be rapidly degraded. Since AHLs can freely diffuse across cell membranes, their concentration increases correspond to the cell density increases. By binding to the transcriptional factor LuxR, the AHLs-LuxR complexes turn on the luxICDABE expression (Figure 3) (28, 32).

Figure 3. A canonical Gram-negative LuxIR-type QS circuits. Red pentagons denote AHL autoinducers. Figure adapted from Ng (28).

2.2. Quorum sensing in V. anguillarum N-acyl homoserine lactone-mediated QS circuits have been identified in V. anguillarum, showing similarities to many other Vibrio species in previous studies (28, 33-35). These circuits contain components for multiple QS phosphoreplay system, and control QS-regulated genes via the transcription factor VanT, which is activated in response to extracellular signaling molecules (35).

In general, at low cell densities, these auto-inducers are also at low concentrations, and the membrane- binding sensors are kinases, VanN, VanQ and CqsS, which start autophosphorylation, and initiate a cascade, transferring phosphate onto the phosphotransferase VanU, which phosphorylates the σ54-dependent response regulator VanO (33, 36). Upon phosphorylation, VanO is activated together with the alternative sigma factor RpoN, together with the RNA chaperone Hfp, which finally destabilizes vanT mRNA, repressing expression of the QS transcriptional regulator VanT (33, 36).

At high cell densities, auto-inducer concentrations reach certain thresholds with vanT expression induced, while VanO is inactivated (35). Specifically, binding of QS-signal molecules to the kinase sensor inhibits kinase activity, which changes the receptor function from kinase to phosphatase activity, leading to the dephosphorylation reactions and finally to inactivate VanO. Thus, QS regulatory RNA (qrr) is not expressed while stabilizing the expression of VanT, leading to the QS-mediated gene regulation (33, 36). Figure 4 is a model of V. anguillarum QS circuits.

Figure 4. V. anguillarum QS-circuits. Solid lines with arrows and bars represent activation and repression of gene expression, respectively. Solid lines with double arrowheads represent the transfer of phosphoryl groups to each other. At low cell densities, auto-inducers are at low concentrations. Upon phosphorylation, VanO is activated together with the alternative sigma factor RpoN, which finally destabilizes vanT mRNA, repressing expression of the QS transcriptional regulator VanT. At high cell densities, autoinducers reach a certain threshold with vanT expression induced, while VanO is inactivated. OM and IM indicate outer membrane and inner membrane, respectively. The P in the circle indicates that the protein is in the phosphorylated state (35).

In addition, several N-Acyl homoserine lactone auto-inducers (N-(3-Oxodecanoyl)-L-homoserine lactone, N- (3-hydroxyhexanoyl)homoserine lactone, and N-hexanoylhomoserine lactone) (Figure 5) have been identified in stationary-phase V. anguillarum cell-free supernatant at concentrations of approximately 8.5, 9.5, and 0.3 nM, respectively (37).

Figure 5. Structures of three different V. anguillarum autoinducers present in stationary phase cell-free culture supernatant, namely, N-(3-Oxodecanoyl)-L-homoserine lactone, N-(3-hydroxyhexanoyl)homoserine lactone, and N-hexanoylhomoserine lactone.

3. Bacterial biofilm formation and its role in infections Until recently, a bacterium cell has been considered as an individual cell without interactions with neighboring cells. This dogma has been challenged by the discovery of the cell-density dependent bioluminescence of the marine bacterium V. fischeri, and bacterial biofilm formation (27, 38, 39). These types of gene regulatory phenomena are generally recognized as QS (25). As discussed above QS regulates multiple bacterial social behaviors, such as bioluminescence (40), sporulation (41), competence (42), virulence (43), motility (44), and phage-host interactions (45, 46), as well as bacterial biofilm formation (47). The development of biofilm and QS are meticulously interconnected, as cooperative bacterial group behaviors in a self-produced extracellular matrix are essential for biofilm formation and development (48). It is, therefore, necessary to better understand the mechanisms of V. anguillarum evolution and pathogenicity to develop better animal models and design new strategies and therapeutics for the control of vibriosis.

During vibriosis infection, bacterial growth in planktonic form is relatively rare. In natural environments and aquaculture, bacterial infection involves adhesion to the host organism in the form of a biofilm, sustaining in the persistence of the infection during the vibriosis outbreak (49, 50). Studies have shown, after the first stages of infection, V. anguillarum formed biofilm-like micro-colonies within the skin mucosal tissues of rainbow trout, causing chronic infection and contributing to disease persistence in the face of antibiotic treatment regimens, due to poor antibiotic penetration, nutrient limitation and slow growth, as well as adaptive stress responses (51, 52). Thus, understanding of the role of bacterial biofilm during infection should help the management of antibacterial therapy of relevant infections.

3.1. Biofilm structure Biofilms are matrix enclosed microbial communities that can be established on a range of biological or non- biological surfaces (53). The complex mixture of hydrated polymers, known as extracellular polymeric substances (EPS), plays an important role in the function of biofilms, including retention of nutrients and water as well as protection from antimicrobial agents such as antibiotics, protozoan grazing, host immune systems and bacteriophages (53). Although polysaccharides are predominant components, EPS also contain a variety of other substances, including protein, extracellular DNA (eDNA), membrane vesicles, and other polymers. For instance, in the model organisms of strain Enterococcus faecalis, Staphylococcus aureus, P. aeruginosa and V. cholerae, eDNA has been shown essential for maintaining the stability of saturated biofilms (54-57). The structural role of eDNA in promoting biofilm formation is highly variable and depends on the bacterial strains and growth conditions as well as the stages of the biofilm. The observation of prophage-stimulated biofilm formation (58, 59), and demonstration that addition of DNase I at the

17 initiation stage of biofilm growth inhibited biofilm formation suggested that prophage mediated lysis results in the release of eDNA enhancing biofilm formation. However, DNase I-treated experiments did not affect phage KVP40-meduated V. anguillarum strain PF430-3’s biofilm formation, suggesting that eDNA released from phage KVP40-mediated lysis did not serve as a critical structural component in the initial stages of biofilm formation (Tan et al., unpublished). However, the results should be interpreted carefully, since further work with more controls, such as DNase I inactivation and biofilm penetration, are needed.

3.2. Biofilm development and variation in biofilm formation Biofilm formation is a multistep process (Figure 6), first involving surface-associated bacterial attachment followed by immobilization, then combined unidirectional movement along the surface, and finally the formation of three-dimensional micro-colonies (60). Meanwhile, these sessile biofilm communities can also rapidly multiply and disperse to planktonic bacterial cells (61).

Figure 6. Five complex development processes involving biofilm maturation. Stage 1-5, initial attachment; irreversible attachment; maturation I; maturation II; and dissemination, respectively. Figure adapted from Monroe’s study (62).

It turns out that there is a whole range of different types of biofilm development that bacteria use for a variety of purposes. For instance, V. cholerae bacterium stops biofilm formation when a certain density of bacterial cells gather together; while other bacterium such as P. aeruginosa bacterium makes biofilms when the bacterial number reaches a certain density threshold.

P. aeruginosa biofilms, which is contrasted with cholera acute infection case, cause devastating chronic lung infections in the immunocompromised hosts (63). QS signaling molecules, on the other hand, activate gene transcriptions which facilitate biofilm formation at high cell densities (64). Another well-known example of biofilm formation at high cell densities is that of E. coli, in which the physiological and morphological development of its biofilms have been well studied. In the post-exponential growth phase of E. coli, while nutrients are no longer optimal yet still available, E. coli cells stop producing flagella, and enter into stationary phase, in a process of cellular aggregation (65). This aggregation imposes diffusion

18 constraints on material exchanges, preventing both entering or leaving the biofilm system, which impedes, for example, antimicrobial substances and the interactions between phage receptor and phage binding proteins (66). However, biofilm sessile cells can also be detached by seeding dispersal, and these detached cells also involve the establishment of new biofilms, which is of fundamental importance to the dissemination of pathogens in the infection model (67).

In V. cholerae cases, biofilm formation is activated at low cell densities (47), in contrast to other bacterial pathogens that induce biofilm formation at high cell densities in the presence of QS signaling molecules. The ability to produce biofilm at low cell densities not only protects the V. cholerae cells against the host immune system and gastric acid, but it also defends against host internal bacteria, such as human gut microbial communities (68). Once the cell densities reach high numbers, bacterial cells stop generating biofilm, since biofilm production takes up valuable resources, which could otherwise be used in growth and cell division (47). At high cell densities, QS-mediated expression of the hap protease gene, was shown to promote release from the epithelium and escape into the environment for successful infection of new hosts (69). The same phenomenon also occurred in the cultures of V. anguillarum strain PF430-3 and phage KVP40 at high cell densities, detachment of cells from aggregates were disseminating into free-living variants, which co-existed with phage KVP40.

Potential application of treatments that promoted dispersal of aggregated cells into the planktonic phase seems attractive, as the results described in previous work indicated that even the use of pro- and anti-QS therapies that function by modulating bacterial chemical communication circuits to weaken the biofilm and lead the sessile cells into planktonic phase, where they could be efficiently killed (70-72). Also, it seems likely that the antibiotics used for biofilm infection could be significantly reduced if biofilms have already partially been disintegrated (73). Thus, detachment of biofilms will ultimately improve the therapeutic effect of biofilm control.

As discussed above, comparisons of differences among V. cholerae, P. aeruginosa and E. coli strains reveal a wide range of biofilm formation patterns, all linked to QS signaling molecules (AI-1 or AI-2) (47, 64, 74). These interspecific differences are likely dependent on the internal environment and related to the amount of competition and the necessity for dispersal.

Intraspecific variations in biofilm formation have also been observed. The biofilm formation of V. anguillarum strains PF430-3 and BA35 in a flow cell device (75) were highly different with respect to structure and stability (Figure 7). For example, an increase in biofilm formation associated with the pellicle on the top of tubes were observed in strain PF430-3; On the contrary, the biofilm stability of V. anguillarum

19 strain BA35 is relatively poor, as no surface pellicle in the liquid cultures could be found. According to the in vitro study of strain BA35, biofilm formation decreased after 2 days. The decrease in the level of biofilm could also be partially attributed to an earlier onset of the dispersal phase because of a rapid increase in biofilm formation, which has been observed in Hosseinidoust’s study (76). In general, biofilm stability is closely correlated with the EPS production (77), as previously visualized by use of fluorescently labeled lectins carbohydrate-containing EPS in P. aeruginosa biofilms (78). Perhaps, EPS production in V. anguillarum strain PF430-3 contributed to the formation of the characteristic colonies, which prevented early dispersing. A similar phenomenon was also mentioned in Fong’s V. cholerae study (79), by deleting vps genes which is required for biofilm formation, many of the mutants exhibited reduced capacity to produce VPS and biofilm formation.

Figure 7. Bacterial biofilm formation of V. anguillarum strain PF430-3 and BA35 after 24 h, samples were stained with LIVE/DEAD BacLight viability stain (Invitrogen) and visualized by confocal scanning light microscope.

3.3. Heterogeneity in biofilms Bacterial biofilms are physiologically and spatially heterogeneous; even within a single species, differential spatial and temporal gene expression occurs due to chemical gradients and adaptation to the local environmental conditions (80-82). The expression of different extracellular molecules could change the surface antigen properties of the cells and thereby promote the formation and maintenance of different types of multicellular communities during the biofilm infection, including different stages between “on” and “off” status (83). These strategies generally were recognized by the function of enabling the individual cells to evade immune system or predator (84). For example, a subset of the population in the planktonic phase may accumulate mutations that enable them to produce more EPS, grow slower, become non-motile and form aggregates (80). These aggregates from the mutants may increase the adhesion capacity to the substratum and function as a physical barrier that protects against attacks and enhances the survival of the entire community. The remaining planktonic members of the community could contribute to different

20 benefits to the community, such as protozoa grazing, phage attack or others may be involved in cellular communication (47, 85, 86). Thus, functional specialization may be promoted through mutations or gene regulations and could also stimulate multicellular development (86).

Despite an increasing knowledge of biofilm structure and properties, many key questions still remain to be answered about essential factors regulating biofilm formations. Several environmental conditions have been proposed to influence biofilm structure. For instance, Kreft’s study (87) has shown that the stability of a biofilm is closely correlated with the EPS production as well as pilus and flagella, i.e. high EPS production resulted in a solid structure of biofilm. Similarly, Nagaoka reported that a high level of EPS production increased viscosity of the medium (88). Moreover, in Reisner’s E. coli K-12 biofilm models, the presence of transfer constitutive IncF plasmids were essential in inducing biofilm forming structures (89). It should be noted that the discussion here is limited to the single-species biofilm formation; additionally, solid-air biofilms and liquid-gas biofilms would probably have their distinct characteristics, which would influence cellular communications (90).

3.4. Diagnosis of biofilms Detection of bacterial biofilms of V. anguillarum, V. cholerae, S. aureus and P. aeruginosa are pivotal in many diagnostic decisions. In addition, the presence of multi-drug resistance V. anguillarum, V. cholerae and P. aeruginosa, and methicillin-resistant S. aureus (MRSA) can pose intractable problems. These pathogens all form culturable biofilms, which can be studied in the laboratory, and much of the progress in our understanding of biofilm structure, regulation and treatment relates to studies of such pathogens (91, 92). Additionally, the problems of biofilms cannot be neglected, since most biofilm samples are either conditionally viable environmental cells (CVEC) or viable but non-culturable (VBNC). Due to these CVECs and VBNCs, traditional methods are inefficient in detecting and identifying certain biofilms. However, molecular methods offer a way to overcome these shortages in traditional methods. Most nucleic acid- based molecular methods for the detection and identification of bacteria begin with the extraction of DNA and/or RNA from the sample to be analyzed or directly identified by fluorescence in situ hybridization (FISH) (93). This extraction and thus diagnosis will be more efficient, and will yield more precise quantification, if the nucleic acids have not been degraded by chemical preservatives or by endonuclease enzymes (91). Also, recent studies have shown that using enrichment media containing autoinducers (AIs) improves resuscitation of dormant V. cholerae in environmental water samples and S. enterica serovar typhimurium and enterohemorrhagic E. coli (92, 94-96).

3.5. Treatment of vibriosis in aquaculture Traditionally, pathogens in aquaculture have been treated with antibiotics. For instance, quinolones is still the first drug of choice for vibriosis in aquaculture environments. This may explain the oxolinic acid (OA), a 4-quinolone resistance found among Vibrio spp. strains isolated from diseased Atlantic cod (97). Moreover, bacterial infections mediated via biofilm formation are persistent and difficult to treat with traditional methods, due to low antibiotic penetration and non- or extremely slow-growing physiological state (98). Besides, a severe problem in the aquaculture industry these days is that even without any apparent disease symptoms, antibiotics are still used routinely and prophylactically, due to lack of sanitary barriers (99). The unconsumed fish pellets and fish feces, may add to excess antibiotic residues in the sediments at the bottom of raising pen (99). For instance, in Thailand, it has been reported by Holmström and colleagues that of 76 shrimp farmers, 56 used antibiotics (100), with more than 10 different antibiotics used prophylactically. Table 1 is an overview of the major classes of antibiotics used in the aquaculture industry adapted from Defoirdt’s study (101). Serious concerns have been raised with respect to the development of bacterial antibiotic resistances and horizontal gene transfer to human pathogenic bacteria, via antibiotic residues. As discussed above, antibiotic use has to be restricted and alternatives for treating bacterial infection must be implemented.

Table 1 (101)

The different classes of antibiotics used in aquaculture, their importance for human medicine and examples of (multi)resistant pathogenic bacteria isolated from aquaculture settings. Importance for Multiple Drug class human medicine Example Resistant bacteria resistance? Isolated from Aminoglycosides Critically important Streptomycin Edwardsiella ictulari Yes Diseased striped catfish (Pangasianodon hypophthalmus), Vietnam Amphenicols Important Florfenicol Enterobacter spp. and Yes Freshwater salmon farms, Pseudomonas spp. Chile Beta-lactams Critically important Amoxicillin Vibrio spp., Aeromonas Yes spp. and Edwardsiella Different aquaculture tarda settings, Australia Beta-lactams Critically important Ampicillin Vibrio harveyi Yes Shrimp farms and coastal waters, Indonesia Fluoroquinolones Critically important Enrofloxacin Tenacibaculum Yes Diseased turbot maritimum (Scophthalmus maximus) and sole (Solea senegalensis), Spain and Portugal Macrolides Critically important Erythromycin Salmonella spp. Yes Marketed fish, China Nitrofurans Critically important Furazolidone Vibrio anguillarum Yes Diseased sea bass and sea bream, Greece Nitrofurans Important Nitrofurantoin Vibrio harveyi Yes Diseased penaeid shrimp, Taiwan Quinolones Critically important Oxolinic acid Aeromonas spp., Yes Pond water, pond sediment Pseudomonas spp. and and tiger shrimp (Penaeus Vibrio spp. monodon), Philippines Sulphonamides Important Sulphadiazine Aeromonas spp. Yes Diseased katla (Catla catla), mrigel (Cirrhinus mrigala) and punti (Puntius spp.), India Tetracyclines Highly important Tetracycline Aeromonas hydrophila Yes Water from mullet and tilapia farms, Egypt Tetracyclines Highly important Oxytetracycline Aeromonas salmonicida Yes Atlantic salmon (Salmo salar) culture facilities, Canada

4. Alternative disease control in aquaculture Multiple commercial vaccines are currently used to protect fish against outbreaks of vibriosis; all of these vaccines consist of inactivated strains of both V. anguillarum serotypes O1 and O2 (102). However, outbreaks of vibriosis caused by serotype O2 have not been completely prevented (22). Moreover, at the larval stage, fish are more vulnerable to V. anguillarum infections, and the vaccine is largely ineffective as their immune system is not fully developed. Therefore, alternative antibacterial strategies are required to be identified and developed.

Global regulators such as autoinducer-1 (AI-1) and autoinducer-2 (AI-2) inhibitors of bacterial physiology have emerged as a potential approach to manipulate bacteria as discussed above (103, 104). These AIs not only have important roles in biofilm formation, virulence and antibiotic resistance, but also have secondary functions, which can be used as pro- or anti-QS/antibiotic/phage therapy synergy for both acute and persistent infection treatment in the future (103, 105-108). All these characteristics can be taken into consideration for the development of future treatment strategies for bacterial infections in aquaculture.

4.1. Bacteriophages Bacteriophages are viruses that parasitize bacteria. Phages are the most abundant microorganisms in the ecosystem, with total numbers estimated to be more than 1030 (109). Bacteriophages are ubiquitous, and are found in marine and freshwater, soils, as well as the intestinal tracts of animals, and are estimated to be on the order of 107–109 per gram dry weight of soils and feces or per milliliter of seawater (109). The ecological functions of bacteriophages also involves playing important roles in e.g. structuring bacterial diversity and succession in the ocean, promoting biogeochemical element cycling and as key drivers of horizontal gene transfer (110).

4.2. Phage classification Bacteriophages are classified by the International Committee on Taxonomy of Viruses (ICTV) based on their morphology and types of nucleic acid. More than 5500 phages have been examined by electronic microscopy, ~96% are tailed (111). These tailed phages, which belong to the order of Caudovirales, can be divided into 3 families (Myoviridae, Siphoviridae, and Podoviridae) (Figure 8). The differences among these three families are: a long or short contractile tail (Myoviridae), a long non-contractile tail (Siphoviridae), and a short, non-contractile tail (Podoviridae).

Figure 8. Transmission electron microscopy (TEM) images of selected vibriophages from three different families, scale bar indicates 200 nm. Phage KVP40, ΦH20, and Φ4-7 belong to Myoviridae, Siphoviridae, and Podoviridae, respectively (18).

4.3. Bacteriophage life cycle Since phages need bacterial hosts to replicate, phage infection may lead to bacterial cell death (lytic life cycle) or to a lysogenic relationship, where both genomes of phage and host replicate together. Specifically, in the lytic life cycle, phage progenies are released from lysis of the bacterial host. New progenies then continue to infect more hosts. In the lysogenic life cycle, temperate phage DNA is integrated into host chromosomes and replicates along with cell division (Figure 9). However, this is not always permanent and phages can be induced from the lysogenic to the lytic life cycle, which is dependent upon environmental signals and the number of infected phages per cell (112), such as DNA damage, UV exposure, mitomycin C, hydrogen peroxide and high MOI, by causing an SOS response (113). Additionally, phages are known to play a critical role in the evolution of pathogenic bacterial species, and as it is particularly true for V. cholerae (114). For example, a major virulence factor of V. cholerae, the cholera toxin (CT), is encoded by ctxAB in the lysogenic phage CTXΦ (115). Likewise, cryptic prophages also were shown to help bacteria cope with adverse environments, such as cell growth, antibiotic resistance, early biofilm formation, as well as environmental stresses (116, 117).

Figure 9. Schematic picture of phage lytic and lysogenic life cycles. Phage adsorption is the first key step in phage proliferation; in order to efficiently bind to the bacterial receptor, phage receptor-binding protein (RBP) is required to specifically interact with bacterial cell surface receptor to enable intracellular DNA injection. In the lytic life cycle, phage DNA replicated separately from host genome, resulting in the destruction of the infected cell and its membrane. In the lysogenic life cycle, phage DNA is integrated into the host genome and can be transferred to daughter cells, until the lytic cycle is induced. However, a portion of these induced cells could enter an abortive lytic cycle by losing prophage and becoming non-lysogens (curing) (112).

4.4. Vibriophages Although viruses have been enumerated, isolated and characterized from the marine environment, there is little information about the specific phage types. Previous seasonal and spatial studies about vibriophages were carried out mainly based on the V. parahaemolyticus hosts, such as vibriophages isolated from the Strait of Georgia (Vancouver, Canada) (118) and Tampa Bay (Florida, USA) (119), as well as V. cholerae prophages such as K139 (120), and filamentous phage CTXΦ, which has been shown to be linked to the bacterial pathogenicity, CT production (121).

Among the best characterized vibriophages is bacteriophage KVP40. KVP40 was originally isolated from polluted seawater off the coast of Japan using V. parahaemolytcius as the host (122). Phage KVP40 is a novel T4-like virulent vibriophage, with a broad-host-range, belonging to the Myoviridae family, and the genome size is around 244 kb, with a G+C content of 42.6%, and a National Center for Biotechnology Information (NCBI) accession number of B_KVP40 AY283928 (123). Vibriophage KVP40 is known to cause infection through the universal outer membrane protein K (OmpK) and has previously been shown to infect

26 more than 8 Vibrio species, including V. anguillarum, V. parahaemolyticus, V. harveyi, V. natriegens, V. cholerae, as well as Photobacterium leiognathi (Table 2) (122, 124).

Table 2. Adapted from Inoue (124)

Distribution of OmpK or its homologs among Vibrio and Photobacterium strains Phage-resistant Species Strain Phage sensitivity a Phage adsoprtion b OmpK-like protein c mutants d V. parahaemolyticus 1010 + + + OmpK- VIB4 - + + VIB8 + + +

VIB9 + + + OmpKd V. alginolyticus VIB20 + + +

VIB31 + + + OmpK- VIB33 - + +

V. natriegens VIB30 + + - OmpKd, OmpK- V. cholerae non-O:1 VIB5 + + + OmpKd, OmpK-, V. mimicus VIB13 + + + OmpK+ V. anguillarum I VIB35 + + + OmpKd VIB36 + + + OmpK+

V. splendidus II VIB37 + + + OmpK- V. fluvialis I VIB7 + + +

VIB18 + + + OmpK- V. vulnifics VIB6 - - + V. harveyi VIB26 - + + V. campbellii VIB27 - - + V. nereis VIB29 - - + V. pelagius I VIB38 - - + V. costicola VIB39 - + + P. leiognathi PHO1 + + + P. angusturn PHO2 - + -

a Sensitivity to KVP40 tested by plaque formation. b More than 10% adsorption of KVP40 as compared with 1010. c OmpK or its homologs detected by immunoblotting. d Phenotypes of KVP40-resistant mutants isolated. The OmpK-like protein was defective (OmpK-), partially defective (OmpKd), or produced normally (OmpK+).

4.5. Application of phages to control fish pathogens: Phage therapy Due to their efficient lysis, lytic phages can potentially be used against bacterial infection, and are much more specific than commonly used antibiotics. Therefore, by using phage therapy, a specific bacteriophage could theoretically be chosen to target a specific pathogen. Because of their host specificity, they would not affect beneficial bacteria (e.g. gut flora), thus reducing the chances of opportunistic infections (125). In 2006, the United States Food and Drug Administration (FDA) approved using bacteriophages on ready-to- eat meat and poultry products in order to kill the Listeria monocytogenes bacteria, giving these products GRAS status (Generally Recognized as Safe) (126). Therefore, properly developed phage products can be invaluable in controlling bacterial infections in various settings.

Recently, there has been a growing number of studies on phage therapy, focusing on a variety of fish pathogens including A. salmonicida in brook trout, Yersinia ruckeri in salmon, Lactococcus garvieae in yellowtail, P. plecoglossicida in ayu, F. psychrophilum in rainbow trout, V. anguillarum in Atlantic salmon and V. harveyi in shrimp (127-134). These studies have demonstrated the potential of specific phages to significantly control pathogen density and, in some cases, reduce fish mortality. For instance, Silva’s study of V. anguillarum and vibriophage showed that the larvae mortality in the infected and treated group was similar to normal levels and significantly lower than the infected but not treated group (135). Moreover, according to Lomelí-Ortega’s study, lytic phage A3S and Vpms1 were also effective to reduce larvae mortality caused by V. parahaemolyticus (136). Similarly, in Vinod’s field trail experiments, treatment with bacteriophage improved larval survival and brought about decline in luminescent V. harveyi counts in hatchery tanks (133).

In addition, studies from V. cholerae (114) have also shown that lytic vibriophages may play an essential role in modulating the seasonal dynamics of cholera in endemic areas, such as the Ganges Delta region of Bangladesh and India. Specifically, the number of cholera patients increased whenever the number of lytic vibriophages in the water decreased, and cholera epidemics tended to end concurrent with large increases in the concentration of vibriophages in the water (114, 137), indicating that lytic vibriophages specific for V. cholerae may limit the severity of cholera outbreaks by killing susceptible bacteria present in the reservoir. Due to their selective killing of susceptible strains, vibriophages are also believed to play a key role in the emergence of new V. cholerae pandemic serogroups or clones (114, 138, 139).

Therefore, it seems a promising strategy to apply vibriophages to gain control of vibriosis infections in fish used for aquaculture. However, successful application of phage therapy in the treatment of vibriosis requires a detailed understanding of phage-host interactions in both planktonic and biofilm forms,

28 especially with regards to anti-phage defense mechanisms in the bacterial hosts, specifically, bacterium V. anguillarum in this Ph.D. study.

5. Bacteriophage resistance mechanisms Predation pressure from bacteriophages is substantial, mainly because their abundance outnumbers microbial cells by an estimated 10-fold in most natural environments (140). Accordingly, bacteriophage lytic infection imposes a strong selection for bacterial mutations/tolerance providing reduced phage susceptibilities or resistance against phage infection. Bacteria have evolved a wide range of different resistance/tolerance mechanisms including 1), preventing phage adsorption; 2), cutting phage nucleic acid; 3), abortive infection through altruistic suicide; and 4), QS-mediated receptor down-regulation, which can make the host immune to the viral infection (141). Similarly, phages can also overcome bacterial resistance by adapting to new receptors, battling restriction-modification systems, evading CRISPR-Cas systems, and escaping abortive-infection mechanisms (142).

5.1. Phage receptor modification Adsorption is a key step recognition between phage receptor-binding protein and phage receptors on the sensitive host cells (143). Most of phage receptors are presented on the bacterial cell walls, such as LamB for phage lambda, the prion outer membrane protein F and C (OmpF and OmpC) for phage T2 and T4, and moreover, the flagella protein for phage phi (144-147). A recent study showed that ϕCb13 and ϕCbK actively interact with the flagellum of the bacterium Caulobacter crescentus and subsequently attach to receptors on the cell pole. Using the flagella or pili to initiate contact with the host cell, increases the likelihood of attachment and successful infection (148). Similarly, we noticed that a large fraction of cells in the BA35+ΦH20 cultures were found to be either non-motile or with impaired mobility, which may imply that flagella play an important role in phage-host interactions. Furthermore, a specific polysaccharide was also implicated as an essential component in adsorption, for instance, phage kh and host L. lactis subsp. cremoris KH, as well as phage 1358 and L. lactis (149, 150). Additionally, according to Seed’s study, V. cholerae lipopolysaccharide O1 antigen functions as a major target of phage ICP1 (151). By using phase variation of O antigen biosynthesis, V. cholerae cells can easily generate variable expression of surface components, which is generally thought to help these organisms evade the immune system and phage predation (151). However, little is known about the requirements of these phage receptors and it is unclear if the polysaccharide was acting as a receptor or if it was facilitating reversible phage binding to a secondary receptor (152, 153).

Mutating phage receptors or producing EPS to block the interaction between phage receptor and phage receptor-binding protein to prevent phage adsorption can be the first step that bacterial cells exhibit in developing resistance/tolerance to avoiding infection (154). Previous studies in Vibrio hosts, showed that mutations or modifications of the outer membrane protein K (OmpK), led to resistance to vibriophage

KVP40 (124). Similarly, in Paper III, ompK in-frame deletion also proved that OmpK acts as a phage binding receptor in V. anguillarum strain PF430-3 (Figure 10). Furthermore, mutations in outer membrane protein A (OmpA) of E. coli K-12 appear to play a role in inhibiting phage infection (155). Phage receptors, in addition to the phage attachment, were involved in the bacterial nutrient intakes, which may be responsible for the morphological changes of the colonies with small size, known as small-colony variants (SCVs) and fitness costs, as have been previously demonstrated experimentally, such as reduced abilities to take up specific nutrients (156) or reduced competitive abilities in general (157, 158). For example, mutation in the LamB phage receptor caused the inability to transport long chain maltodextrin across the outer membrane (159). In Paper II, phage ΦH20-resistant mutant of strains BA35 was found to have reduced ability to utilize a number of carbon resources in the BIOLOG GN2 test.

Additionally, Park (160) found that bacteriophage-resistant mutants of P. plecoglossicida lacked virulence for ayu. In addition, in a successful phage therapy experiment of E. coli infection in mice and calves, the resistant mutants isolated from the treatment were the less virulent k-1 type mutants (161). In Paper III, the function of phage KVP40 binding receptor OmpK still remained unknown, but, it has been suggested to be involved in bile salt resistance and iron acquisition (162). Studies on vaccines have shown that OmpK could also function as a subunit vaccine against V. anguillarum infection (162), because the outer membrane has been reported as selectively permeable or serves as a receptor as well as adhesions in most Gram-negative bacteria for colonization (163, 164).

Figure 10. Spot assay for testing host specificity of phage KVP40 against wild-type, QS mutants (ΔvanT and ΔvanO) and ompK mutants (ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK) as well. Five μl serial 100-fold dilutions of phage KVP40 lysate (1, 109; 2, 107; 3, 105; 4; 2, 103 PFU ml-1) were shown by spot titration onto top agar lawns of the indicated strains (Paper III).

The role of spatial refuge in stabilizing bacteria-phage interactions has been observed in many ecosystems, especially, micro-colonies and biofilms, as discussed in previous studies (165-167), and even in the marine environments, such as marine snow and sediments. Because of the low dispersal rates in the heterogeneous environments, by creating ephemeral refuge may directly/indirectly block phage receptor from phage attack by non-mutation-based mechanisms (165-168); as also shown in V. anguillarum strain PF430-3 protected against phage KVP40 infection by increasing cell aggregation and biofilm formation, allowing coexistence rather than coevolution, finally promoting the stability of phage-host systems by reducing the risk of lytic phage attacks.

5.2. Cutting phage nucleic acids Once the nucleic acid has been injected into the bacterial cell, the restriction-modification (R-M) systems protect the bacterium by cutting invading DNA into pieces (169). When unmethylated phage DNA enters a cell harboring R-M systems, restriction enzymes, thereby, rapidly degrade the foreign genetic material functioning as a prokaryotic immune system (170). For instance, in Bacillus subtilis Marburg nonB mutated strain, nonsense mutation on ydiB was found to be related to the restriction system targeting sequence of BsuMR, which was identical to XhoI (CTCGAG) (171, 172). However, according to Krüger’s study (173), R-M systems are not always perfect, for instance, phages and plasmids can acquire host modifications to avoid restriction endonuclease, which highlights an evolutionary arms race between bacterial host and phages (142).

5.3. CRISPR/Cas bacterial immune system cleaves bacteriophage DNA Another mechanism recently described is CRISPR (Clustered Interspaced Short Palindromic Repeats) and the CRISPR-associated (cas) genes system, in which a CRISP-cas loci was identified composing of 21-48 bp direct repeats interspaced by non-repetitive spacers (26-72 bp) of similar length (174, 175). By using this immunity system to acquire at least one new repeat-spacer unit at the 5’ end of the repeat-spacer region of a CRISPR locus that targets foreign nucleic acids, bacteria can efficiently protect themselves from including phage DNA and plasmids (175, 176).

5.4. Abortive infection (ABi) system The study of the ABi system began 50 years ago, and even now, the mechanisms underlying the infection are still not completely understood (141). The bacterial cell can increase the chances of its own population survival by using the abortive infection system, where phage infection leads to the death of infected bacterial cells. The system is characterized by a normal infection start (i.e., the phage adsorbs and injects its DNA into the host cell), followed by an interruption of the replication, transcription or translation, leading to the release of little or no new phage progenies (141, 177). Recent studies in B. subtilis showed that the

Marburg strain remained resistant to phage SP10 due to a NonA-mediated aborted infection system, acted as a second layer of protection against phage SP10 infection, specifically, the overexpression of nonA gene terminated cell growth with reduced efficiency of colony formation and respiration activity (172).

5.5. Regulation of phage-host interactions by extracellular signaling molecules A recent study has demonstrated a new mechanism of phage resistance in which, bacteria can coordinate their receptor gene expression upon the environmental QS signal, to avoid the risk of infection at high cell densities (45). Since phages require a host to replicate, it follows that the predation pressure is relatively higher at a high cell density status compared to sparsely populated environments. Hence, if bacterial hosts could regulate their anti-phage mechanisms based on cell densities, they could easily reduce their susceptibilities to infection during high cell densities, while avoiding the metabolic burden of maintaining elevated anti-phage defenses during growth at low cell densities. For instance, E. coli possesses the ability to use AHLs to reduce its susceptibility to at least two phages, phage λ and phage χ (45). In the phage λ case, phage receptor LamB was shown 40% down-regulation compared to the untreated controls without AHLs (45). In addition, high concentration of universal communication molecule AI-2 (100 μM) was demonstrated to induce virulence genes and transfer them via phage release in E. faecalis V583ΔABC (178). Intriguingly, a recent study from Hargreaves (179) identified Clostridium difficile phage θCDHM1 encoded an intact QS operon. Similarly, QS-associated genes have also been identified in several phages of Psedomonas spp (180, 181), and function as acylhydrolase to break down AHLs, was identified in the Iodobacter-phage ϕPLPE (182). Although it remains unknown whether any of these genes are of functional importance, it certainly opens the exciting possibility that some phages may actively interfere with the QS in the bacterial communities.

5.6. Implications of phage protection mechanisms: Phage-host coexistence and co-evolution As complexity arises within populations of bacterial resistant mutants, it may help mutants survive better or have more offspring. If so, this complexity will be favored by natural selection and spread through the bacterial population. However, most mutations with phenotypic effects are harmful, such as reducing the competitiveness of the mutant strains, as most phage receptors involve nutrient intake or pathogenicity, a receptor-deficient mutant will have a slower growth rate or reduced virulence (160, 161, 183-185). That is, phage resistant mutants with those traits will tend to be wiped out before reproducing, taking the deleterious traits out of bacterial communities. Therefore, the non-mutation defense mechanisms among these phage hosts may suggest phage-host co-existence interactions, rather than the classical phage-host co-evolutionary arms race, known as Red-Queen theory.

The Red-Queen hypothesis was first formed by Van Valen in order to explain the “law of extinction” (186). According to the previous phage-host interaction studies, virulent phages managing to coexist with their bacterial host leads to continuous variations and selections towards the adaptation of bacterial hosts by evolving resistance to current phages and phage evolve to counter resistance. It has been reported that the arms race has a huge impact on global nutrient cycling, on climate, on the evolution of the biosphere, and on the evolution of virulence pathogens (187). However, this theory was also criticized because the evolution rates between phage and host are not symmetrical. Recent studies showed that, in soil, phages seem ahead of the bacterial hosts in the evolutionary arms race (188). In the natural environment, at each stage of the arms race, one could become extinct, and without one, phage and bacteria coexistence, therefore, does not even exist. For instance, one of the typical examples of “Cheshire Cat” ecological dynamics is the haploid phase of Emiliania huxleyi provided an escape mechanism that involved separation of meiosis from sexual fusion in time, to ensure that genes of dominant diploid clones were passed on to the next generation in a virus-free environment (189). Thus, it is important to understand both evolutionary and non-evolutionary mechanisms (such as gene regulation) that can regulate phage-host interactions in the future.

Uncovering anti-phage defense mechanisms is essential for understanding phage-host dynamics and for application of phages in disease control, as they reflect the remarkable diverse interactions between bacterial hosts and viruses and play a key role as agents shaping microbial community structure. As the predation pressure from phages is a key determinant of the size and composition of bacterial populations, understanding of the potential factors that govern phage-bacterial interactions will be important in any context where the goal is to control the growth of a microbial population including, for example, the treatment of bacterial infections, development of effective probiotics, production of cultured dairy products, or manipulation of the human microbiome to prevent or treat life-style diseases.

Aims of this Ph.D. project The aim of this Ph.D. project was to investigate the interactions between vibriophages and their hosts V. anguillarum, and to ultimately improve the efficiency of phage therapy.

The objectives of this project were:

To isolate and characterize vibriophages and V. anguillarum and investigate the occurrence of phage susceptibility patterns. (Paper I)

To explore phage-host interactions in two V. anguillarum strains, and the resistance/tolerance mechanisms responsible for the different outcomes of these two different systems. (Paper II)

To determine whether AHLs-mediated Quorum Sensing regulates anti-phage defenses in V. anguillarum strain PF430-3. (Paper III)

A summary of this Ph.D. project

Paper I Vibriophages and their interactions with the fish pathogen Vibrio anguillarum

The use of phage therapy to control bacterial pathogens may offer an effective method to reduce vibriosis infection in fish or shellfish maintained in aquaculture. Phage therapy is a promising alternative to the use of antibiotics and thus avoids the risks associated with antibiotics abuse and overuse. However, a detailed understanding of phage-host interactions is needed to evaluate the potential of this technique.

As the first approach, we collected and isolated 24 V. anguillarum strains and 13 Vibrio sp. strains, which represented considerable temporal (20 years) and geographic (9 countries) variations. Irrespective of their origins of isolation, further characterizations based on phylogenetic (16S rRNA) and physiological (BIOLOG GN2 and API 20NE) fingerprints, revealed that they were quite similar.

Ten phages were isolated from Danish water samples with different Vibrio hosts. In addition to the Danish phage isolates, a broad-host-range vibriophage KVP40 was also included in this study. Phages were further characterized by genome sizes, morphologies, and host-range analysis. Together, 11 phages represented three different families (Myoviridae, Siphoviridae, and Podoviridae), which covered all the host ranges of 37 Vibrio strains.

Despite the occurrence of unique susceptibility patterns of the individual host isolates, key phenotypic properties related to phage susceptibility are distributed worldwide and have been maintained in the global Vibrio community for decades. Unfortunately, the phage susceptibility pattern of the isolates did not show any relation to the physiological relationships, demonstrating that similar phage susceptibility patterns may occur across a broad phylogenetic spectrum and with large physiological differences among Vibrio communities.

Subsequently, culture experiments were conducted with two V. anguillarum strains (BA35 and PF430-3) and their corresponding phages (ΦH20 and KVP40), representing strong lytic potential initially followed by rapid regrowth of phage-resistant and phage-tolerant subpopulations, which is an obstacle to successful phage therapy. Different bacteriophage resistance/tolerance mechanisms underlined by these two specific phage-host interactions were not revealed in this study.

Paper II Vibriophages differentially influence biofilm formation by Vibrio anguillarum strains

In this study, we explored in vitro phage-host interactions in two V. anguillarum strains (BA35 and PF430-3) and their corresponding phages (ΦH20, Siphoviridae and KVP40, Myoviridae), respectively, during growth in the settings of micro-colonies, biofilms, as well as the free-living phase.

We sought to figure out the resistance/tolerance mechanisms responsible for the different phenomena between these two phage-host systems.

For strain BA35 and phage ΦH20, a strong phage control of the phage-sensitive population and subsequent selection for the phage-resistant mutants with fitness cost, implicates a co-evolutionary arms race may exist between bacterial hosts and their predators. This then serves as a potential mechanism to promote diversity of resistant subpopulations and phage ΦH20 through stable co-evolution and mutations.

For strain PF430-3 and phage KVP40, addition of phage KVP40 to strain PF430-3 cultures resulted in an increase in the levels of biofilm/micro-colony/aggregate formation, especially, during the initial stage. By creating physical spatial refuge between host and parasite, the phage can potentially allow long-term coexistence between strain PF430-3 and phage KVP40. In addition, we were unable to isolate any single colonies with full and permanent resistance to phage KVP40. Hence, it may be speculated that the development of this transient protection is due to the fact that phage KVP40 infects more than 8 Vibrio species using phage receptor OmpK, which is widely distributed in the Vibrio community. Thus, resistance mutations are either very rare or have significantly negative or even lethal effects on host fitness. In that case, development of alternative strategies to genetic mutations in the OmpK receptor would be of strong selective advantage, perhaps contributing to the evolution of the protection mechanism.

Overall, the specific mechanisms of phage resistance/tolerance were not identified in this study between these two phage-host systems, and further studies are needed to continue to investigate the mechanism of resistance/tolerance towards these two phages. Taken together, these data demonstrated highly variable phage protection mechanisms among Vibrio communities, emphasizing the challenges of using phages to control vibriosis in aquaculture, and adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.

Paper III Quorum Sensing determines the choice of anti-phage defense strategy in Vibrio anguillarum

Since phage and host have a long co-existence and co-evolutionary development, it is generally believed that bacteria can effectively defend against phage predation by using various means and with limited fitness cost to cope with further selection pressure. Alternating different protection strategies at different stages perhaps lessens the metabolic burden, facilitating specific subpopulations to prevail in phage-host systems. As bacterial densities vary overtime from sparsely populated environments to highly dense bacterial populations, phage predation pressure is not constant. By altering their anti-phage activities upon population density (phase-dependent defense), bacteria could actively reduce their susceptibility to infection during growth at high cell densities. Vibriophage KVP40 is known to cause infection through the universal outer membrane protein K (OmpK) and has previously been shown to infect more than 8 Vibrio species; by using spatial refuge and a quorum sensing-mediated receptor down-regulation would decrease the metabolic burden in these Vibrio communities.

In this study, we revealed the roles of spatial refuge and cell-cell signaling in shaping phage-host interactions. Two V. anguillarum PF430-3 QS mutants were constructed, ΔvanT-locked in low cell density and ΔvanO-locked in high cell density, allowing us to explore the role of QS for protection against phage infection. Specifically, ΔvanT became more susceptible to phage KVP40 by enhancing biofilm formation, and thus creating spatial refuge, providing protection to the host against phage infection. In contrast, ΔvanO became more resistant by down-regulating the phage receptor OmpK to reduce its susceptibility to phage infection with no bacterial aggregates. Further, wild-type strain culture experiments showed a strong negative correlation between AHLs production in the supernatant and phage receptor ompK gene expression, which was consistent with previous results. The data presented here, therefore, suggest that VanO negatively regulates the expression of VanT, which in turn directly or indirectly represses the expression of essential phage receptor OmpK (Figure 11).

Figure 11. Models for the mechanism and function of QS regulation and biofilm formation and phage receptor ompK expression in V. anguillarum. Solid arrows represent positive effects, while solid T bars represent negative effects.

Future perspectives The studies presented in this Ph.D. dissertation provide new insights into phage-host interactions of the important fish pathogen V. anguillarum and their vibriophages, shedding light on the relationships between phage infection pressure, biofilm development, quorum sensing and bacteriophage resistance/tolerance mechanisms in V. anguillarum. However, there are lots of unsolved issues in this study, which raise questions regarding future experiments.

Paper I Thirty seven Vibrio strains were collected with considerable temporal (20 years) and geographical (9 countries) differences in their origins of isolation, which provides us with knowledge on spatial and temporal distributions of the pathogen which may prove useful when assessing global outbreaks of vibriosis. However, these strains are still insufficiently characterized. For instance, phage susceptibility patterns of the isolates did not show any similarities to the physiological relationships obtained from BIOLOG GN2 and 16S rRNA profiles, and thus further studies linking phage susceptibility to phenotypic and genotypic characteristics would be relevant.

First, by selecting vibriophage resistant mutants among the potential hosts, we could identify cellular components that are involved in the bacterial receptor for the specific phages, as previously shown in Davidson’s study in B. anthracis (gamma) phage γ receptor GamR (190). Further studies will be focused on potential phage receptor identification and will determine whether there is any correlation between phage susceptibilities and phage receptor types.

Secondly, one of the most important issues in phage therapy is the ability to obtain phages capable of infecting the causal pathogen; hence, the isolation step is a critical element. The isolation of vibriophages is considered relatively difficult, and there is a need for new and more efficient isolation methods.

Thirdly, physical and chemical factors, such as temperature, acidity, and ions have shown to affect phage persistence (191). A better understanding of storage and stability of phage stock, i.e. mechanisms and rates of phage decay, are also essential for a successful future phage therapy, not only for those interested in pharmaceutical and agricultural applications, but also for laboratory phage studies.

Lastly, most bacterial pathogens contain prophages or phage remnants integrated into bacterial chromosomes (192). As these prophages may carry virulence genes, it is essential to obtain a better understanding of the content and genetic properties of prophages integrated in V. anguillarum strains. During the lytic phage cycle, prophage induction can be triggered, which makes lytic phage stock

39 contaminated with the induced prophages that were previously resident in the host strains. Thereby, the data we obtained from the host range assay could be related to the prophage from the host of the lytic phage. The argument of prophage here suggests that phage host range can be optimized by using prophage free hosts for lytic phage proliferation in the future. We are currently whole genome sequencing our full collection of V. anguillarum and vibriophages, which will allow such a detailed analysis of prophage contents.

Paper II The studies from Paper II demonstrated that two closely related V. anguillarum strains BA35 and PF430-3 displayed highly variable phage protection mechanisms, during growth in the settings of micro-colonies, biofilms, as well as the free-living phase.

In general, biofilm stability is closely correlated with EPS production (87), and we hypothesized that EPS production in strain PF430-3 contributed to the formation of the characteristic micro-colonies, which protected the strain PF430-3 from phage infection by creating spatial refuge. Therefore, further experiments on EPS quantification and identification remain to be carried out. Previous studies on P. aeruginosa have shown fluorescently labelled lectins could be used in combination with epifluorescence microscopy, allowing the visualization and characterization of carbohydrate-containing EPS in biofilm matrices (78). This could lead us to the next step in determining and quantifying EPS production. Moreover, vps in-frame deletion mutants could be constructed to test whether phage-induced biofilm/aggregate formation is mediated by EPS production at the genetic level. Additionally, to better understand the genetic regulatory network, bacterial transcriptomes could provide us with the bacterial strain's immediate genetic response to phage exposure, enabling an assessment of the transcriptional pattern, such as genes involved in polysaccharide production and flagella assembly (193). Furthermore, it is relevant to determine whether phage KVP40-induced aggregates are related to host strain PF430-3 or to the phage KVP40 and could be tested by combining phage KVP40 with susceptible V. anguillarum strains which have been characterized in Paper I.

As previously mentioned, isolating vibriophages is tedious and isolation of a natural vibriophage which is both specific to a certain host and expresses a relevant depolymerase is thus likely very low. Therefore, using synthetic biology to engineer our broad-host-range phage KVP40 to express the most effective EPS- degrading enzymes specific to the Vibrio targeting biofilms may be a way forward, as has been successfully conducted in an E. coli study (194). This would allow us to develop a diverse library of biofilm-dispersing phages rather than rely on isolating vibriophages from the environment.

Albeit the specific mechanism of resistance of strain BA35 was not identified in Paper II, the observation that a large fraction of phage resistant mutants of BA35 were found to be either non-motile or having severely impaired motility, which implies that flagella may play an important role in the infection of V. anguillarum strain BA35 by phage ΦH20. This was consistent with recent observations that the flagellum was involved in the initial infection of C. crescentus and S. enterica serovar typhimurium phages (148, 195). Future work on whether phage-resistant mutants BA35 have mutations or down-regulations in genes involved in both flagella and potential phage receptors should be carried out. Lastly, time-shift experiments

(196) will be conducted to gain new insights into the underlying processes of antagonistic co-evolution between BA35 and phage ΦH20, if any.

Paper III It is well known that mutations to phage resistance frequently have maladaptive pleiotropic effects (197, 198). It should be advantageous to avoid maintaining constantly elevated anti-phage defenses, if bacteria would benefit from subjecting their anti-phage defenses to QS-control. Thus, future studies will focus on whether QS-mediated OMP down-regulation gives rise to a reduction in relative fitness to elucidate the regulatory pathway connecting QS-signaling to OMP down-regulation in V. anguillarum. Then it should be possible to construct mutants, in which regulation of the relevant OMPs is uncoupled from QS, without affecting the remainder of the QS regulon. The relative fitness of these mutants relative to the otherwise isogenic parent strains will be measured in the absence of vibriophage.

Ultimately, to fully characterize the regulatory pathway underlying QS-mediated OmpK down-regulation mechanisms, which have not been identified in Paper III, we will determine whether QS transcription factor VanT binds directly to the promoter of putative targets, such as ompK and vps. Specifically, we will set up a VanT Electrophoretic mobility shift assay (EMSA) experiment to test our hypothesis. Using this approach would allow us to potentially expand the currently modest number of described targets mediated by VanT in the literature.

Finally, the evidence that QS regulates phage receptor expression may potentially be used actively by quenching QS signaling, hence preventing receptor down-regulation. However, in the case of V. cholerae, at low cell densities, when the concentrations of the extracellular AIs are low, the expressions of virulence factor toxin-co-regulated pilus, CT and biofilm formation are activated; at high cell densities, the accumulation of two QS autoinducers (CAI-1 and AI-2) repress the expression of virulence factors and biofilm formation (43, 104). If the close-related V. anguillarum strains behave similarly, it would be problematic to synergize phage therapy and quorum quenching to prevent vibriosis infection. Further, it would be interesting to target potential phage receptors which are not regulated by QS signaling molecules, and by combining specific phages and QS molecules (CAI-1 and AI-2) may offer a synergistic antimicrobial effect to prevent vibriosis infection in aquaculture industries.

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Accompanying papers

Vibriophages and Their Interactions with the Fish Pathogen Vibrio anguillarum

Demeng Tan, Lone Gram and Mathias Middelboe Appl. Environ. Microbiol. 2014, 80(10):3128. DOI: 10.1128/AEM.03544-13. Published Ahead of Print 7 March 2014. Downloaded from

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Demeng Tan,a Lone Gram,b Mathias Middelboea Marine Biological Section, University of Copenhagen, Helsingør, Denmarka; Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmarkb

Vibrio anguillarum is an important pathogen in aquaculture, responsible for the disease vibriosis in many ﬁsh and invertebrate Downloaded from species. Disease control by antibiotics is a concern due to potential development and spread of antibiotic resistance. The use of bacteriophages to control the pathogen may offer a non-antibiotic-based approach to reduce vibriosis. A detailed understanding of the phage-host interaction is needed to evaluate the potential of phages to control the pathogen. In this study, we examined the diversity and interactions of 11 vibriophages, 24 V. anguillarum strains, and 13 Vibrio species strains. Together, the host ranges of the 11 phages covered all of the tested 37 Vibrio sp. host strains, which represented considerable temporal (20 years) and geographical (9 countries) differences in their origins of isolation. Thus, despite the occurrence of unique susceptibility patterns of the individual host isolates, key phenotypic properties related to phage susceptibility are distributed worldwide and maintained in the global Vibrio community for decades. The phage susceptibility pattern of the isolates did not show any rela- http://aem.asm.org/ tion to the physiological relationships obtained from Biolog GN2 proﬁles, demonstrating that similar phage susceptibility patterns occur across broad phylogenetic and physiological differences in Vibrio strains. Subsequent culture experiments with two phages and two V. anguillarum hosts demonstrated an initial strong lytic potential of the phages. However, rapid regrowth of both phage-resistant and phage-sensitive cells following the initial lysis suggested that several mechanisms of protection against phage infection had developed in the host populations.

ibriosis is one of the most prevalent and devastating diseases cases, reduce fish mortality. Therefore, there is growing evidence on April 29, 2014 by Copenhagen University Library Vin marine aquaculture, causing substantial mortality and that phages can be applied to control diseases in aquaculture. economic losses in both fish and shellfish cultures worldwide (1). The use of bacteriophages to control specific pathogens is com- The disease is caused by the marine pathogen Vibrio (Listonella) plicated by the phenotypic and genotypic complexity of both anguillarum, which infects Ͼ50 different species of fish and shell- phage and host communities, which may consist of multiple co- fish, with severe implications for the marine fish-rearing industry existing strains with different host range and susceptibility pat- (1, 2). Vaccines against vibriosis have been developed and have terns (14, 15). In order to establish efficient phage control of a been widely successful in grown fish (3). However, the larval stage diverse host community, a large and well-characterized collection is especially vulnerable to V. anguillarum infections, as the im- of phages is required which, in combination, covers a broad range mune system is not fully developed and vaccines are not effective. of hosts. Traditionally, antibiotics have been widely used in antimicrobial In this study, a collection of Vibrio sp. strains and vibriophages prophylaxis and treatment of vibriosis in aquaculture, despite was isolated from Danish aquaculture farms and characterized concern about the development and dispersal of antibiotic resis- along with a collection of 20 V. anguillarum strains and one lytic V. tance in the pathogen community. Tetracycline and quinolones anguillarum phage of different temporal and geographic origins. are still the first drugs of choice, and multiple cases of antibiotic Patterns in phage and host phenotype, genotype, morphology, resistance have been reported for V. anguillarum in aquaculture and phage-host ranges and interactions were studied to establish a environments (4, 5). Therefore, there is a need for the develop- collection of well-characterized vibriophages and to evaluate their ment of alternative methods to control and prevent V. anguilla- potential for controlling the pathogen. rum infection in aquaculture. Bacteriophages are natural and abundant biological entities MATERIALS AND METHODS in the marine environment, playing important roles in, e.g., struc- Bacterial strains. The collection consisted of 20 V. anguillarum strains turing bacterial diversity and succession in the ocean and promot- provided by Technical University of Denmark (DTU, Denmark); V. ing biogeochemical element cycling, and being key drivers of anguillarum strain PF430-3, derived from strain PF4, which was originally horizontal gene transfer (6). The strong lytic potential of bacteriophages against specific bacterial hosts has led to an increasing interest in the therapeutic use of lytic phages to control pathogenic Received 29 October 2013 Accepted 5 March 2014 bacterial infections in aquaculture. There has been a growing Published ahead of print 7 March 2014 number of studies on phage therapy focusing on a variety of fish Editor: K. E. Wommack pathogens, including Aeromonas salmonicida in brook trout, Yer- Address correspondence to Mathias Middelboe, [email protected]. sinia ruckeri in salmon, Lactococcus garvieae in yellowtail, Pseu- Supplemental material for this article may be found at http://dx.doi.org/10.1128 domonas plecoglossicida in ayu, Flavobacterium psychrophilum in /AEM.03544-13. rainbow trout, V. harveyi in shrimp, and V. anguillarum in salmon Copyright © 2014, American Society for Microbiology. All Rights Reserved. (7–14). These studies have demonstrated the potential of specific doi:10.1128/AEM.03544-13 phages to significantly control pathogen density and, in some

3128 aem.asm.org Applied and Environmental Microbiology p. 3128–3140 May 2014 Volume 80 Number 10 Isolation and Characterization of Vibriophages

TABLE 1 The 37 Vibrio sp. strains used in the study API 20NE BIOLOG 16S phylogenetic Reference Numbera Strain Source BLAST result (accession no.) Homology (%) code GN2c groupd or source 1* 87-9-117 Rainbow trout, Finland Vibrio anguillarum (CP006699.1) 96 7577745 1 1 24, 49 2* BA35 Sockeye salmon, USA Vibrio anguillarum (CP006699.1) 95 7577745 1 1 49 3* 51/82/2 Rainbow trout, Germany Vibrio anguillarum (CP006699.1) 97 7577745 1 1 24, 49 4* T265 Salmon, UK Vibrio anguillarum (CP006699.1) 97 7577745 1 1 24, 49 5* 91-8-178 Turbot, Norway Vibrio anguillarum (CP006699.1) 97 7577745 1 1 24, 49 6* 87-9-116 Rainbow trout, Finland Vibrio anguillarum (CP006699.1) 97 7577745 1 1 24, 49 7* 601/90 Sea bass, Italy Vibrio anguillarum (CP006699.1) 96 7577745 1 1 This study 8* 178/90 Sea bass, Italy Vibrio anguillarum (CP006699.1) 96 7577745 1 1 24, 49 9* 261/91 Sea bass, Italy Vibrio anguillarum (CP006699.1) 97 7577645 1 1 24, 49 Downloaded from 10b PF430-3 Salmon, Chile Vibrio anguillarum (KF150778.1) 96 7477745 1 1 17

11* A023 Turbot, Spain Vibrio anguillarum (CP006699.1) 95 7577345 1 1 49 12* 4299 Unknown Vibrio anguillarum (KF150778.1) 96 5576744 1 1 50 13* LMG 12010 Unknown Vibrio anguillarum (CP006699.1) 95 7577344 1 1 49 14* 850610–1/6a Rainbow trout, Denmark Vibrio anguillarum (CP006699.1) 97 7577745 1 1 49 15* 90-11-287 Water sample, Denmark Vibrio anguillarum (CP006699.1) 96 7577745 1 1 24, 51 16* 91-7-154 Trout, Denmark Vibrio anguillarum (CP006699.1) 97 7577745 1 1 49 17* S2 2/9 Rainbow trout, Denmark Vibrio anguillarum (FJ824662.1) 98 7577745 1 1 49 http://aem.asm.org/ 18* 90-11-286 Rainbow trout, Denmark Vibrio anguillarum (KF150778.1) 97 7576745 1 1 49 19* HWU53 Rainbow trout, Denmark Vibrio anguillarum (CP006699.1) 97 7576745 1 1 49 20* 6018/1 Rainbow trout, Denmark Vibrio anguillarum (CP006699.1) 97 7576745 1 1 49

21* 9014/8 Rainbow trout, Denmark Vibrio anguillarum (CP006699.1) 96 7577745 1 1 49 22 VA1 Salmon, Denmark Vibrio anguillarum (KF150778.1) 96 7577745 1 1 This study 23 VA2 Salmon, Denmark Vibrio anguillarum (KF150778.1) 95 7577745 1 1 This study 24 VA3 Salmon, Denmark Vibrio anguillarum (KF150778.1) 96 7576745 1 1 This study 25 VSP1 Salmon, Denmark Vibrio splendidus (KC884584.1) 96 7430044 2 2 This study 26 VSP2 Salmon, Denmark Vibrio cyclitrophicus (KC185413.1) 97 7520044 2 2 This study

27 VSP3 Salmon, Denmark Vibrio cyclitrophicus (KF488567.1) 97 7430044 2 2 This study on April 29, 2014 by Copenhagen University Library 28 VSP8 Denmark Vibrio cyclitrophicus (KF488567.1) 95 7430044 2 2 This study 29 VSP10 Salmon, Denmark Vibrio cyclitrophicus (KC185413.1) 97 7430044 2 2 This study 30 VSP12 Denmark Vibrio cyclitrophicus (KF488567.1) 96 7430044 2 2 This study

31 VSP4 Sole, Denmark Vibrio shilonii (JX999944.1) 95 7420044 2 3 This study 32 VSP5 Sole, Denmark Vibrio sp. (EU517619.1) 97 7476744 3 3 This study 33 VSP6 Denmark Vibrio sp. (FM957468.1) 96 7476744 3 3 This study 34 VSP7 Denmark Vibrio sp. (FM957468.1) 97 7476744 3 3 This study 35 VSP9 Denmark Vibrio sp. (KC178920.1) 97 7476744 3 3 This study 36 VSP11 Denmark Vibrio sp. (KC178920.1) 97 7476744 3 3 This study 37 VSP13 Denmark Vibrio sp. (FM957471.1) 97 7476744 3 3 This study a Strains marked by an asterisk were provided by Technical University of Denmark (DTU). b The strain PF430-3 was provided by the Hellenic Centre for Marine Research. c Group numbers according to the BIOLOG GN2 dendrogram. d Group numbers according to 16S rRNA phylogenetic tree (see Fig. S1 in the supplemental material).

␮ isolated in Chile (16) but was reisolated after long-term (2 weeks) cultur- (dNTPs), primers (12.5 M each), MgCl2 (25 mM), hot start polymerase, ing; and 16 Vibrio sp. strains recently isolated from Danish aquaculture and water. PCR amplification was carried out on a thermal cycler (Applied farms (Table 1). The 16 recent Danish isolates were obtained from 18 Biosystems) for 5 min at 95°C for denaturation, followed by 35 cycles of 45 water samples collected from two fish farms (Venøsund and Maximus, s at 95°C, 1 min at 51°C, and 2 min at 72°C and final extension at 72°C for Denmark) between May 2012 and August 2012. Aliquots of water samples 7 min. PCRs were verified using 1% agarose gels stained with ethidium (200 ml) were mixed with 200 ml 2ϫ LB medium (12106; MO-BIO) and bromide. PCR products were subsequently sequenced in both forward agitated at room temperature for 24 h. For isolation of Vibrio sp. strains, and reverse directions (Beijing Genomic Institute [BGI]). 100-␮l subsamples from serial dilutions were plated on thiosulfate citrate A phylogenetic analysis based on neighbor joining was performed on bile salt sucrose agar (TCBS; 221872; Difco) incubated at 30°C for 24 h. 16S rRNA gene sequences. The original sequences were manually cor- Single colonies were then isolated and subsequently pure cultured by re- rected, trimmed, and aligned using the BioEdit sequence alignment tool peated streaking on TCBS agar. These isolates were then further charac- (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Each of the compiled terized to determine if they were V. anguillarum (see below). All of the sequences were compared to the available databases using the basic local strains were kept at Ϫ80°C in LB medium with sterile glycerol (15%, alignment search tool (BLAST) to determine phylogenetic affiliations. vol/vol). Mega 5.1 software (http://www.megasoftware.net/) was used to align all 16S rRNA sequencing. Bacterial genomic DNA was extracted using a 37 sequences. Overhanging ends were removed to allow direct compari- commercial genomic DNA purification kit (Clontech). The 16S rRNA son of sequences with the same length (695 bp). The phylogenetic rela- gene was amplified by universal primers 27f (5=-AGAGTTTGATCCTGG tionship was bootstrapped 1,000 times, and the gaps were treated by pair- CTCAG-3=) and 1492r (5=-GGTTACCTTGTTACGACTT-3=). PCR was wise deletion. performed in a 24-␮l reaction mixture, which contained genomic DNA Phenotypic analysis of bacterial strains. Biochemical tests were per- template, 10ϫ PCR buffer, 2 mM deoxynucleoside triphosphates formed using an API 20NE system (bioMérieux) according to the instruc-

May 2014 Volume 80 Number 10 aem.asm.org 3129 Tan et al.

tions of the manufacturer. The inoculum was distributed into test strips provide a quantitative measure of the phage lytic potential, the efficiency of which were then incubated at 30°C for 48 h. Biochemical reactions were plating (EOP) for each phage on each phage-sensitive strain was determined read based on color development, translated into numerical codes, and by measurement of the number of PFU produced on individual hosts from a interpreted with the API 20NE database. given phage stock, as quantified by plaque assay (19). The sensitivity of the strains to the vibriostatic compound agent 0129 Purification of bacteriophage genome DNA. Aliquots (500 ␮l) of (2,4-diamino-6,7-diisopropylpteridine phosphate) (Oxoid) was deter- high-titer (ca. 109 PFU mlϪ1) bacteriophage stocks were further concen- mined by the disk diffusion method (17) at both 10 and 150 ␮g per disk trated by centrifugation (1,000 ϫ g, 25 min) in 30-kDa ultrafiltration with incubation for 24 h at 30°C. Amicon Microcon spin devices (Millipore), and then the membrane was Biolog GN2 MicroPlates (Biolog) containing 95 different carbon washed three times by 50 ␮l 100ϫ Tris-EDTA buffer (TE buffer; 10 mmol sources were used to test the ability of the strains to use different carbon Tris-Cl, 1 mmol EDTA, pH 8) (1,000 ϫ g for 18 min each). After the sources. Briefly, pure cultures of bacterial strains were grown on LB agar washing, the phages were eluted with 20 ␮l 100ϫ TE buffer by centrifu- Downloaded from plates for 24 h at 30°C and subsequently transferred to 0.9% saline buffer gation of the inverted membrane into a new tube (300 ϫ g for 5 min). To (9 g NaCl in 1 liter distilled water) and washed twice to remove medium denature phage proteins and release the phage DNA, the concentrated phages 8 (8,000 ϫ g for 10 min). From this bacterial suspension (ca. 10 CFU were heated to 70°C for 10 min and then cooled on ice for subsequent deter- Ϫ1 ␮ ml ), 100- l aliquots were inoculated into each well in duplicate plates mination of phage genome size. To confirm the concatemer phenomena of a and incubated in the dark at 30°C for 48 h. Data were analyzed based on number of phages (phage ⌽H2, ⌽H8, and ⌽H20), additional extraction of visual inspection of tetrazolium violet development as an indicator of DNA from these phages was performed using a QIAprep spin M13 kit metabolic activity. (Qiagen) according to the manufacturer’s instructions. Bacteriophage isolation. The water samples for bacterial isolation Phage genome size. The bacteriophage genome sizes were determined http://aem.asm.org/ were also used for propagation of phages for subsequent isolation and by pulsed-field gel electrophoresis (PFGE) using the CHEF-DRIII pulsed- purification of new vibriophages. Four different protocols were used (18). field electrophoresis system (Bio-Rad) (14). Phage DNA was run using 1% ␮ (i) A 100-ml water sample was sterile filtered (0.22 m; Millipore) and agarose gels in 0.5ϫ Tris-borate-EDTA (TBE) buffer (10ϫ TBE, 890 mM ϫ mixed with 100 ml 2 LB medium containing a mixture of 5 random Tris, 20 mM EDTA, 890 mM boric acid, pH 8.3) with two different in- Vibrio strains (Table 1) to stimulate proliferation of phages specific to the strument settings for small and large genome sizes. For the large genome added strains. After 24 h of incubation at room temperature, bacteria in sizes (Ͼ50 kb), switch times were ramped from 50 to 90 s for 24 h and a the lysate were killed by chloroform (20 ␮lmlϪ1), followed by centrifu- ϫ voltage of 6 V/cm, whereas switch times ranged from 0.5 to 5 s and a total gation (10,000 g, 10 min) to remove bacterial debris. (ii) One-hun- run time of 15 h for small genomes (Ͻ50 kb). An 8- to 48-kb molecular dred-ml unfiltered water samples were mixed with 100 ml 2ϫ LB broth size DNA marker and a Lambda DNA ladder (Bio-Rad) were used as and incubated at room temperature for 24 h to stimulate proliferation of on April 29, 2014 by Copenhagen University Library standards. The gels were stained with 0.01% (vol/vol) SYBR green I (In- phages specific to cooccurring Vibrio species host strains in the water vitrogen) in 0.5ϫ TBE for 1 h in the dark and then washed with distilled sample. Aliquots of enrichment samples (50 ml) were then treated with water for 30 min prior to visualization of the bands using the ChemiDoc chloroform and centrifuged as described above, and the supernatant was MP imaging system (Bio-Rad). mixed with individual Vibrio strains (Table 1) in a second round of en- Bacteriophage morphology by TEM. For transmission electron mi- richment as described above. (iii) Five-ml water samples were filtered croscopy (TEM) analysis of phage isolates, the phage stocks were further (0.22 ␮m pore size), and the filtrate was diluted and used directly for concentrated by ultracentrifugation (100,000 ϫ g, 90 min, 20°C; 70.1Ti; plaque assay (19) without previous enrichment. (iv) Two-hundred-ml Beckman), followed by resuspension of the pellet in 100 ␮l SM buffer. water samples were filtered (0.22 ␮m), and phages were then concentrated Formvar-carbon-coated copper grids were placed on top of the phage using 30-kDa centrifugation filters (Amicon ultra 15 protein; Millipore) concentrates, allowing the phages to adsorb for 20 min. The grids were and stored at 4°C until further use. ␮ ␮ The presence of Vibrio-specific phages in the water samples described then stained with 10 l 2% sodium phosphotungstate (pH 7.4; 0.02- m- above was tested against all 37 Vibrio sp. strains by spotting 5-␮l samples pore-size filters) for 2 min. Excess stain was removed by touching the edge on lawns of individual strains using the double-layer agar method (18). In of the grids with filter paper, and the grids were washed with several drops the case of cell lysis in the spotted area, phages were scraped off the plate of distilled water and allowed to dry on the filter paper for 15 min. The and transferred to SM buffer (50 mM Tris-Cl, pH 7.5, 99 mM NaCl, 8 mM grids were observed with a JEM-2100 transmission electron microscope (JEOL) operated at 80 kV. MgSO4, 0.01% gelatin). These phage concentrates were then diluted, and the presence of phages was verified by plaque assay (19). For purification One-step growth curve. To characterize phage life cycle characteris- of individual phages, single plaques were isolated with a sterile pipet and tics, one-step growth experiments were performed for two of the phage transferred to SM buffer. This procedure was repeated at least three times isolates using marine broth (MB; 2216; Difco) at a low multiplicity of to ensure purification of individual phages. infection (MOI; i.e., the initial ratio of phage particles to host cells), fol- To obtain high-titer phage stocks, 5 ml SM buffer was added to plates lowing a protocol modified from that of Ellis and Delbrück (21). For ⌽ 8 Ϫ1 with confluent lysis, and the top agar was shredded with an inoculation phage H20, 1 ml of exponentially growing bacteria (10 CFU ml ) was 8 Ϫ1 loop. The mixture was transferred to a sterile tube and incubated on a mixed with 100 ␮l lytic phage stock (10 PFU ml ; MOI of 0.1) and shaker for at least2hatroom temperature to release phage particles and incubated at room temperature for 20 min to allow the phages to adsorb then centrifuged (10,000 ϫ g, 10 min, 4°C). The supernatant was filtered to the host according to procedures described by Hidaka and Tokushige through a 0.22-␮m membrane (Millipore), and the phage stock was (22). Bacteria were then pelleted (7,000 ϫ g, 10 min), and the nonad- stored at 4°C in the dark. In addition to the phage isolates obtained from sorbed phages in the supernatant were discarded. The pellet was then Danish aquaculture farms, a broad-host-range vibriophage (KVP40) (20) resuspended in 1 ml MB and diluted to an appropriate phage titer (104 Ϫ was kindly provided by the Hellenic Centre for Marine Research. PFU ml 1). Two 100-␮l subsamples of culture were collected at regular Phage host range and EOP. The host range of each phage isolate was intervals for 60 min for enumeration. In one subsample, free phages were determined using a spot assay against all of the available Vibrio sp. strains immediately quantified by plaque assay (18). The other subsample was (Table 1). Aliquots of 5 ␮l phage stock (phage titer between 106 and 109 pretreated with 10 ␮l chloroform to measure the release of intracellular PFU mlϪ1) were spotted on lawns of the potential host strain prepared as phages and then centrifuged at 10,000 ϫ g for 1 min prior to phage enu- 4 ml top agar inoculated with 500 ␮l bacterial cultures in mid-log phase (ca. meration. For phage KVP40, the same procedure was applied, except that 108 CFU mlϪ1). After 24 h of incubation at 30°C, the spots were assessed by for this phage, the stock was diluted to 107 PFU mlϪ1, reducing the MOI the clarity of plaques and scored as transparent, turbid, or no inhibition. To to 0.01.

3130 aem.asm.org Applied and Environmental Microbiology Isolation and Characterization of Vibriophages

Susceptibility of V. anguillarum to phage infection at different acid, whereas the remaining 11 biochemical tests gave various re- MOIs. The lytic potentials of two selected phages, ⌽H20 and KVP40, sults (data not shown). against V. anguillarum were examined at MOIs of 0.1, 1, and 10 in liquid Biolog ﬁngerprint. The phenotypic diversity of and relation- MB cultures during infection of V. anguillarum BA35 and PF430-3, re- 7 9 Ϫ1 ship between the 37 strains were explored by studying the differ- spectively. Serial dilutions of the phage stocks (ca. 10 to 10 PFU ml ) ences in carbon resource utilization, which showed a clustering of were inoculated in duplicate 50-ml mid-log-phase cultures of host bacteria in MB at a density of ϳ107 CFU mlϪ1. For each experiment, duplicate the strains into three primary phenotypic groups (Fig. 1). Cluster control cultures without phage addition were also established. The effects 1 contained 24 strains, including all of the strains in the DTU of phage lysis on host cell density were monitored by regular measure- collection, the PF430-3 strain, and three of the Danish isolates

ments of the optical density at 600 nm (OD600) during the 24-h incuba- (VA1, VA2, and VA3), whereas the remaining 13 Danish isolates tions. Phage abundance was quantified in one of the experiments (MOI, were grouped into clusters 2 and 3 (Fig. 1). Overall, the number of Downloaded from 0.1) from the number of PFU obtained on lawns of host strains by plaque substrates used varied from 54 (strain BA35) to 22 (strain VSP10) assay (19) periodically over 24 h. At various time points, individual colo- of the 95 tested substrates, with the highest number of available nies were isolated from the cultures on TCBS plates and subsequently substrates found in cluster 1 (33 to 54) and the lowest in cluster 2 purified through 2 to 3 rounds of reisolation of single colonies to determine changes in phage susceptibility in the host community during incu- (22 to 38). All of the 22 substrates used by strain VSP10 could also bation. be used by all other strains, suggesting that the utilization of these Statistical analysis. For all of the dendrograms, UPGMA (un- 22 substrates was a fundamental property of Vibrio strains. weighted-pair group method with arithmetic mean) construction exper- Bacteriophage enrichment and isolation. Ten different bac- iments and Dice coefficients were calculated between pairs of each strain teriophage isolates (Table 2) were obtained from Danish waters http://aem.asm.org/ or phage and grouped by UPGMA and 100 bootstrap replicates using the using different enrichment protocols. Phages ⌽H20, ⌽H8, dendrogram construction utility DendroUPGMA (Biochemistry and ⌽S4-7, and ⌽S4-18 were isolated by following protocol I, using a Biotechnology Department, Rovira i Virgili University, Tarragona, Spain) mixture of Vibrio sp. strains as hosts for enrichment, whereas (http://genomes.urv.cat/UPGMA/index.php). phages ⌽H1, ⌽H2, ⌽H4, ⌽H5, ⌽H7, and ⌽2E-1 were isolated using a single Vibrio sp. strain for enrichment (protocol II). RESULTS Phage genome sizes, as determined by PFGE, showed large Isolation of Vibrio strains. A total of 16 Vibrio sp. strains isolated differences among the 11 phages and covered a 20-fold range,

from Danish aquaculture were susceptible to phage KVP40; there- from 11 kb to 244 kb (Table 2). An interesting phenomenon was on April 29, 2014 by Copenhagen University Library fore, these 16 strains were tentatively classified as closely related the systematic observation of several distinct bands appearing on Vibrio sp. strains. Of the total of 37 strains examined, 35 were the PFGE gels for phage ⌽H20, ⌽H8, and ⌽H2 despite up to 5 ␮ sensitive to the vibriostatic agent O129 at both 10 and 150 g, rounds of phage purification from a single plaque. For example, whereas two isolates (VSP8 and VSP9) were resistant to O129 at ⌽ ␮ PFGE analysis of phage H20 revealed seven bands, ranging from concentrations of up to 150 g. The 37 Vibrio sp. strains used in 50 to 291 kb, with each band size increasing by 50 kb, and phage this study covered a considerable spatial variability, with isolates ⌽H8, for which two bands of 50 kb and 100 kb, respectively, were originating from the Mediterranean Sea (Spain and Italy), the Bal- observed (Fig. 2; also see Fig. S2 in the supplemental material). tic Sea (Finland and Germany), the North Sea (Norway and Host range and EOP. The host range and lytic potential of the United Kingdom), the Atlantic Ocean (United States), the south 11 vibriophages were tested against 24 V. anguillarum and 13 Pacific Ocean (Chile), and inner Danish waters; the range in time Vibrio sp. strains by spotting 5 ␮l of a serially diluted phage stock of isolation covered ϳ20 years (23). onto top agar containing the potential host strain. Together, the Identification of Vibrio strains. Based on 16S rRNA gene sim- collection of phages was able to infect all 37 strains; however, there ilarity, 24 of the isolates were presumptively identified as V. anguillarum according to NCBI BLAST analysis (NCBI BLAST; were large differences in host range pattern and lytic efficiency among the phages (Fig. 3). Six phages (⌽H2, ⌽H8, ⌽H20, ⌽S4-7, http://www.ncbi.nlm.nih.gov/BLAST/)(Table 1), including the ⌽ ⌽ entire DTU collection, the Chilean strain (PF430-3), and 3 Danish S4-18, and 2E-1) exhibited a narrow host range, infecting only water isolates (VA1, VA2, and VA3), whereas the remaining 13 Dan- the host used for its isolation and perhaps one or two additional ⌽ ⌽ ⌽ ⌽ ish isolates yielded identification as V. cyclitrophicus, V. slendidus, V. strains. Phages H1, H7, H4, H5, and KVP40, on the other shilonii, and Vibrio spp. (Table 1). A phylogenetic tree based on the hand, were characterized by broader host ranges covering both 16S rRNA gene sequence analysis showed three main groups: local host isolates and isolates obtained from geographically dis- group 1 (V. anguillarum), containing 20 DTU strains, strain tant locations. Conversion of the phage host range pattern into a PF430-3, and three Danish V. anguillarum isolates (VA1, VA2, dendrogram using the Dice coefficients comparing algorithm and VA3); group 2 (V. splendidus and V. cyclitrophicus), contain- showed some distinct clusters of the phages and bacterial strains ing six Danish water isolates (VSP1, VSP2, VSP3, VSP8, VSP10, (Fig. 3). Generally, phages with the same genome size had similar and VSP12); and group 3 (V. shilonii and Vibrio spp.), containing host ranges, for example, the ⌽H1 and ⌽H7 pair and the ⌽H4 and the remaining strains (VSP4, VSP5, VSP6, VSP7, VSP9, VSP11, ⌽H5 pair, which had pairwise identical genome sizes and also and VSP13) (see Fig. S1 in the supplemental material). clustered together according to host range (Fig. 3). Interestingly, API 20NE fingerprinting. Eleven different identities (Table 1) the phages with the largest genomes, i.e., ⌽H1, ⌽H7 (194 kb), and were generated from the 37 strains using API 20NE test. For all of KVP40 (244 kb), also had the largest host range, infecting many of the strains, positive results were obtained for potassium nitrate, the same hosts, despite the fact that they were isolated from Dan- D-glucose, esculin ferric citrate, 4-nitrophenyl ␤-D-galactopyra- ish (⌽H1 and ⌽H7) and Japanese (KVP40) locations (24). Phage noside, malic acid, and oxidase reactions, and negative reactions susceptibility pattern, on the other hand, did not show obvious were found for urea, capric acid, adipic acid, and phenylacetic connection to the other genotypic or phenotypic relationships.

May 2014 Volume 80 Number 10 aem.asm.org 3131 Tan et al. Downloaded from http://aem.asm.org/ on April 29, 2014 by Copenhagen University Library

FIG 1 Phenotype dendrogram derived by UPGMA cluster analysis of all 37 strains in the Biolog GN2 proﬁles. The data were scored and compared by using Dice coefﬁcient algorithm. The scale bar indicates the dissimilarity.

Phage morphology. Phage morphology was examined by es: Siphoviridae (⌽H8 and ⌽H20), with ﬂexible tails, Podoviridae TEM for all 11 phages (Table 2 and Fig. 4). According to the (⌽H2, ⌽2E-1, and ⌽S4-18), with short tails, and Myoviridae International Committee on Taxonomy of Viruses (ICTV), three (⌽H1, ⌽H4, ⌽H5, ⌽H7, ⌽S4-7, and KVP40), possessing an ico- morphological groups of phages were represented by the 11 phag- sahedral head and contractile tails with tail ﬁbers and relatively

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TABLE 2 Method of isolation, morphological characteristics, and genome size of vibriophages isolated from Danish aquaculture farms and phage KVP40 (Japan) Genome Head diam Head length Tail diam Tail length Reference Family Phage Isolation protocola size (kb) Host (nm) (nm) (nm) (nm) or source Myoviridae KVP40 Unknown 242 PF430-3 76 120 30 120 32 Myoviridae ⌽H1 II 194 4299 107 129 19 233 This study Myoviridae ⌽H7 II 194 90-11-286 117 129 22 226 This study Myoviridae ⌽S4-7 I 145 VSP10 81 80 26 58 This study Myoviridae ⌽H4 II 95 90-11-287 72 80 22 229 This study Myoviridae ⌽H5 II 95 51/82/2 63 77 24 222 This study Downloaded from Siphoviridae ⌽H8 I 50 T265 114 127 19 237 This study Siphoviridae ⌽H20 I 50 BA35 54 65 12 144 This study Podoviridae ⌽S4-18 I 45 VA1 63 64 12 13 This study Podoviridae ⌽2E-1 II 42 VSP8 53 46 8 10 This study Podoviridae ⌽H2 II 11 A023 89 82 15 32 This study a I, phage isolation method using a single enrichment step with a mixture of potential host strains; II, phage isolation method using two steps of enrichments with a single strain. For further information, see Materials and Methods. http://aem.asm.org/ large genomes (25)(Fig. 4 and Table 2). Interestingly, some the two phages, with a strong dependence on the initial MOI for vibriophages with different genome sizes, e.g., ⌽H2 and ⌽S4-18, phage control of the host population (Fig. 5). In batch cultures at belong to the same family. an MOI of 0.01, both phage KVP40 and ⌽H20 showed rapid Phage-host interactions. Life cycle characteristics were deter- propagation during the first 10 h of incubation, resulting in phage mined for phages ⌽H20 and KVP40 from one-step growth curves titers stabilizing around 1010 and 109 PFU mlϪ1, respectively (Fig. during incubation with their host strains BA35 and PF430-3, re- 5). The host growth rates in the control cultures were 0.241 Ϯ spectively. Despite different morphologies and genome sizes, 0.002 hϪ1 and 0.231 Ϯ 0.007 hϪ1 for PF430-3 and BA35, respec- phages ⌽H20 and KVP40 had quite similar burst sizes (70 and 80 tively, and both strains reached stationary phase after 10 h. In the PFU per cell, respectively) and identical eclipse (20 min) and la- phage-amended cultures, phages were able to control growth of on April 29, 2014 by Copenhagen University Library tent periods (25 min). the host for 5 to 6 h followed by regrowth of the host population. The parallel long-term (24 h) infection experiments with Accordingly, decreasing the initial MOI resulted in a delayed phages ⌽H20 and KVP40 and the hosts BA35 and PF430-3, re- and less pronounced decrease in the OD600 (Fig. 6). Regrowth spectively, at different MOIs also showed similar lytic potential of occurred in all cultures, and after 20 h the OD600 stabilized at 0.6 to 0.7, corresponding to 65 to 75% of the final OD600 in the control cultures. In addition to the OD600 measurements, bacterial isolates were obtained during the incubations for further analysis of changes in phage susceptibility in the host population (Fig. 6). In the BA35-plus-⌽H20 cultures, the occurrence of the bacteriophage-resistant isolates varied considerably over time and between treatments. According to the spot test results, the emergence of the bacteriophage-resistant bacteria was faster at high than at low MOI. Within the first hour after incubation, resistant strains were isolated at an MOI of 10, whereas resistant phenotypes were not detected at MOIs of 1 and 0.1. At high MOI, only resistant isolates were obtained after7hofincubation. At the lower MOIs, resistant isolates dominated during the regrowth phase (7 h). However, partially sensitive bacteria (i.e., reduced sensitivity compared to the sensitive wild-type strain) were isolated from 8 h onwards and constituted 40 to 50% of the isolates after 24 h. All of the resistant and partially sensitive strains isolated from the phage-amended BA35 culture after 24 h formed smaller colonies with lower growth rates on TCBS plates than those seen in the control cultures without phages. For the partially sensitive and resistant BA35-derived isolates, a second round of purification and subsequent susceptibility test were carried out. Interest- ingly, all of the partially sensitive strains returned to their sensitive phenotype after purification in the absence of phages, whereas all of the completely resistant isolates remained resistant. In the parallel experiment with V. anguillarum strain PF430-3 FIG 2 PFGE analysis of selected phage genomes with concatemers (⌽⌯2, ⌽⌯8, and ⌽⌯20), with switch times ranging from 0.5 to 5 s and a total run and phage KVP40, no completely resistant isolates were obtained time of 15 h. from any of the treatments during the incubation, and the colony

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FIG 3 Host ranges of 11 phages (columns) against 37 Vibrio sp. strains (rows). Black boxes indicate clear plaques, gray boxes indicate turbid plaques, and white boxes indicate no infection. Numbers express the efficiency of plating, i.e., the fraction of infectivity of the phage stock on a given host compared to the infectivity on the original host of isolation (isolation efficiency of the host was 100%). Dendrograms were derived by UPGMA cluster analysis. The data were scored and compared by using the Dice coefficient algorithm. The scale bar indicates the dissimilarity. morphology did not change over time. Instead, all of the isolates vibriophages and 37 Vibrio sp. strains (Table 1 and 2), covering were sensitive or partially sensitive strains, and after 8 h all ob- considerable temporal and spatial variability. Further, two V. an- tained isolates were fully sensitive to KVP40. In contrast to the guillarum strains, BA35 and PF430-3, and the specific phages control cultures, visible biofilm was formed in the KVP40- ⌽H20 and KVP40 were selected for more detailed studies of phage amended cultures, and some isolates formed aggregates when lytic potential and phage-host interactions. subsequently grown in liquid culture. For the aggregating and Diversity of V. anguillarum strains. The V. anguillarum partially sensitive PF430-3-derived isolates, a second and third strains examined in the present study (Table 1) showed close sim- round of purification and subsequent spot assay showed that all ilarity according to 16S rRNA gene sequences irrespective of time strains returned to the original phenotype and were fully sensitive or place of isolation. Thirty-seven of the isolates which grouped to phage KVP40. together covered a period of isolations of Ͼ20 years and a wide range of geographical locations (Chile, Spain, Italy, Germany, DISCUSSION United Kingdom, Norway, Finland, Denmark, and the United Successful application of phage therapy in the treatment of vibri- States) and fish species (turbot, trout, sea bass, and salmon). This osis requires detailed knowledge of the diversity and distribution suggests that the 16S rRNA gene phylogeny of V. anguillarum is of the phage susceptibility properties of Vibrio sp. pathogens as- highly uniform and stable in time and space. Thus, the method is sociated with vibriosis as well as a collection of well-characterized unable to discriminate between potentially pathogenic and non- phages that covers host diversity. In this study, we characterized 11 pathogenic V. anguillarum strains or resolve differences in V.

3134 aem.asm.org Applied and Environmental Microbiology Isolation and Characterization of Vibriophages Downloaded from

FIG 4 TEM pictures of selected phages. Phages were stained with 2% sodium phosphotungstate. Scale bar, 200 nm. http://aem.asm.org/ anguillarum communities across spatial and temporal scales. Sim- from rainbow trout farms and salmon farms in Germany and the ilarly, genetic fingerprinting methods (enterobacterial repetitive- United States, respectively, both were infected by phages ⌽H4 and element intergenic consensus sequence [ERIC] PCR) showed that ⌽H5, isolated from a Danish turbot farm 20 years later. This dem- Vibrio sp. isolates from coastal British Columbia did not group onstrates that despite the occurrence of unique susceptibility pat- according to geography, suggesting genetic homogeneity among terns of individual isolates, key phenotypic properties related to the environmental strains (26). Previous studies have, however, phage susceptibility are distributed worldwide and maintained in revealed a large degree of microheterogeneity in 16S rRNA genes the global V. anguillarum community for decades. on April 29, 2014 by Copenhagen University Library within Vibrio sp. strains (27), limiting the use of 16S rRNA genes Phage isolation and diversity. The 10 vibriophages isolated for phylogenetic studies in Vibrionaceae. Higher taxonomic reso- from Danish fish farms all had unique host ranges when tested lution has been obtained using multilocus sequence analyses against the collection of 37 Vibrio strains. All three major morpho- (MLSA) of six protein-coding genes (28); however, this method logical families, Myoviridae, Podoviridae, and Siphoviridae, were also focuses on variations in housekeeping genes and is applicable represented, and the phages ranged in genome size from 11 to 194 mainly for identification of Vibrio strains at the species level. kb, distributed in 6 distinct groups (Table 2). The phage isolates The V. anguillarum isolates clustered in distinct groups based showed large differences in host specificity, and together the Dan- on the Biolog GN2 profiles and differed from the other identified ish phages were able to infect and lyse 30 of the 37 strains, covering clusters of Vibrio strains. In contrast, the phage susceptibility pat- most of the phylogenetic, geographical, and temporal variation tern of the V. anguillarum isolates did not show any connection to that characterized the host isolates. This is interesting, as it sug- the species or physiological relationships obtained from Biolog gests that vibriophage infective properties are not restricted to profiles. In fact, phage susceptibility patterns did not even group local cooccurring host communities but rather widely distributed according to the species clusters defined for the 37 strains. This across large spatial and temporal scales, as also suggested by pre- suggests that phenotypic traits related to sensitivity to phages are vious observations of an ocean-scale distribution of genetically not resolved by the methods used in this study. This supports related vibriophages (31). previous studies of Cellulophaga baltica (29), V. parahaemolyticus The two broad-host-range phages isolated in this study (⌽H1 (15), F. psychrophilum (14), and Escherichia coli (30), which have and ⌽H7) both belonged to the Myoviridae family and had rela- all demonstrated a high degree of phenotypic diversity in host tively large genomes (194 kb). This is consistent with the host susceptibility at the strain level that was uncoupled from the over- range pattern observed for F. psychrophilum phages, where the all genetic relationships among the host isolates. Thus, our results large-genome phages (Ͼ90 kb) had substantially broader host confirm highly variable patterns in phage sensitivity even within ranges than small-genome phages (Ͻ12 kb) (29). In accordance relatively narrow phylogenetic and phenotypic groups of bacteria, with this, the previously isolated large-genome vibriophages and at the same time they demonstrate similar susceptibility pat- KVP40 (244 kb) (32) and ␸pp2 (246 kb) (33) also have very broad terns across a broad phylogenetic scale in Vibrio spp., emphasizing host ranges covering several species of Vibrio. KVP40 is known to the complexity of phage-host interactions in this group. cause infection through the common outer membrane protein As observed for the physiological fingerprints, there was over- OmpK and has previously been shown to infect eight Vibrio spe- lap in the phage susceptibility patterns for V. anguillarum strains cies, including V. anguillarum, V. parahaemolyticus, and V. chol- isolated across the large temporal and spatial scales and from dif- erae (20). The presence of several copies of genes encoding pro- ferent fish species represented in the current study. For example, teins associated with phage tail or tail fibers in the KVP40 genome the strains 90-11-286 and VA3, which were isolated from Danish suggested an increased flexibility in host range adaptation, in- rainbow trout and salmon farms, respectively, with 20 years be- creasing host range (32). Despite the fact that KVP40 was origi- tween isolations, were both infected by the same 3 phages (⌽H1, nally isolated in Japan Ͼ20 years ago (24), it still infected approx- ⌽H7, and KVP40). Similarly, strains VIB88 and BA35, isolated imately 40% of the Vibrio sp. isolates obtained from Danish

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⌽ FIG 5 (A) Optical density (OD600) in cultures of V. anguillarum BA35 amended with phage H20 at MOIs of 0 (control), 0.1, 1, and 10 and a corresponding Ϫ1 abundance of PFU ml in the treatment at an MOI of 0.1. (B) Optical density (OD600) in cultures of V. anguillarum PF430-3 amended with phage KVP40 at MOIs of 0 (control), 0.1, 1, and 10 and the corresponding PFU abundance in the treatment at an MOI of 0.1. Error bars represent standard deviations from all experiments carried out in duplicate. aquaculture, conﬁrming its cross-species host range, and it main- Generally, vibriophages were widespread in Danish aquacul- tained infective properties across large temporal and spatial scales. ture, as phages were isolated from all of the investigated farms Thus, the characteristic of the new phage isolates ⌽H1 and ⌽H7 independently of outbreaks of vibriosis. However, the fact that adds to the emerging view of a global distribution of large-ge- phages could not be isolated without previous propagation in en- nome, broad-host-range vibriophages. richment cultures indicated that densities of speciﬁc vibriophages

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FIG 6 Phage susceptibility properties of V. anguillarum isolates obtained from cultures enriched with phages at different MOIs. (A to C) V. anguillarum strain BA35 plus phage ⌽H20 at an MOI of 10 (A), 1 (B), or 0.1 (C). (D to F) V. anguillarum strain PF430-3 plus phage KVP40 at an MOI of 10 (D), 1 (E), or 0.1 (F). “Sensitive” indicates that isolates were fully sensitive to the phage. “Resistant” indicates that isolates were not susceptible to phage ⌽H20 infection in the spot test. “Partially resistant” indicates that isolates had a reduced susceptibility to the phage. “Aggregates” indicates that isolates were forming aggregates, which protected against phage infection.

May 2014 Volume 80 Number 10 aem.asm.org 3137 Tan et al. were relatively low in the fish farms. Some samples from which Vibrio strains with reduced sensitivity, dominated the bacterial popula- sp. hosts were isolated did not harbor their corresponding phages, tion after7hintheculture containing strain BA35 and phage suggesting that specific hosts and lytic phages did not always co- ⌽H20. Interestingly, however, phage-resistant strains were not exist in the same environment. However, it should be noted that isolated after exposure of strain PF430-3 to phage KVP40, as has the filtration of the water samples and chloroform treatment prior also been observed in other phage infection studies with hosts to phage isolation may have resulted in the loss of phages (18, 34). such as Salmonella enterica (42), S. enterica serovar Oranienburg In all isolate-based studies of phage diversity, the results are (43), and E. coli O157:H7 (44). In the current experiment with biased by the choice of host strains used for isolation, as each strain PF430-3, phage exposure led to the formation of bacterial individual strain will only target a subset of the infective phages aggregates, and all of the isolated colonies had maintained full present in the sample. In the current study, broad-host-range sensitivity to the phage after purification. These results suggest Downloaded from phages ⌽H1 and ⌽H7 were isolated using strains from the global that different mechanisms were responsible for the regrowth of Vibrio species collection, whereas the more host-specific phages, cells in the presence of phages following lysis of the initial sensitive such as ⌽2E-1, ⌽S4-18, and ⌽S4-7, were isolated using a cooc- population. The BA35 and phage ⌽H20 interaction results in a curring Vibrio sp. host obtained from the same sample. As the succession toward phage-resistant strains, whereas the strain evolution of a broad host range in phages is believed to be accom- PF430-3 apparently is protected against phage KVP40 infection by panied by a fitness cost, i.e., a reduced infective efficiency in its aggregation. The underlying mechanism providing bacterial pro- original hosts (35), it is likely that the observed differences in tection against phages via aggregation, allowing the coexistence of phage isolation pattern reflect different phage properties. There- lytic phages and sensitive strains, is not revealed in the present http://aem.asm.org/ fore, we speculate that highly specific phages are efficient, oppor- study. However, previous studies have shown that phage-induced tunistic phages which propagate rapidly in response to the growth lysis can promote the formation of bacterial biofilms (45), which of their specific hosts and will dominate in samples where their may offer protection against phage infection via the exopolysac- host is present. Consequently, these phages are the most likely to charide (EPS) matrix covering the biofilm (46) or the presence of be isolated using cooccurring hosts as target strains. Broad-host- bacterial refuges in the spatially heterogeneous environment (47). range phages, on the other hand, may follow a K-strategy (36), More recently, Høyland-Kroghsbo et al. (48) found that N-acyl- maintaining lower densities but for longer periods; therefore, they L-homoserine lactone (AHL) quorum-sensing signals in E. coli may be more likely to be pulled out from a sample using non- caused a transient reduction in the phage adsorption rate and on April 29, 2014 by Copenhagen University Library cooccurring or rare hosts. Due to these differences in isolation suggested this as a mechanism allowing coexistence of lytic phages pattern, we do not know to what extent the isolated phages are and sensitive hosts. Such mechanisms may also have contributed representative of the local phage community. to the observed coexistence of sensitive strains of PF430-3 and the PFGE provides a good discrimination for phage genome sizes lytic phage KVP40 in the current study. and clearly demonstrates a large variability in genome size within Potential for phage therapy in aquaculture. One of the main the group of vibriophages. Several phage genomes, including challenges in using bacteriophages to control pathogens in aqua- ⌽H20, ⌽H8, and ⌽H2, yielded several bands in PFGE analysis, culture is to cover the diversity and temporal and spatial variations representing multiplications of its own genome size. These bands in pathogen composition, as well as the phage resistance that may may represent concatemers of the phage genome produced during rapidly develop. A detailed characterization of phage properties DNA replication, as previously demonstrated in ␭ phage (37). and understanding of phage-host interactions are essential re- Also, concatemer formation of phage DNA has been observed as a quirements for the successful application of phage-based patho- result of DNA cleavage during heating and subsequent reassembly at ambient temperature (38). The possibility of coinfection of gen control. The local occurrence of both specific and broad-host- other phages still cannot be ruled out; however, we did not see range phages which, in combination, were able to infect all of the different phage morphotypes during TEM analysis of single iso- tested host strains, covering large geographical and temporal lates. A previous report of different phage morphotypes associated scales, suggested that vibriophage-host systems are ubiquitously with multiple bands on a PFGE gel, however, suggested that coin- distributed. In a phage therapy context this is promising, as it fection of more than one phage may be an explanation for the suggests the possibility of establishing a phage library with broad occurrence of several genome sizes on a PFGE gel of a purified application against V. anguillarum communities worldwide. phage (29). Further genomic analyses are required to elucidate the However, the fast development of phage resistance during expo- origin of the multiple bands obtained in the PFGE analyses. sure to specific phages and the indication that different mecha- Phage-host interactions. The in vitro bacterial challenge assay nisms provided resistance in different hosts also emphasized that with V. anguillarum strains BA35 and PF430-3 demonstrated sig- the susceptibility to phages is a dynamic property in the Vibrio sp. nificant but temporary phage control of both strains which was hosts. Consequently, phage resistance mechanisms and dynamics strongly dependent on the initial MOI. Even at the highest MOI, and the implications for host properties are important to address the host bacteria were not completely eliminated and regrowth of in future exploration of phage control of V. anguillarum in aqua- cells following phage infection suggested the emergence of phage- culture. resistant strains after 5 to6hofincubation. Lytic bacteriophages have previously been shown to select for the emergence of resis- ACKNOWLEDGMENTS tant strains in culture studies with marine heterotrophic bacteria This study was supported by the Danish Strategic Research Council (39–41), where phage-resistant strains rapidly replaced the sensi- (ProAqua; project 12-132390), by a grant from the Danish Council for tive wild type following exposure to phages. Independent Research (FNU 09-072829), and by the EU-IRSES-funded Isolation and characterization of bacterial isolates during the project AQUAPHAGE. experiments confirmed that completely resistant strains, and We thank Pantelis Katharios, Hellenic Centre for Marine Research, for

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3140 aem.asm.org Applied and Environmental Microbiology

Figure S1. Neighbour-joining tree based on 16S rRNA gene sequencing showing the phylogenetic relationship between 37 strains listed in table 1 and V. harveyi (AY747308.1, NCBI) and V. parahaemolyticus (FJ594056.1, NCBI). Phylogenetic relationships were bootstrapped 1,000 times. The scale bar represents the number of nucleotide substitutions per site.

Figure S2: PFGE analysis of all the 11 phage genomes. Ladders indicate DNA size in standards (kb)

1 Vibriophages differentially influence biofilm formation by Vibrio anguillarum strains

2 Demeng Tan, Amalie Dahl and Mathias Middelboe*

3 Marine Biological Section, University of Copenhagen, Helsingør, Denmark

5 *correspondence: [email protected]

6 Running title: The interaction of vibriophages and biofilms

9 Abstract

10 Vibrio anguillarum is an important pathogen in marine aquaculture, responsible for vibriosis.

11 Bacteriophages can potentially be used to control bacterial pathogens; however, successful

12 application of phages requires a detailed understanding of phage-host interactions at both free-

13 living and surface-associated growth conditions. In this study, we explored in vitro phage-host

14 interactions in two different strains of V. anguillarum (BA35 and PF430-3) during growth in micro-

15 colonies, biofilms, and free-living cells. Two vibriophages, ΦH20 (Siphoviridae) and KVP40

16 (Myoviridae), had completely different effects on the biofilm development. Addition of phage ΦH20

17 to strain BA35 showed efficient control of biofilm formation as well as in liquid cultures. The

18 interactions between BA35 and ΦH20 were thus characterized by a strong phage control of the

19 phage-sensitive population and subsequent selection for phage-resistant mutants. Addition of phage

20 KVP40 to strain PF430-3 resulted in increased biofilm development, especially during the early stage.

21 Subsequent experiments in liquid cultures showed that addition of phage KVP40 stimulated the

22 aggregation of host cells, which protected the cells against phage infection. By the formation of

23 biofilms, strain PF430-3 created spatial refuges that protected the host from phage infection and

24 allowed coexistence between phage-sensitive cells and lytic phage KVP40. Together the results

25 demonstrate highly variable phage protection mechanisms in two closely related V. anguillarum

26 strains, thus emphasizing the challenges of using phages to control vibriosis in aquaculture, and

27 adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.

33 Introduction

34 Vibrio anguillarum is a marine pathogenic bacterium causing vibriosis, a fatal hemorrhagic

35 septicemia, which contributes to significant mortalities in fish and shellfish aquaculture worldwide

36 (1-3). The persistence of Vibrio pathogens in aquaculture has been attributed to their ability to form

37 biofilms with increased tolerance to disinfectants and antibiotics (4, 5). Moreover, the first stage of

38 infection involves biofilm-like micro-colonies in the skin tissue, causing chronic infection (5).

39 Recently, bacteriophages have been suggested as potential agents of pathogen control in

40 aquaculture, and the controlling effects of phages have been explored for a number of fish

41 pathogens (6-8). Successful application of phages to reduce vibriosis-related mortality has been

42 demonstrated (9, 10). The capabilities of some phages to produce depolymerases, which hydrolyze

43 extracellular polymers in bacterial biofilms, have made the use of bacteriophages particularly

44 relevant in the treatment of biofilm-forming pathogens, as demonstrated in biofilms of

45 Pseudomonas aeruginosa (11), Escherichia coli (12), and Staphylococcus aureus (13).

46 Apart from the potential physical and chemical barrier provided by biofilms, the use of

47 bacteriophages to control pathogens is challenged by the development and selection for phage-

48 resistant or phage-tolerant subpopulations. A broad range of resistance mechanisms have evolved in

49 bacteria (14) and the lytic infection of phage-susceptible bacteria has shown to drive a functional

50 and genetic diversification of the host population based on the rapid emergence of phage-resistant

51 mutants (15, 16). A deeper understanding of temporal and spatial dynamics of phage-biofilm

52 interactions and the development of phage-resistance in these systems is therefore essential for

53 assessing the potential of using phages in disease control.

54 In this study, we quantified the influence of phages on biofilm formation in two strains of V.

55 anguillarum, both during the initial stages of attachment and micro-colony formation and in more

56 developed biofilms. The results showed large intraspecific differences in phage-biofilm interactions

57 of two V. anguillarum phage-host systems, and revealed the presence of highly different protective

58 mechanisms against phage infection, adding further complexity to the use of phages to control

59 vibriosis.

60 Materials and Methods

61 Bacterial strains and bacteriophages

62 Bacteriophages and hosts used in this study are listed in Table 1. Two V. anguillarum strains were

63 used in this study: strain PF430-3, originally isolated from salmonid aquaculture in Chile (17) and

64 strain BA35, originally isolated from sockeye salmon in USA (18). Phage KVP40 which infects PF430-3

65 is a broad-host-range phage originally isolated from Japan (19, 20) and phage ΦH20, infecting BA35,

66 was isolated from Danish aquaculture (21).

67 Effects of phages on initial bacterial attachment and micro-colony formation

68 To quantify the effect of phages on micro-colony formation of the two strains, 10 µl of diluted mid-

69 log-phase cultures (106 CFU ml-1) was filtered onto 20+ replicate 25 mm 0.2 µm polycarbonate filters

70 and incubated on LB agar in 6-well plates (22). Two ml phage stock (ca. 108 PFU ml-1) was added to

71 half the filters, the other half serving as controls with addition of 2 ml SM buffer (50 mM Tris-Cl, pH

72 7.5, 100 mM NaCl, 8 mM MgSO4, 0.01% gelatin). Duplicate filters of phage amended and controls

73 were collected every hour for 8 h, transferred to a 25 mm filtration manifold (Millipore) and stained

74 with 0.5 % SYBR Gold (Invitrogen) for 10 min, followed by 3 x rinsing with Milli-Q water. The filters

75 were then mounted on a microscope slide and stored frozen until quantification by epifluorescence

76 microscopy using Cell M image analysis software (Olympus) for determination of colony area.

77 Phage-biofilm interactions

78 Effects of phage ΦH20 and KVP40 on the formation and disruption of V. anguillarum biofilms were

79 monitored for the two strains BA35 and PF430-3 in two different experimental approaches: In the

80 pre-treated experiment, duplicate sets of polypropylene plastic tubes (Sarstedt) with 5 ml marine

81 broth (MB, Difco) were inoculated with 100 μl overnight bacterial culture and 100 μl phage stock

82 (final concentration 107 CFU ml-1 and 106 PFU ml-1, multiplicity of infection, MOI=0.1) simultaneously.

83 In the post-treated experiments, the bacteria were allowed to grow in 5 ml MB for 10 days after

84 inoculation to establish a biofilm. After 10 day incubation, the liquid was removed and the tubes

85 were rinsed with MB. Then 5 ml MB was added along with 100 μl phage stock (final concentration

86 106 PFU ml-1), and the tubes were incubated for another 7-8 days.

87 In both experiments, the tubes were incubated at 30 °C and duplicate tubes were sacrificed daily for

88 measurement of biofilm formation, and density of free-living bacteria and phages. Measurements of

89 OD600nm were used to estimate free-living bacterial abundance and determination of phage

90 concentration in the free-living phase was done by plaque assay (23). Biofilm was quantified

91 according to O'Toole (24) with some modifications. Briefly, the liquid was removed, and tubes were

92 rinsed twice with artificial seawater (ASW, Sigma). Then 6 ml 0.4% crystal violet (Sigma) was added

93 to each tube and after 15 min, stain was removed. Tubes were washed with tap water in order to

94 remove excess stain and left to dry for 5 min. Six ml of 33% acetic acid (Sigma) was added and left

95 for 5 min to allow the stain to dissolve. The absorbance was measured at OD595nm.

96 In addition to the measurements above, we also quantified the biofilm-associated infective phages

97 in the post-treated experiment after disruption of the biofilm by sonication. First, the liquid was

98 removed and the tube rinsed 3 times with ASW. Then, 6 ml ASW was added to completely cover the

99 biofilm, and the tube was sonicated for 10 s at 80 amplitude (Pulse on 1 s and Pulse off 1 s, Misonix

100 sonication bath) to dislodge bacteria and phages from the biofilm matrix. Serial dilutions were

101 performed and phage concentration was quantified by plaque assay (23).

102 Phage-host interactions in the free-living phase: detection of aggregate formation

103 The effects of phages ΦH20 and KVP40 on the growth and aggregate formation of their respective

104 host strains, BA35 and PF430-3, during free-living growth in liquid cultures were examined.

105 Overnight cultures were inoculated in 50 ml MB with their respective phage at a MOI of 0.1. The

106 flasks were incubated at room temperature with agitation; samples were collected for

107 determination of the presence of cell aggregates by epifluorescence microscopy. For epifluorescence

108 microscopic analysis of cell aggregation, subsamples were filtered onto 0.45 μm polycarbonate

109 membrane filters (Whatman) and stained with SYBR Gold (0.5%, Invitrogen) for 10 min. Bacteria and

110 phages on the filters were distinguished based on their dimensions and/or their relative brightness.

111 Phage susceptibility and physiological fingerprint of bacterial isolates

112 In order to determine changes in phage susceptibility, a total of ~180 cells were isolated from phage-

113 treated micro-colony experiments, pre-treated biofilm experiments and free-living samples and

114 analyzed for phage susceptibility pattern. For colony isolation, samples were in all cases plated onto

115 Thiosulfate Citrate Bile Salt Sucrose agar (TCBS, Difco) and incubated for 24 h. From these plates,

116 single colonies were isolated and purified by 3 rounds of re-isolation and then transferred to 4 ml

117 MB for subsequent phage susceptibility analysis. Phage susceptibility of the isolated strains was

118 determined by spot tests (21).

119 BIOLOG GN2 Micro-Plates (BIOLOG) containing 95 different carbon sources were used to test the

120 ability of selected phage-amended isolates to use different substrates following manufacturer’s

121 instructions (21). For strain PF430-3, the procedure was slightly modified as bacterial lysate

122 (bacterial culture subjected to phage-induced lysis) and aggregates were collected by centrifugation

123 at a lower speed (3,000×g, 5 min), and washed twice with 0.9% saline buffer.

124 Adsorption experiments with fluorescently labelled phages

125 Phage adsorption to selected isolates was tested by adding SYBR Gold labelled phages to cultures

126 followed by visual inspection phage binding to the cells using epifluorescence microscopy, following

127 the protocol of Kunisaki (25) with some modifications. Aliquots of 600 μl phage lysate were digested

128 by addition of DNase I (1 μl DNase I +3 μl RDD buffer, Qiagen) at 37°C for 2 h and then stained with

129 SYBR Gold (final concentration 5×, Invitrogen) overnight at 4 °C. Ten μl chloroform was then added

130 to inactivate DNase I. The phage stock was then further filtered through a 30 kDa ultrafiltration spin

131 devices (Millipore) at 1,000×g for 90 min to remove the free SYBR Gold. The SYBR Gold-labelled

132 phages were added to the mid-log wild-type cells and phage lysate, respectively, at a MOI of

133 approximately 10 and incubated at room temperature for 20 min. Samples were pelleted at

134 12,000×g for 3 min, and the pellet was re-suspended in a small volume of SM buffer, mixed with pre-

135 heated 0.5% agarose (w/v) (Bio-rad) around 45°C and transferred to gel-coated slide glass.

136 Statistical analysis

137 All statistics were performed using the Student's paired t-test (two-tailed) (OriginPro 8.6) to

138 determine the significance of the differences between phage amended and control cultures.

139 Differences were considered to be significant if P<0.05.

140 Results

141 Initial biofilm formation and short-term effects of phages

142 The two V. anguillarum strains formed micro-colonies with highly different morphologies in the

143 absence of their corresponding phages. Strain BA35 formed simple single-layered micro-colonies,

144 where individual cells could easily be identified whereas strain PF430-3 formed complex 3-

145 dimensional volcano-shaped structures (Fig. 1). Phage ΦH20 showed an efficient control of micro-

146 colonies BA35 according to the microscope observations, reducing total colony area from 41,000

147 µm2 per mm2 filter on the control filters to 5,000 µm2 per mm2 filter after 6 h incubation, and leaving

148 mainly single cells on the filters following phage exposure (data not shown). For strain PF430-3, the

149 individual colonies were unaffected by phage exposure, whereas single cells were lysed by phage

150 KVP40, leaving mainly colonies on the filters 6 h post addition (Fig. 1).

151 Effects of phages on biofilm formation

152 Establishment and growth of biofilm produced by the two V. anguillarum strains BA35 and PF430-3

153 in cultures pre-treated with phage ΦH20 and KVP40, respectively, was examined over a long-term

154 experiment in parallel to control cultures without phages. The OD600nm of free-living bacteria in the

155 control cultures reached maximum values of 0.72±0.01 and 0.98±0.01 for BA35 and PF430-3,

156 respectively, after 2 days (Fig. 2A and 2C). A gradual minor decrease was then observed for BA35,

157 whereas the density of PF430-3 decreased to ~50 % of the maximum value at day 9 (Fig. 2A and 2C).

158 The presence of the phages had different effects on the free-living cell density of the two strains.

159 For strain BA35, the density reached a level of 70 % of the control cultures after 2 days and remained

160 at this level (only significantly lower at day 3 (p=0.025) and 6 (p=0.009)) throughout the experiment

161 (Fig. 2A). In the cultures with strain PF430-3, addition of phage KVP40 significantly reduced the

162 optical density of the culture (0.008≤p≤0.027 when comparing the OD values at individual time

163 points from day 2 onwards) to approximately 25% of the corresponding values in the control

164 cultures (Fig. 2C). Phage concentration reached peaks of around 1.3×1010±3.5×108 and

165 3.4×108±7.1×106 PFU ml-1 for ΦH20 and KVP40, respectively at day 1 (Fig. 2A and2C). Interestingly,

166 in the PF430-3+KPV40 cultures, phage concentrations decreased significantly (p=0.007) from day 1

167 to day 2 then stabilizing around 106 PFU ml-1 (Fig. 2C). Likewise, the concentrations phage ΦH20

168 decreased significantly (p=0.0055) from day 1 to day 5 and then remained at a titer above

169 2.2×109±2.8×108 PFU ml-1 (Fig. 2A).

170 The biofilm development in response to phage addition also showed completely different patterns in

171 the two strains with either inhibition (BA35) or stimulation (PF430-3) of biofilm formation relative to

172 control cultures. In the BA35+ΦH20 pre-treated cultures, phage ΦH20 effectively prevented biofilm

173 formation, causing a 2-fold decrease in the biofilm biomass relative to the control level (Fig. 2B). In

174 the PF430-3+KVP40 pre-treated cultures, phage KVP40 addition resulted in almost a 3-fold increase

175 relative to the control (Fig. 2D). In the control cultures, there was a steady increase in biofilm density

176 for strain PF430-3, whereas a fast initial establishment of biofilm of strain BA35 was gradually

177 reduced during the 7-day period (Fig. 2B and 2D).

178 To assess the effect of phages on fully matured biofilms, 10-day biofilms were challenged with their

179 corresponding phages measuring the same parameters as above. As in the pre-treatment

180 experiments, phage addition significantly reduced the BA35 biofilm (3-4 fold reduction) within 2

181 days (p=0.046, when comparing OD values from day 1 and 2) and slightly stimulated the production

182 of biofilm in PF430-3, relative to controls (Fig. 3B and 3D).

183 For the free-living populations, the input of nutrients associated with the addition of phages (and

184 corresponding addition of medium in the controls) caused an increase in bacterial concentration in

185 the control cultures (Fig. 3A and 3C). Similar to the pre-treated cultures, the OD600nm decreased over

186 time in the control cultures, with the strongest decrease observed in the PF430-3 cultures. The

187 presence of phages, however, imposed a significant 4-5 fold decrease in concentration of free-living

188 cells in both experiments (0.003≤p≤0.046, when comparing the OD values at individual time points

189 from day 2 onwards), except for day 5 (Fig. 3A and 3C).

190 The concentration of free-living phages reached a peak around 3×109±2.8×107 and 5.8×108±2.8×107

191 PFU ml-1 for ΦH20 and KVP40, respectively, after 24 h (Fig. 3A and 3C) followed by a gradual

192 decrease in the concentration of phage KVP40 (Fig. 3C). The biofilm-associated concentration of

193 phage KVP40 constituted less than 10% of the free-living population and decreased dramatically

194 after the peak (Fig. 3D).

195 Effects of phage KVP40 on bacterial aggregation in liquid cultures

196 In the BA35 and BA35+ΦH20 cultures, no aggregate formation was observed and phage addition

197 reduced the density of free-living cells relative to the control as a result of cell lysis (Fig. 4). Motility

198 of BA35 seemed to be affected by phage addition as cells in the phage-added cultures had reduced

199 or lost motility after 24 h (Supplementary video 1 and 2). In the PF430+KVP40 cultures, on the other

200 hand, aggregates of multi-layered cell clusters embedded in a matrix were observed after 24 h (Fig. 4

201 and Supplementary video 3 and 4).

202 Bacteriophage resistance and physiological fingerprint

203 For strain BA35, all the isolates obtained from the micro-colony experiment were resistant to phage

204 infection and approximately 30 % of the free-living and biofilm-associated isolates were sensitive to

205 ΦH20 (Table S1). The resistant colonies were small-colony variants on TCBS plates. In contrast, all

206 the isolates from KVP40-exposed PF430-3 cultures were fully or partially susceptible to phage KVP40

207 and no completely KVP40-resistant mutants were isolated (Table S1). Colony morphologies of PF430-

208 3 isolates were similar to the wild-type PF430-3. Spot assays showed that all partially resistant

209 isolates regained full sensitivity to phage KVP40 after purification by re-streaking of single colonies

210 on agar plates.

211 The phenotypic diversity and implications of resistance for the metabolic properties of the isolates

212 following phage exposure were explored by obtaining a metabolic fingerprint of selected isolates

213 using BIOLOG GN2 assay (Fig. S1). For strain BA35, the phage-resistant isolate had lost the ability to

214 use 10 of the 54 substrates that could be metabolized by wild-type BA35 cells, corresponding to a 19%

215 reduction of the capacity substrate utilization. In contrast, none of the PF430-3 isolates obtained

216 after phage exposure showed any changes in physiological fingerprint according to the BIOLOG

217 profile (Fig. S1).

218 To further examine the mechanisms of resistance/tolerance against phages in the two strains, SYBR

219 Gold-labelled phages ΦH20 and KPV40 were incubated with wild-type cells and phage lysate.

220 Fluorescently labeled phage ΦH20 attached to the sensitive cells of the strain BA35 (Fig. 5A),

221 whereas no phage-attachment was observed in phage-resistant mutants (Fig. 5B). In the parallel

222 experiment with fluorescently labelled KVP40 phages, the phages were immobilized in the

223 aggregates and did not attach to the cell surface (Fig. 5C and 5D).

224 Discussion

225 Phage effects on micro-colony formation and initial biofilm development

226 The microscopic observations of micro-colony formation of cells attached to a polycarbonate filter

227 surface revealed that the two strains formed highly different types of micro-colonies, with large

228 implications for the phage-host interactions. The phage-resistant isolates obtained from BA35

229 cultures exposed to ΦH20 maintained resistance upon re-culturing, suggesting that resistance was a

230 result of genetic mutations which prevented phage infection. In contrast to the strong lytic effect of

231 ΦH20 and corresponding reduction in micro-colony formation of strain BA35, phage KVP40 did not

232 significantly affect the size of individual PF430-3 micro-colonies. A possible explanation could be that

233 the complex 3-dimensional structure of PF430-3 micro-colonies provided protection from phage

234 infection by creating physical barriers, once formed.

235 Long-term effects of phages on biofilm formation from liquid cultures of different V. anguillarum

236 strains

237 The strong controlling effects of phage ΦH20 on the strain BA35 biofilm observed both in the pre-

238 treated and post-treated conditions supported the microscopic observations that the BA35 biofilm is

239 a relatively simple structure which is easily accessible for phage adsorption. Also in the free-living

240 phase, phage ΦH20 was able to control the phage susceptible BA35 population, but at these

241 conditions, phage-resistant mutants replaced the sensitive population within the first 24 h. The

242 interactions between ΦH20 and strain BA35 thus seemed very similar for attached and free-living

243 cells, suggesting that the flat and smooth biofilm structure did not offer protection against phage

244 infection.

245 In contrast, addition of phage KVP40 to strain PF430-3 resulted in an enhanced biofilm formation in

246 both experiments, and particularly during the early stage of biofilm development. At the same time,

247 the free-living bacterial population was efficiently controlled and maintained at an OD level of <25 %

248 of the maximum density in the control cultures, in both the pre-treated and post-treated PF430-3

249 experiments. This suggested a coupling between the phage-mediated decrease in density of free-

250 living cells and the corresponding cell aggregate formation and subsequent increase in biofilm

251 formation.

252 Potential mechanisms of phage protection in V. anguillarum strains

253 While the dominance of phage resistant strains following phage exposure suggested mutational

254 changes as the mechanism of phage protection in strain BA35, the interaction between strain PF430-

255 3 and phage KVP40 pointed towards aggregation and biofilm formation as a protection against

256 phage infection. This mechanism of protection was supported by a number of observations: 1),

257 addition of fluorescently labelled KVP40 phage to aggregates and free-living cells of PF430-3 showed

258 that KVP40 was trapped in the aggregate matrix and did not adsorb to the host cells embedded in

259 the aggregates; 2), the complex 3-dimensional structure of the micro-colonies observed for PF430-3

260 was also consistent with the hypothesis that the aggregation provided a refuge for phage-sensitive

261 bacteria (26-28); and 3), the presence of a fraction of free-living phage-sensitive cells outside the

262 aggregates may in part be explained by the entrapment of phages in the aggregates which reduced

263 the phage encounter rate, possibly allowing co-existence of small populations of free-living phages

264 and free-living sensitive cells.

265 In combination with the reduced phage-sensitivity found in a number of the isolates, the

266 immobilization of free-living cells in aggregates and subsequent attachment to surfaces provides an

267 efficient refuge for cells exposed to phage attack, thus adding to the multiple and complex

268 protection mechanisms found in bacteria (14, 29, 30). Whether the aggregation is a direct response

269 to phage exposure, e.g. through quorum sensing (QS)-mediated gene regulation which is well known

270 from V. anguillarum (31-33), or is an indirect effect of phage-lysis of free-living cells, thus adding a

271 strong positive selection pressure for aggregate-forming phenotypes needs to be elucidated (30).

272 It should be noted that successful phage amplification depends not only on the host cell lysis, but

273 also on the phages able to bind to the susceptible bacterium. Polysaccharide depolymerases have

274 been reported in various phages, which allow phages to access phage receptors in biofilm-associated

275 cells (34), Consequently, the generality of protection mechanisms observed in the present study is

276 unknown, as other phages may be able to infect the biofilm-forming strain PF430-3. It is often

277 assumed that bacteriophage resistance is associated with a cost (35, 36) due to reduced abilities to

278 take up specific nutrients (37) and reduced competitive abilities in general (38, 39). The BIOLOG

279 fingerprint of resistant isolates of strain BA35 supported such a link between phage-resistance and

280 reduced physiological performance, as the resistant mutants had a reduced ability to utilize a

281 number of substrates. The absence of observed physiological changes in the phage-exposed isolates

282 of strain PF430-3 relative to the wild-type strain , on the other hand supported the suggestion that

283 phage tolerance in strain PF430-3 was not caused by mutational changes but rather was due to

284 protection by aggregation. It should be emphasized however, other fitness costs than those resolved

285 by the BIOLOG assay may have been present in strain PF430-3.

286 Interestingly, the free-living isolates obtained from KVP40-amended PF430-3 cultures, which had

287 reduced phage susceptibility, all recovered full sensitivity after re-growth in the absence phage

288 KVP40. This indicated that the phage tolerance was a transient phenotype, as has also been

289 observed in other phage infection studies including hosts such as V. cholerae (40), S. enterica (41), S.

290 oranienburg (42), and E. coli O157:H7 (41, 43), and suggests that down-regulation of phage receptor

291 gene expression may also play a role in phage susceptibility in strain PF430-3 (44).

292 In conclusion, our data demonstrated completely different mechanisms of protection against phages

293 in two strains of V. anguillarum and showed that these intraspecific differences strongly influenced

294 the outcome of the phage-host interactions with respect to 1) the ability of phages to control free-

295 living host populations; 2) the ability of phages to control the development and stability of biofilm; 3)

296 the phage-driven diversification of bacterial populations; and 4) the co-existence and co-evolution of

297 phages and hosts. However, additional studies are needed to explore whether the different

298 responses to phage exposure are general properties of the strains or are linked to the specific

299 phages.

300 Implications for phage-control of the pathogen

301 In a phage therapy context, the interaction between strain BA35 and phage ΦH20 suggests that

302 phage control of strain BA35 potentially would prove useful, since the phage efficiently inhibited

303 biofilm formation both on short and longer time scale as well as proved efficient in disrupting

304 already established biofilms. The phage-derived stimulation of biofilm formation in strain PF430-3

305 may, on the other hand, suggests that phage KVP40 would be inefficient in controlling this strain in

306 aquaculture, and perhaps rather stimulate its pathogenic impact by inducing biofilm formation.

307 Our study thus confirms that successful application of phage therapy in the treatment of biofilm

308 requires detailed understanding of phage-host interactions, and emphasizes that the complexity and

309 diversity of phage-host interactions even within the same species of pathogen is a challenge for

310 future use of phages in disease control.

311 Acknowledgements

312 This study was supported by grants from the Danish Strategic Research Council (ProAqua project 12-

313 132390), the Danish Council for Independent Research (FNU 09-072829) and the EU-IRSES-funded

314 project AQUAPHAGE (269175). We thank Pantelis Katharios, Hellenic Centre for Marine Research,

315 for kindly providing V. anguillarum PF430-3 and vibriophage KVP40 and Lone Gram, Technical

316 University of Denmark, for kindly providing V. anguillarum BA35. Jeanett Hansen is acknowledged

317 for technical support.

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427

428 Figure Legends

429 Figure 1. Fluorescent microscopic images of micro-colony formation of strain BA35 (left two columns) 430 and PF430-3 (right two columns) at different time points in the absence and presence of phage 431 ΦH20 and KVP40, respectively. Samples were stained with 0.5% SYBR Gold for 10 min.

432 Figure 2. Culture density and biofilm formation of V. anguillarum strain BA35 and PF430-3 in cultures 433 pre-treated with phage ΦΗ20 and KVP40, respectively, and control cultures. A) Optical density 434 (OD600nm, left y-axis) of free-living cells (strain BA35) in the presence and absence of phage ΦΗ20, 435 and the corresponding free-living phage ΦΗ20 concentration (PFU ml-1, right y-axis); B) Strain BA35 436 biofilm formation in the presence and absence of phage ΦΗ20, quantified by crystal violet (OD595nm, 437 left y-axis); C) Optical density (OD600nm, left y-axis) of free-living cells (strain PF430-3) in the presence 438 and absence of phage KVP40 and the corresponding free-living phage KVP40 concentration of (PFU 439 ml-1, right y-axis) ; and D) Strain PF430-3 biofilm formation in the presence and absence of phage 440 KVP40, quantified by crystal violet (OD595nm, left y-axis). Error bars represent the actual ranges from 441 all experiments carried out in duplicate.

442 Figure 3. Culture density and biofilm formation of V. anguillarum strain BA35 and PF430-3 in 443 cultures post-treated with phage ΦΗ20 and KVP40 respectively, and control cultures. A) Optical 444 density of free-living cells (strain BA35) (OD600nm, left y-axis) in the presence and absence of phage 445 ΦΗ20, and the corresponding phage free-living ΦΗ20 concentration of (PFU ml-1); B) Strain BA35 446 biofilm formation in the presence and absence of phage ΦΗ20, quantified by crystal violet (OD595nm, 447 left y-axis) and corresponding biofilm-associated phage concentration (PFU ml-1, right y-axis); C) 448 Optical density (OD600nm, left y-axis) of free-living cells (strain PF430-3) in the presence and absence 449 of phage KVP40, and the corresponding free-living phage KVP40 concentration of (PFU ml-1, right y- 450 axis); and D) Strain PF430-3 biofilm formation in the presence and absence of phage KVP40, 451 quantified by crystal violet (OD595nm, left y-axis) and the corresponding biofilm-associated phage 452 concentration of (PFU ml-1, right y-axis). Error bars represent the actual ranges from all experiments 453 carried out in duplicate.

454 Figure 4. Fluorescence microscopic examination of aggregate formation in V. angillarum strains BA35 455 and PF430-3 in the presence and absence of phages. No aggregate formation was observed in BA35 456 and BA35+ΦH20 cultures, and phage addition reduced the concentration of free-living cells relative 457 to the control . In the PF430+KVP40 cultures, phage-induced aggregates were observed.

458 Figure 5. Visualization of phage adsoprtion by SYBR Gold-labelled phages under phase-contrast 459 epifluorescence microscope. A, wild-type BA35 cells with SYBR Gold-labelled phage ΦH20 (phase- 460 contrast+fluorescence); B, BA35 phage lysate with SYBR Gold-labelled phage ΦH20 (phase- 461 contrast+fluorescence); C, PF430-3 phage lysate with SYBR Gold-labelled phage KVP40 (phase- 462 constrast); and D, PF430-3 phage lysate with SYBR Gold-labelled phage KVP40 (phase- 463 contrast+fluorescence).

464

Table 1. Bacteriophages and hosts Phage Bacterial host (source) Phage receptor Genome Family Phage source KVP40 PF430-3 (Chile) (17) outer membrane 244 kb Myoviridae seawater, Japan (19, 20) protein K (OmpK) ΦH20 BA35 (USA) (18) unknown 50 kb Siphoviridae seawater, Denmark (21)

Table S1. Susceptibility of isolates of V. anguillarum strains BA35 and PF430-3 to phages ΦH20 and KVP40, respectively, obtained from microlony experiment, pre-treated free-living culture experiment and biofilm experiment. ND, not done.

Time (h) % phage sensitive cells Micro-colonies Free-living cells Biofilm PF430-3 BA35 PF430-3 BA35 PF430-3 BA35 8 100 0 ND ND ND ND 18 100 0 ND ND ND ND 24 100 0 100 30 100 ND 46 100 0 ND ND ND ND 120 ND ND 100 25 100 32

Supplementary Figure S1. Carbon substrate utilization of different V. anguillarum strains (BA35 and PF430- 3). BA35 WT, BA35 wild-type; BA35 R, BA35 resistant isolate; PF430-3 WT, PF430-3 wild-type; PF430-3 S, PF430-3 Sensitive isolate; and PF430-3 L, PF430-3 bacteria and bacteriophage lysate. 1 Quorum sensing determines the choice of anti-phage defense strategy in Vibrio anguillarum

2 Demeng Tana, Sine Lo Svenningsenb, Mathias Middelboea*

4 aMarine Biological Section, bSection for Biomolecular Sciences, Department of Biology, University 5 of Copenhagen, Copenhagen, Denmark

6 *Correspondence: [email protected]

22 Abstract

23 Selection for phage resistance is a key driver of bacterial diversity and evolution, and phage-host 24 interactions may therefore have strong influence on the genetic and functional dynamics of 25 bacterial communities. In this study, we found that an important, but so far largely overlooked, 26 determinant of the outcome of phage-bacterial encounters in the fish pathogen Vibrio 27 anguillarum is bacterial cell-cell communication, known as quorum sensing. Specifically, V. 28 anguillarum PF430-3 cells locked in the low-cell-density state (ΔvanT mutant) express high levels 29 of the phage receptor OmpK; resulting in a high susceptibility to phage KVP40, but achieve 30 protection from infection by enhanced biofilm formation. By contrast, cells locked in the high-cell- 31 density state (ΔvanΟ mutant) are almost completely unsusceptible due to quorum-sensing- 32 mediated down-regulation of OmpK expression. The phenotypes of the two quorum sensing 33 mutant strains are accurately reflected in the behavior of wild-type V. anguillarum, which (i) 34 displays increased OmpK expression in aggregated cells compared to free-living variants in the 35 same culture, (ii) displays a clear inverse correlation between ompK mRNA levels and the 36 concentration of N-acyl homoserine lactone quorum-sensing signals in the culture medium, and (iii) 37 survive mainly by one of these two defense mechanisms, rather than by genetic mutation to 38 phage resistance. Taken together, our results demonstrate that V. anguillarum employs quorum 39 sensing information to choose between two complementary anti-phage defense strategies. 40 Further, the prevalence of non-mutational defense mechanisms in strain PF430-3 suggests highly 41 flexible adaptations to KVP40 phage infection pressure, possibly allowing the long-term co- 42 existence of phage and host.

49 Importance

50 Comprehensive knowledge on bacterial anti-phage strategies and their regulation is essential for 51 understanding the role of phages as drivers of bacterial evolution and diversity. In an applied 52 context, development of successful phage-based control of bacterial pathogens also requires 53 detailed understanding of the mechanisms of phage protection in pathogenic bacteria. Here we 54 demonstrate for the first time the presence of QS-regulated phage defense mechanisms in the fish 55 pathogen V. anguillarum and provide evidence that QS regulation allows V. anguillarum to 56 alternate between different phage protection mechanisms depending on population cell density. 57 Further, our results demonstrate the prevalence of non-mutational defense mechanisms in the 58 investigated V. anguillarum strain, which allow flexible adaptations to a dynamic phage infection 59 pressure.

73 Introduction

74 Vibrio anguillarum is a marine pathogenic bacterium which causes vibriosis in numerous fish and 75 shellfish species leading to high mortalities and economic losses in aquaculture worldwide (1). The 76 use of bacteriophages to control bacterial infections in aquaculture has gained increased attention 77 the past years and successful application of phages to reduce vibriosis-related mortality has been 78 demonstrated (2). Development of a phage-based treatment is, however, challenged by the wide 79 variety of anti-phage defense strategies observed in bacterial hosts (3). There is clear evidence 80 that genetic mutation, normally causing disruption or modification of phage receptors in the host 81 membrane, plays an important role in preventing phage infection in some cases (4-8). Such 82 genetic changes may impose a fitness cost to the host cell as disruption of phage receptors can 83 reduce the uptake of certain substrates (9-11). Alternative defense mechanisms which do not 84 involve mutational changes have been described and also play a role in V. anguillarum (Tan et al., 85 unpublished). Aggregate formation and production of exopolysaccharides was suggested to 86 provide protection against infection in V. anguilllarum strain PF430-3 (7, Tan et al., unpublished). 87 Recently, a more flexible protection mechanism was discovered in Escherichia coli, involving a 88 temporary down-regulation of phage receptor production in response to N-acyl-L-homoserine 89 lactone (AHL) cell-cell signaling molecules (12). This mechanism is controlled by quorum sensing 90 (QS), i.e., the ability of bacteria to regulate gene expression according to population density via 91 the production and subsequent detection of extracellular signaling molecules (13).

92 Little is still known about the mechanisms of phage protection in natural Vibrio communities. The 93 universal outer membrane protein K (OmpK) has previously been shown to be the infection site 94 for vibriophage KVP40, which infects more than 8 Vibrio species, including V. anguillarum, V. 95 parahaemolyticus, V. harveyi and V. cholerae (14, 15). Mutations in this protein have been 96 reported in V. parahaemolyticus R4000 upon exposure to KVP40 (14), but were not detected in 97 KVP40-amended cultures of V. anguillarum PF430-3 (7, Tan et al., unpublished). Thus, other 98 mechanisms for preventing infection by KVP40 in the Vibrio community may also be prevalent.

99 AHL-mediated QS circuits have been identified in many Vibrio species including V. anguillarum (13, 100 16, 17). V. anguillarum controls QS-regulated genes via the transcription factor VanT, which is 101 activated in response to extracellular signaling molecules (18, 19). At low autoinducer

102 concentrations (i.e. low cell density), the response regulator VanO becomes activated by 103 phosphorylation, and represses the expression of VanT. At high cell densities, autoinducer 104 concentrations increase and bind to membrane-bound receptors (18, 19). At a certain threshold 105 concentration, VanO is dephosphorylated, and VanT expression is induced, allowing gene 106 regulation within the QS regulon (18, 19). Several N-Acyl homoserine lactone autoinducers have 107 been identified in stationary-phase V. anguillarum spent culture supernatant (20).

108 As densities of bacterial populations vary from sparsely populated environments to highly dense 109 populations in nutrient-rich environments, and phages require a bacterial host in order to 110 multiply, phage predation pressure is not constant and may be expected to correlate with the 111 density of the bacterial host population, among other factors. Thus, bacteria could potentially 112 benefit from altering their anti-phage strategies depending on the perceived population density, 113 thereby minimizing the metabolic burden often associated with resistance by genetic mutation (3, 114 9, 21, 22).

115 In this study, we identified a potent anti-phage defense mechanism in V. anguillarum, and show 116 that different anti-phage strategies prevail at different population densities. At high cell density 117 conditions, QS-mediated down-regulation of the OmpK receptor reduced phage adsorption and 118 rendered individual cells almost unsusceptible to phage infection. At low cell density conditions, 119 on the other hand, OmpK expression was unaffected by QS and the individual cells were fully 120 susceptible to infection. However, at these conditions, we have shown in a previous study that 121 aggregation of the cells prevents phage from reaching the OmpK receptor (7, Tan et al., 122 unpublished). In neither case was phage protection associated with ompK mutation. Overall, the 123 study shows that QS controls the choice of anti-phage defense strategy in the examined V. 124 anguillarum strain PF430-3 suggesting the presence of dynamic, temporary adaptations to phage 125 infection pressure, while still securing the ability to produce a functional OmpK receptor. In a 126 phage therapy context, these results are highly relevant, as a combination of phage-based and 127 anti-QS targeted treatments could enhance the efficiency of phage control of vibriosis.

128 Results

129 We have recently demonstrated that addition of phage KVP40 to V. anguillarum strain PF430-3 130 resulted in increased cell aggregation and biofilm formation, which provided protection against

131 phage infection (7, Tan et al., unpublished). However, at high cell densities, detachment of cells 132 from aggregates into free-living variants were observed, which co-existed with phages (Tan et al., 133 unpublished), suggesting that alternative defense mechanisms also played a role under these 134 conditions. Isolation and subsequent re-culturing of these free-living cells in the absence of KVP40 135 showed that the re-cultured cells had regained sensitivity to phage KVP40 (7), suggesting that the 136 initial protection was not caused by mutational changes but rather a temporary protection 137 mechanism. To test if an extracellular factor might be involved in the regulation of the phage- 138 induced aggregation phenotype, we examined the effect of adding cell-free spent culture fluid 139 from high-cell-density cultures of strain PF430-3 to freshly inoculated cultures of the same strain 140 in the presence or absence of phage KVP40 by phase-contrast microscopy (Fig. 1, left panels). 141 Intriguingly, the phage-induced aggregation phenotype of PF430-3 was completely inhibited by 142 the presence of the cell-free spent culture fluid, suggesting that an extracellular factor(s), possibly 143 a QS signaling molecule, is involved in the regulation of phage defense in V. anguillarum PF430-3.

144 Synthetic AHL-induced down-regulation of phage production

145 V. anguillarum have been shown to produce at least three different AHL QS autoinducers, namely 146 N-(3-hydroxyhexanoyl) homoserine lactone, N-(3-oxodecanoyl) homoserine lactone, and N- 147 hexanoylhomoserine lactone (20). To further evaluate the role of QS for phage-host interactions in 148 V. anguillarum, the effect of synthetic AHL addition on phage KVP40 production was quantified in 149 strain PF430-3 (Fig. S2). The results showed that the presence of synthetic AHL autoinducers in the 150 medium reduced phage KVP40 production by 1.5 to 2-fold after 2 h relative to control cultures 151 without AHL addition (Fig. S2, squares). In parallel cultures grown without phage addition, cell 152 growth was unaffected by AHL addition (Fig. S2, circles).

153 KVP40 efficiency of plating on QS mutants of PF430-3

154 In order to examine the effects of QS on the interaction between phage KVP40 and V. anguillarum 155 PF430-3, two otherwise isogenic QS mutants were constructed which represent cell behavior at 156 high (ΔvanO) or low (ΔvanT) cell densities, respectively. Since the phosphorylated VanO protein 157 represses the QS regulator VanT at low cell densities, ΔvanO displays full expression of VanT at all 158 densities, and is therefore locked in a high-cell-density phenotype (18). By contrast, a ΔvanT 159 mutant has lost the ability to regulate QS-associated functions and is therefore locked in a low-

160 cell-density phenotype. Use of the two extreme QS-phenotypes of ΔvanT and ΔvanO mutants in 161 the current study allowed us to explore the role of QS-mediated phage protection in V. 162 anguillarum PF430-3. We first tested the infectivity of phage KVP40 on the wild-type V. 163 angullarum PF430-3 strain, and the constructed mutants ΔvanT, ΔvanO, ΔompK, ΔvanT ΔompK, 164 and ΔvanO ΔompK by examining the efficiency of plating (EOP, the number of plaques obtained on 165 each host from a given phage input). Relative to the wild-type strain, the phage susceptibility of 166 the ΔvanT mutant had increased by 53% (p< 0.05, n=2), and the plaque morphology had changed 167 from turbid to clear (Fig. S3A). Additionally, the ΔvanO mutant had become less susceptible to 168 phage infection with a reduction of 37%, (p<0.01, n=2) relative to wild-type (Fig. S3B). As expected, 169 ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK had all become resistant to KVP40 confirming that 170 ΟmpK is the phage receptor (Fig. S3Α).

171 Phage-host interactions in liquid cultures

172 Further assessments of QS-mediated regulation of phage infectivity in 10 h infection experiments 173 with phage KVP40 and 3 hosts (wild-type, ΔvanT, and ΔvanO), showed strong effects of the 174 mutational changes on phage-host interactions. Phage KVP40 reduced cell density in the wild-type 175 and ΔvanT cultures within 10 h incubation by 60% and 75%, respectively, relative to the OD of 176 control cultures without phages, with significantly lower OD values in the ΔvanT culture than in 177 the wild-type culture (0.62 and 0.88 after 10 h, respectively) (p<0.01, n=2; Fig. 2A). In the ΔvanO 178 culture, on the other hand, OD was not significantly affected by phage addition (Fig. 2A). In 179 accordance with these results, we observed rapid phage propagation in the wild-type and ΔvanT 180 cultures where KVP40 abundance stabilized at ~1010 PFU ml-1, whereas the phage production was 181 ~100-fold lower in the ΔvanO culture (Fig. 2B). We note that the minimum OD values of 0.33 and 182 0.15 after 5 h in the phage-treated wild-type and ΔvanT cultures, respectively, was followed by a 183 regrowth of cells during the remainder of the incubation (Fig. 2A), despite the abundance of 184 KVP40 phage.

185 Aggregation of cells in the two QS mutants following phage KVP40 exposure was assessed by 186 phase-contrast microscopy (Fig. 1, center and right panels; see also Fig. S1). Similar to the wild- 187 type shown in Figure 1, addition of phages led to increased aggregation in the ΔvanT mutant 188 cultures. In fact, at 24 h most cells occurred in large aggregates in the ΔvanT mutant cultures,

189 while a fraction of free-living cells was still detectable in the wild-type culture (Fig. 1 and Fig. S1). 190 In the ΔvanO culture, on the other hand, phage addition did not result in formation of aggregates 191 (Fig. 1 and Fig. S1), suggesting that the QS pathway is involved in the regulation of the phage- 192 induced aggregation phenotype. Furthermore, the addition of cell-free spent culture fluid, which 193 prevented aggregation of the wild-type culture, did not prevent aggregation of the ΔvanT mutant 194 cultures upon exposure to phage, suggesting that the inhibitory factor present in the cell-free 195 culture fluid prevents aggregation via the QS pathway (Fig. 1). However, addition of the synthetic 196 AHL autoinducers did not mimic the inhibitory effect of adding cell-free spent culture fluid on 197 phage-induced aggregation in wild-type PF430-3 (data not shown).

198 Effects of phage KVP40 and QS on biofilm formation

199 Based on the indications of phage-driven stimulation of biofilm formation in the short term liquid 200 culture experiments, a more systematic quantification of the effects of phage KVP40 on biofilm 201 formation in the three strains was performed. In the control cultures without phages, the biofilm 202 formation of the wild-type and ΔvanT strains increased for 5 days and then remained constant at a

203 level of OD595nm = 1.5-1.75, as measured by crystal violet staining of the biofilm. In ΔvanO cultures, 204 the biofilm developed at a slower rate but reached the same end point as the other strains after 8 205 days (Fig. S4A). Pretreatment of the cultures with KVP40 enhanced biofilm formation in all 3

206 strains. The OD595nm in wild-type and ΔvanT cultures reached values of 2.8 after 3 days, whereas

207 OD595nm in the KVP40-treated ΔvanO cultures increased gradually during the incubation to a 208 maximum of 2.2 after 8 days (Fig. S4A). Phage concentration in the liquid above the biofilm 209 increased rapidly and stabilized around 109 PFU ml-1 in both wild-type and ΔvanT cultures after 1 210 day. In the ΔvanO+KVP40 cultures, phage concentration increased at a slower pace until day 3, 211 where it also stabilized at ~109 PFU ml-1 (Fig. S4B).

212 Together, the results from these experiments demonstrate a reduced susceptibility to phage 213 KVP40 in the ΔvanO mutant despite the fact that this mutant aggregates less and forms biofilm 214 more slowly than the wild type, suggesting that V. anguillarum PF430-3 may obtain protection 215 against KVP40 via a separate mechanism in the high-cell-density QS mode mimicked by the ΔvanO 216 mutant.

217 Phage adsorption rate

218 To address the question of how ΔvanO mutant cells reduce their susceptibility to KVP40, we first 219 measured the rate of adsorption of KVP40 to the wild-type and QS mutants host cells. The 220 adsorption rate of wild-type, ΔvanT, and ΔvanO was calculated as 6.7x10-10 ± 8.49x10-11 ml-1 min-1, 221 8.1x10-10 ± 9.2x10-11 ml-1 min-1, and 3.8×10-10 ± 1.4x10-11ml-1 min-1, respectively. Thus, the rate of 222 adsorption of phage KVP40 to ΔvanO was significantly lower than that observed for the wild-type 223 (p<0.04, n=2) and ΔvanT (p<0.02, n=2) strains.

224 ompK expression in cultures of wild type and QS mutant cells

225 To further examine the mechanism underlying the differences in adsorption rates among wild- 226 type and QS-mutants (ΔvanT and ΔvanO), and to link these differences to QS regulation, we 227 quantified the expression of phage KVP40 receptor ompK in 9 different cultures (wild-type, ΔvanT, 228 ΔvanO, wild-type+KVP40, ΔvanT+KVP40, ΔvanO+KVP40, ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK) 229 by quantitative real-time PCR (qPCR). The relative ompK expression levels of wild-type and ΔvanT 230 strains were approximately 4 times higher than the relative ompK expression in the ΔvanO strain 231 suggesting a down-regulation of ompK, when the cells are locked in the regulatory state mimicking 232 high cell densities (Fig. 3A). Purification and separation of outer membrane proteins by SDS-PAGE 233 (Fig. 3B) using three ompK mutants (ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK) as negative 234 controls confirmed the presence of the OmpK receptor (26 kDa) in wild-type and ΔvanT strains 235 and the QS-mediated OmpK receptor down-regulation in the ΔvanO strain (14). In agreement with 236 the gene expression data, OmpK appeared to be slightly more abundant in the ΔvanT strain than 237 in the wild-type strain. SDS-PAGE outer membrane protein analysis also demonstrated that other 238 outer membrane proteins than OmpK, which potentially function as phage receptors, were down 239 regulated in the QS mutant ΔvanO as well (Fig. 3B, OmpK-indicated by arrowhead).

240 ompK mRNA levels in the free-living and aggregated cell fractions

241 We have shown above that the ΔvanT mutant displays increased aggregation, and contains high 242 levels of ompK, whereas the ΔvanO mutant displays very little aggregation, but instead reduces its 243 susceptibility to phage KVP40 by expressing low levels of OmpK. Next, we verified whether these 244 two phenotypes are also correlated in the wild-type PF430-3 strain. We fractionated cultures 245 (wild-type+KVP40, ΔvanT+KVP40 and ΔvanO+KVP40) into a free-living and an aggregate fraction 246 by centrifugation, and compared relative ompK mRNA levels in the two fractions using qPCR (Fig.

247 4). The analysis revealed that ompK gene expression varied significantly between aggregated and 248 free-living cells in the wild-type strain, with approximately 2-fold higher ompK mRNA levels in the 249 bacteria located in aggregates than in the fraction containing free-living cells (Fig. 4). Moreover, it 250 should be noted that the applied centrifugation procedure does not result in complete separation 251 of the aggregates from the free-living cells; hence the difference in ompK mRNA levels between 252 the two fractions is likely underestimated. Interestingly, ompK mRNA levels were also higher in the 253 aggregated fraction of the ΔvanO mutant cells than in the corresponding free-living fraction, 254 although the ompK levels in both fractions were still much reduced compared to the wild-type or 255 ΔvanT cultures. Thus, even in the absence of a functional QS pathway, free-living cells express less 256 ompK mRNA than aggregated cells, which could either reflect the existence of a redundant VanO- 257 independent regulatory mechanism, or simply reflect a phage-mediated enrichment of cells with 258 low ompK mRNA expression among the surviving free-living cells. Unfortunately, we were unable 259 to collect free-living cells from ΔvanT cultures grown in the presence of phage, as any free-living 260 cells were lysed by the phage and the remaining cells were all aggregated. However, in agreement 261 with the data shown in Figure 3A (ompK mRNA levels in the presence of phage KVP40), ompK 262 mRNA levels were up-regulated in the ΔvanT aggregates even compared to the wild-type 263 aggregated fraction.

264 Temporal changes in ompK expression and AHLs production in wild type V. anguillarum PF430-3

265 In order to determine whether ompK expression was in fact repressed by a cell density-dependent 266 factor secreted by the V. angullarum PF430-3 wild-type strain, simultaneous measurements of 267 ompK expression and extracellular AHL levels were performed during growth of the wild-type 268 strain in batch culture in the absence of phages. Interestingly, phage receptor ompK expression 269 and AHL levels were significantly negatively correlated (r=0.811, p≤0.001, n=2) during the first 12 h. 270 Thus, in accordance with the previous experiment, ompK expression was 3-4 times higher at the 271 low cell density around 2 h, than at high-cell density at 6 h (Fig. 5). AHL production reached a 272 maximum at 6 h and decreased after 8 h, concomitant with an increase in ompK expression. After 273 16 h a sharp decline in ompK expression was observed.

274 Examination of potential fitness costs in ΔompK mutants

275 Throughout our studies of the interaction of phage KVP40 with V. anguillarum strain PF430-3, we 276 did not once observe a single case of resistance to phage infection due to mutation of ompK. By 277 contrast, cells that survived exposure to phage KVP40 always multiplied to produce offspring that 278 had regained phage sensitivity (7, Tan et al., unpublished), Therefore, we speculate that mutation 279 of ompK may be associated with a significant fitness cost. The physiological role of OmpK is not 280 known, but BIOLOG GN2 physiological fingerprints of the wild-type strain and the ΔompK mutant 281 showed that the ΔompK mutant lost the ability to utilize glucose-1-phosphate, glucose-6- 282 phosphate, cis-aconitic acid, L-alaninamide, and L-alanine as growth substrates, suggesting that 283 indeed OmpK may play an important role for V. anguillarum metabolism (Fig. S5). We were, 284 however, unable to detect a change in the relative abundance of the ΔompK mutant compared to 285 wild-type after 8h of growth in mixed culture. Thus, loss of the OmpK receptor does not directly 286 affect the competitive ability of strain PF430-3 under our experimental conditions (Fig. S6).

287 Discussion

288 Given the recent findings that bacteria may employ QS to reduce phage receptor expression 289 during conditions of high infection risk (12), we aimed at exploring the role of QS in regulating 290 susceptibility to the broad-host-range phage KVP40 in V. anguillarum strain PF430-3 through 291 down-regulation of the phage receptor OmpK. The reduced susceptibility to phage KVP40 in V. 292 anguillarum strain PF430-3 after addition of cell-free supernatant or synthetic AHLs, provided the 293 first indications of a QS-regulated mechanism of phage protection. This was further supported by 294 our subsequent studies of phage susceptibility in the two constructed V. anguillarum QS mutants, 295 ΔvanT and ΔvanO. These results showed that phage susceptibility and adsorption was reduced in 296 the ΔvanO and enhanced in ΔvanT relative to the wild type, directly confirming that QS played a 297 key role in the protection against KVP40 infection. The >100–fold higher phage production and 3- 298 fold lower bacterial density in cultures of ΔvanT relative to ΔvanO after phage addition thus 299 emphasized that QS signaling in the high-cell-density phenotype mediated an efficient protection 300 against phage infection.

301 Measurements of the ompK expression further resolved the underlying mechanisms of QS- 302 mediated phage protection. Thus, the significant (4-fold) reduction in ompK expression in the 303 ΔvanO and the verification that an OmpK receptor deficient mutant (ΔompK) was resistant to

304 phage KVP40 provided direct evidence for QS-mediated down-regulation of ompK expression as an 305 important mechanism for protection against phage infection in V. anguillarum.

306 Interestingly, our results also showed that down-regulation of ompK expression was not the only 307 QS-regulated phage defense mechanism in the investigated V. anguillarum strain. Cell aggregation 308 and formation of a biofilm in response to phage addition in the wild-type strain suggested that the 309 transformation from a free-living life form to growth in a biofilm was also a mechanism of 310 protection against phage infection, as also supported by a previous study (Tan et al., unpublished). 311 The exact mechanism by which phages induce cell aggregation, however, remains to be discovered. 312 While the production of extracellular polymers by the host to create a physical barrier against 313 phage infection has been suggested previously (3, 23), the current results suggest that this 314 mechanism is QS-controlled in V. anguillarum. Addition of phage KVP40 strongly stimulated cell 315 aggregation and biofilm formation in the wild-type and low-cell-density mutant (ΔvanT), whereas 316 no aggregation was observed in the high-cell-density mutant (ΔvanO). Hence the data suggest that 317 cell aggregation and biofilm formation is an important phage defense mechanism at low cell 318 densities where QS regulation of ompK expression is inefficient. Consequently, we show that V. 319 anguillarum PF430-3 alternates between these two types of phage defense mechanisms and that 320 QS regulates the choice of strategy by up-regulating one mechanism (removal of OmpK receptor) 321 while down-regulating another mechanism (cell aggregation) at high cell densities. Whether the 322 overall reduction in OmpK receptors at high-cell-density conditions results in a general decrease in 323 the number of receptors per cell in the population, thus reducing the encounter rate between 324 phages and OmpK receptors, and/or if OmpK down-regulation generates a fraction of cells 325 completely without receptors (i.e. resistant cells) is not known. However, the presence of turbid 326 plaques in the ΔvanO mutant upon exposure to KVP40 in spot assays indicated that some of the 327 cells were, in fact, temporarily fully resistant to the phage. The presence of two distinct phage 328 defense mechanisms in wild-type cells was confirmed by the observation that ompK expression 329 was reduced in free-living cells relative to cells embedded in aggregates. Together with the 330 significant negative correlation between extracellular AHL concentration and ompK expression 331 during growth of the wild-type strain in batch cultures, this demonstrated the dynamic nature of 332 phage defense and emphasized that the obtained results were not restricted to the extreme 333 phenotypes of the QS mutants. Interestingly, ompK expression was in fact enhanced in phage-

334 amended wild-type and ΔvanT cultures relative to control cultures without phages. OmpK is a 335 porin-like protein which has been suggested to be involved in bile salt resistance, as well as iron 336 acquisition (24). We show here that OmpK also plays a role for the ability of V. angulillarum to use 337 glucose-1-phosphate, glucose-6-phosphate, cis-aconitic acid, L-alaninamide, and L-alanine as 338 growth substrates. We speculate therefore that up-regulation of ompK in aggregated cells may be 339 an adaptation to stimulate nutrient uptake in an aggregate environment where supply of specific 340 nutrients may be limited, or for enhancing the bile salt resistance of the cell aggregates inside the 341 host. However, in the short-term competition experiment we carried out, the ΔompK mutant was 342 able to reach the same density as the wild-type strain in a mixed culture. Further studies are 343 needed to determine the physiological role(s) of OmpK and hence the cost of its repression or loss.

344 We note that in the wild-type cultures of V. anguillarum PF430-3, AHLs accumulate as the culture 345 grows to high cell densities, but they largely disappear later in stationary phase (Fig. 5). This 346 phenomenon has been observed previously in cultures of Yersinia pseudotuberculosis and 347 Pseudomonas aeruginosa, grown in LB medium, and was found to be caused by pH-dependent 348 lactonolysis (25). We found, however, that the pH of the marine broth cultures used here 349 remained stable around 7.6 for at least 13 hours (data not shown). Alternative explanations for 350 the disappearance of the AHLs in stationary phase are the possible production of an AHL lactonase 351 enzyme as reported in Bacillus spp. (26) or the uptake of AHLs for use as a source of energy, 352 carbon and nitrogen, as reported for Variovorax paradoxus (27). Additional studies are required to 353 determine the mechanism responsible for AHL removal in V. anguillarum strain PF430-3.

354 One surprising observation from the current study was that QS reduced biofilm formation (Fig. S4). 355 Biofilm formation is in many bacterial pathogens found to be up-regulated by QS and stimulated at 356 high cell density promoting virulence (28). This has also been observed in V. anguillarum (strain 357 NB10), where a low-cell-density mutant (ΔvanT) showed significantly lower biofilm formation than 358 the wild-type strain (29).The reason for this contrasting effect of QS on biofilm formation in PF430- 359 3 is not clear. V. anguillarum strain NB10 showed limited susceptibility to phage KVP40 and 360 produced turbid plaques (data not shown), suggesting that KVP40 is not an important predator on 361 that strain; hence other anti-phage strategies may be favored. VanT expression is known to 362 regulate physiological responses required for survival and stress response (18), and in the

363 investigated strain PF430-3, this seems to include shifting from aggregation to ompK down 364 regulation in response to increased cell density. In addition, since total protease activity correlates 365 with VanT expression in V. anguillarum strain NB10 (29), it may be speculated that at high cell 366 densities, when VanT is fully expressed, VanT activates protease activity to promote the release of 367 cells from aggregates, in a manner similar to the action of the VanT homologue HapR in V. 368 cholerae, where QS also down-regulates biofilm formation (30, 31). By QS-mediated ompK gene 369 regulation, aggregate-associated bacteria may thus have evolved a mechanism of detaching a 370 subpopulation of free-living cells with reduced phage susceptibility which can survive in phage 371 dense environments, thus allowing further spreading of vibriosis infections. In any case, the results 372 support previous indications of large differences in phage defense strategies among different 373 strains of V. anguillarum (Tan et al., unpublished) and emphasize the need for further exploring 374 whether the mechanisms described here are a general phenomenon in bacteria or are limited to a 375 subset of V. anguillarum strains.

376 Phages are known to be key drivers of bacterial evolution by selecting for phage-resistant mutants, 377 and it has been proposed that phage-host interactions lead to either an arms race dynamics of 378 antagonistic host and phage co-evolution, or a fluctuating selection dynamics involving frequency- 379 dependent selection for rare host and phage genotypes (32, 33). In this study, however, we found 380 evidence for an alternative to these scenarios, as the defense mechanisms identified do not 381 involve mutational changes or complete elimination of phage susceptible host cells. The described 382 anti-phage mechanisms in V. anguillarum thus represent more flexible adaptations to dynamic 383 changes in phage and host densities, adding to the complexity of phage-host co-evolutionary 384 interactions and phage-driven genetic and phenotypic changes in bacterial populations. Since 385 mutational changes often result in a loss of fitness for the host (9, 10), the mechanisms described 386 here represent a potential fitness advantage for the host. It may be speculated that the 387 development of this temporary protection mechanism in V. anguillarum PF430-3 is related to the 388 fact that the OmpK receptor is widely conserved among Vibrio and Photobacterium species, and 389 thus probably executes an important function(s) in the cell. It is likely, therefore, that mutations in 390 ompK could have significantly negative effects on host fitness in natural habitats, in which case 391 development of alternative phage-defense strategies that leave the ompK gene intact would be of 392 strong selective advantage. Further studies are needed, however, to confirm this hypothesis.

393 Successful application of phage therapy in the treatment of vibriosis requires detailed knowledge 394 of the phage-host interactions and the regulation of anti-phage strategies in Vibrio. Our results 395 add to the suite of known phage defense mechanisms and their regulation in marine bacteria and 396 further emphasizes that the complexity of phage-host interactions pose a challenge for future use 397 of phages in disease control. On the other hand, the evidence that QS regulates phage receptor 398 expression may potentially be used actively by quenching QS signaling, hence preventing receptor 399 down-regulation. In support of that, QS inhibitors have been shown to impede expression of 400 virulence factors (34) and recently the addition of modified T7 phages producing quorum- 401 quenching enzymes have resulted in inhibition of biofilm formation, suggesting that the use of QS- 402 inhibitors may be a promising strategy in future antimicrobial therapy (35).

403 Materials and Methods

404 Bacterial strains and bacteriophages

405 The bacterial strain V. anguillarum PF430-3 was originally isolated from salmonid aquaculture in 406 Chile (36), and phage KVP40 which infects PF430-3 is a broad-host-range phage originally isolated 407 from Japan (37) (Table S1). Basic characterization of phage KVP40 and its interactions with PF430- 408 3 has been provided recently (7). All the constructed mutant strains derived from V. anguillarum 409 PF430-3 wild-type strain and plasmids used in this study are listed in supplementary Table S1. E. 410 coli S17-1 (λpir) was used as the donor strain for transfer of plasmid DNA into V. anguillarum 411 PF430-3 by conjugation (29, 38). Antibiotics were used at the following concentration: 100 μg ml-1 412 ampicillin, 25 μg ml-1 chloramphenicol (for E. coli), and 5 μg ml-1 chloramphenicol (for V. 413 anguillarum) (29, 38).

414 DNA manipulation and mutant construction

415 For mutant constructions, in-frame deletions were made in the vanT, vanO, and ompK genes by 416 allelic exchange as first described by Milton and Croxatto (29, 38). The upstream DNA sequence of 417 the vanT gene was amplified from wild-type V. anguillarum PF430-3 by PCR using primers vT1 and 418 vT2, which introduced a BglII site. The downstream DNA sequence of vanT was amplified using 419 primers vT3 and vT4, which introduced a SacI site. These two fragments, which contain a 15-nt 420 overlap of identical sequence, were used as template for a second PCR using primers vT1 and vT4.

421 The PCR product was digested with BglII and SacI and cloned into the BglII and SacI sites of pDM4, 422 creating pDM4vanT. In-frame deletion of vanT in PF430-3 was confirmed by PCR using primers vT1 423 and vT4 and subsequent gel electrophoresis. To construct plasmid pDM4vanO and pDM4ompK, 424 and vanO and ompK mutants, we used the same method described for vanT above. The primers 425 are listed in supplementary Table S2.

426 Examination of fitness loss in the ompK mutant

427 In an attempt to search for potential functions of phage receptor OmpK and hence implications of 428 its down regulation or loss, BIOLOG GN2 Micro-Plates (BIOLOG) containing 95 different carbon 429 sources were used to test the ability of wild-type strain and ΔompK mutant to use different 430 substrates following the manufacturer’s instructions. Further, the potential reduction in 431 competitive abilities of the ΔompK mutant was examined in competition assays. Briefly, wild-type 432 and ΔompK bacterial cells were mixed in a ratio of 1:1 in sterilized seawater and then quantified 433 over time. Subsamples were collected every 1-2 h for 8 h and diluted and plated on LB plates. 434 Subsequently, 20 colonies were picked from each time point and identified as either wild-type or 435 ΔompK by PCR with Primers 1340 F and 1340R listed in Table S2.

436 Effects of cell-free supernatant enrichment on KVP40 phage infection

437 Cell-free supernatant from wild-type V. anguillarum PF430-3 cultures was prepared from a mid- 438 log-phase culture of V. anguillarum PF430-3 in Marine Broth (MB) , which was centrifuged 439 (10,000×g for 10 min), sterile filtered (0.2 μm, Millipore) and subsequently added to freshly 440 inoculated cultures of wild-type, ΔvanT and ΔvanO, with and without phage KVP40, for 441 examination of the potential effects of V. anguillarum PF430-3 -produced autoinducer molecules 442 on the lytic effects of the phage KVP40. Images were obtained after 24 h incubation.

443 Effects of synthetic AHLs on phage production in V. anguillarum PF430-3 wild type

444 Synthetic AHLs (obtained from Sigma and Nottingham University), N-hexanoylhomoserine lactone 445 (C6-HSL), N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL) and N-(3- 446 Oxododecanoyl)-L-homoserine lactone (3-oxo-C10-HSL) were added to V. anguillarum PF430-3 to 447 examine their effects on phage susceptibility. Briefly, the AHLs were dissolved in acidified ethyl 448 acetate (0.1% v/v acetic acid), mixed and added to a glass test tube to a final concentration of 10

449 μM and placed at room temperature for complete evaporation of the solvent, as described 450 previously (12, 39). For the control cultures without AHLs, test tubes containing only ethyl acetate 451 were prepared in parallel. Overnight cultures of wild-type cells were pelleted and resuspended, 452 and aliquots of 100 μl cell suspension were transferred to 5 ml 3% NaCl buffer in tubes containing 453 AHLs or the solvent control, and incubated at 30 °C for 1 h. The phage susceptibility assay was 454 then initiated by addition of phage KVP40 at an average phage input (API) of 1. Parallel control 455 cultures without phages were also established. Samples were collected every hour for 7 h for 456 quantification of possible effects of AHL on cell growth in the absence of phage (colony forming 457 units, CFU) and on phage production (plaque forming units, PFU).

458 Phage-host interactions and effects on cell aggregation in wild-type and QS-mutants

459 The susceptibility of each strain (wild-type, ΔvanT, ΔvanO, ΔompK, ΔvanT ΔompK, and ΔvanO 460 ΔompK) to phage KVP40 was gauged by the efficiency of plating. Briefly, a fixed quantity of phage 461 obtained from the same phage stock (KVP40) was mixed with identically grown bacterial cells of 462 each genotype, and the plaque forming units were quantified by plaque assay (40). The 463 experiments were performed in triplicate.

464 The lytic potential of phage KVP40 against wild-type and QS-mutants (ΔvanT and ΔvanO) was 465 tested at an API of 1 in duplicate 100 ml liquid MB cultures and parallel control cultures without

466 phage. The effect of phage-mediated lysis on host cell density was monitored by regular OD600nm 467 measurement over the 10 h incubation. Phage concentration was quantified by plaque assay 468 periodically (40).

469 For visual inspection of cell densities and aggregate formation, additional aliqouts were collected 470 at 24 h, and visualized by phase-contrast microscopy using an oil immersion objective (Olympus 471 BX61). The images shown in Figure 1 and Figure S1 represent random fields on the microscope 472 slide from the 24 h samples.

473 Effects of phage addition on biofilm formation in wild-type and QS-mutants

474 Biofilm formation was monitored in wild-type, ΔvanT, and ΔvanO cultures following addition of 475 phage KVP40 using methods described previously (41) with some modifications. Briefly, 13 ml 476 polypropylene plastic tubes filled with 5 ml MB were inoculated with 100 μl overnight bacterial

477 inoculum and 100 μl phage stock (108 CFU ml-1 and 106 PFU ml-1, API=0.01) and incubated without 478 shaking along with parallel control cultures without phages. For each experiment, duplicate tubes 479 were examined daily for quantification of phage abundance in the liquid phase (40) and biofilm 480 biomass was quantified by crystal violet staining using a standard protocol (42). For biofilm 481 quantification, the liquid was removed, and tubes were rinsed twice with artificial seawater (ASW, 482 Sigma). The biofilm was stained with 0.4% crystal violet (Sigma) for 15 min and the tubes were 483 washed with tap water to remove excess stain. An aliquot (6 ml) of 33% acetic acid (Sigma) was

484 added and left for 5 min to allow the stain to dissolve. The absorbance was measured at OD595nm.

485 Bacteriophage adsorption rate

486 The rates of adsorption of phage KVP40 to wild-type, ΔvanT, and ΔvanO cells were determined by 487 mixing phage KVP40 and exponentially growing host cultures at an API of 0.01 and incubating at 488 room temperature for 32 min with agitation. Aliquots were removed periodically, centrifuged at 489 18,000×g at 4°C for 2 min, and the supernatants were immediately diluted to prevent additional 490 adsorption. The un-adsorbed phage particles were quantified by plaque assay (40).

491 Outer membrane protein K (OmpK) preparation and SDS-PAGE analysis

492 OMP preparation was made essentially according to Wang (43) with some modifications. Briefly, 493 cells were pelleted from 25 ml overnight cultures of all the strains (wild-type, ΔvanT, ΔvanO, 494 ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK), resuspended in 4.5 ml water, and sonicated on ice 495 (100 amplitude, 3 min). To solubilize the cytoplasmic membranes, sarkosyl (N-lauroyl sarcosine) 496 was added to a final concentration of 2%, and incubated at room temperature for 30 min. To 497 pellet the outer membranes, the mixture was ultra-centrifuged (400,000 rpm for 1 h at 4 °C, SW 498 55 Ti, Beckman). The pellet was washed with ice-cold water and ultra-centrifuged again (400,000 499 rpm for 30 min at 4 °C, SW 55 Ti, Beckman). The pellet was resuspended in 100 μl 100 mM Tris-HCl 500 (pH 8) and 2% SDS buffer, and the proteins were separated on a 10% SDS-polyacrylamide gel and

501 stained with Coomassie blue.

502 Fractionation of V. anguillarum cultures in aggregate and free-living cell fractions

503 In order to examine ompK gene expression patterns in aggregates and free-living cells, respectively, 504 cultures were fractionated by centrifugation and ompK expression was determined in the 505 aggregate and non-aggregate fractions. Cultures of V. anguillarum wild-type and QS mutants were 506 grown in MB in the presence of phage KVP40 at an API of 1 at RT for 12 h, and the aggregates 507 were collected by centrifugation (1,000×g, 10 min). The supernatant was transferred to a new 508 tube and centrifugation was repeated. After the second round of centrifugation, the free-living 509 cells in the supernatant was transferred to a new tube and collected by centrifugation at 10,000×g 510 for 10 min. Pellets of cell aggregates and free-living cells were collected and stored at -80 °C for 511 subsequent RNA extraction.

512 AHL signal levels in cultures of wild-type V. anguillarum PF430-3.

513 An overnight culture of the wild-type strain was diluted in MB to the final OD600nm of 0.04 and 514 grown at 30 °C for 20 h with agitation. Samples were taken every 2 h for determination of AHL 515 production and for extraction of RNA for ompK mRNA quantification. AHL biosynthesis was 516 assayed using E. coli SP436 reporter strain (44). E. coli SP436 harbors a plasmid-borne fusion of the 517 V. fischeri lux operon to gfp, and responds to AHL by expressing green fluorescence protein (GFP) 518 as described previously (44). Briefly, 50 μl cell-free supernatant was mixed with 150 μl LB medium 519 and 10 μl of an overnight culture of SP436 (100 μg ml-1 ampicillin), and further incubated at 30 °C 520 for 5 h with agitation using a FLUOstar Omega microtiter plate reader (BMG Labtech) with gain 521 setting of 1150.

522 RNA extraction and cDNA synthesis

523 For quantification of ompK expression in the wild-type and QS mutants (ΔvanT and ΔvanO), RNA 524 was extracted from cells pelleted after 9 h incubation in MB for the experiments shown in Figure 5, 525 or as described above for the experiments shown in Figure 3 and 4. Total RNA was extracted using 526 Trizol (Invitrogen) and chloroform extraction as described previously (45). Genomic DNA was 527 removed by adding DNase I according to the manufacturer’s protocol (Fermentas). RNA was 528 stored at -80 °C until further use.

529 Reverse transcription was performed with the Thermo Scientific Revert Aid First Strand cDNA 530 Synthesis Kit as described by the manufacturer (Thermo). The first cDNA strand was obtained

531 using random hexamer primers with 1000 ng of DNase I-treated RNA. Control samples (-RT) were 532 treated identically except that the reverse transcriptase enzyme was omitted.

533 Real-time PCR gene expression quantification

534 The relative expression levels of ompK and recA (as the endogenous control) were determined by 535 quantitative RT-PCR performed in a CFX96 real-time PCR detection system (Bio-Rad), using 536 SsoAdvanced SYBR Green Supermix (Bio-Rad), and the gene-specific primers listed in 537 supplementary Table S2 (46). qPCR reactions were set up in 25 μl containing 0.2 μM of primers, 538 and SYBR Green qPCR mix with the following program, 30 s at 95 °C for denaturation, followed by 539 40 cycles of (4 s at 95 °C, 30 s at 55°C). Melting curve analysis was performed from 65 °C to 95 °C

540 with an increment of 0.5 °C for 5 s. The comparative CT method was used for relative 541 quantification of RNA (47).

542 Acknowledgements

543 The study was supported by the Danish Council for Strategic Research (ProAqua project 12-132390) 544 and the EU-IRSES-funded project AQUAPHAGE (269175). We thank Debra Milton, Umeå University, 545 for providing plasmid pDM4 and E. coli S1-17 and Pantelis Katharios, Hellenic Centre for Marine 546 Research, for providing V. anguillarum PF430-3 and vibriophage KVP40. We are grateful to Nina 547 Molin Høyland-Kroghsbo for many helpful discussions.

548 References

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664 Figure Legends

665 Figure 1. Visualization by phase-contrast microscopy of V. anguillarum strains PF430-3 (wild-type, 666 ΔvanT and ΔvanO) in the presence or absence of phage KVP40 in either fresh Marine Broth or cell- 667 free spent culture fluid obtained from a mid-log phase culture of wild-type V. anguillarum PF430-3.

668 Figure 2. (A) Optical density (OD600nm) of cultures of V. anguillarum wild-type and QS mutants 669 (ΔvanT and ΔvanO) in the presence or absence of phage KVP40 at API of 1 were measured at 1 h 670 intervals over a 10-h period incubation. (B) Corresponding abundance of PFU ml-1 were quantified 671 by plaque assay over a 10-h period incubation in wild-type+KVP40, ΔvanT+KVP40 and 672 ΔvanO+KVP40 cultures, respectively. Error bars represent standard deviations from all 673 experiments carried out in duplicate.

674 Figure 3. (A) ompK gene expression of wild type and QS mutants (ΔvanT and ΔvanO) in the 675 presence or absence of phage KVP40. (B) SDS-PAGE analysis of the outer membrane protein K 676 (OmpK) with Coomassie blue staining. Lane M, protein marker is shown on the right; lane 1-6, 677 wild-type, ΔvanT, ΔvanO, ΔompK, ΔompK ΔvanT, and ΔompK ΔvanO, respectvely. Phage receptor 678 ompK mRNA transcript levels were quantified by real-time PCR. “Relative gene expression” 679 corresponds to the level of ompK mRNA after normalization to the level of recA mRNA in the same 680 sample. Error bars represent standard deviations (duplicate samples).

681 Figure 4. Fractionation experiment. For bacterial aggregates (A), samples were harvested at 682 1,000×g for 10 min. For free-living bacteria (F), samples were harvested at 10,000×g for 10 min. 683 Phage receptor ompK mRNA transcript levels were quantified by real-time PCR. Relative gene 684 expression was normalized against the gene expression level of recA. Error bars 685 represent standard deviations (duplicate samples).

686 Figure 5. AHL assay and ompK expression quantification in the wild-type strain over time. Aliquots 687 of cultures were withdrawn every 2 h during growth for measurement of AHL production and 688 ompK gene expression by RT-PCR as described in the legend to Figure 3. Error bars 689 represent standard deviations (duplicate samples).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5 TABLE S1. Bacterial strains, bacteriophage and plasmids

Strains, phage or plasmids Genotype or relevant markers E. coli S17-1 thi pro hsdR hsdM+recA RP4-2-Tc::Mu-Km::Tn7 λpir SY327 Δ(lac-pro) argE(Am) rif malA recA56 λpir SP436 Apr V. anguillarum PF430-3 wt, isolated from Chile ΔvanT ΔvanT ΔvanO ΔvanO ΔompK ΔompK ΔompK ΔvanT ΔompK ΔvanT ΔompK ΔvanT ΔompK ΔvanT Plasmids Cmr; suicide vector with an R6K origin (pir requiring) and sacBR genes from pDM4 Bacillus subtilis pDTvanT Cmr; pDM4 derivative containing vanT fused in-frame pDTvanO Cmr; pDM4 derivative containing vanO fused in-frame pDT ompK Cmr; pDM4 derivative containing ompK fused in-frame r pAHL-GFP Ap , luxR-PluxI-gfpmut3 Phage KVP40 Genome 244 kb, Myoviridae, isolated from Japan

Table S2 Primers

Name Primers (5'-3) qPCR (gene) recA F-CTAGTCGAAATTTTTTCGACACAGC R-GGCTCGTTAAATTTTTTATCGACTCTT ompK F-TAGAGATGGAATTTGGCGG R-TTTGTCACTGCTTGGGTT vanT deletion vT1 F-TTTAGATCTCATCACTTCAACTGTATGTTTCATACTGTTC vT2 R-AAGTTAAGCCTAGTGCATGAGTTGTTAATCCTTGCCAATGAC vT3 F-CACTAGGCTTAACTTCCTAGTCTC vT4 R-TTTGAGCTCCGGTTTGGCTATCGAGATGG vanO deletion vO1 F-TTTAGATCTGAGATCGCCAAACTAGAAGGG vO2 R-TCTCTATGAGTTCTCGTCAGGTTGCATATTCTAGCCATT vO3 F-TTTGAGCTCCGGTTTGGCTATCGAGATGG Vo4 R-TTTGAGCTCCGGTTTGGCTATCGAGATGG ompK deletion oK1 F-TTTAGATCTGTAAGCCCCTAATTGAGT oK2 R-TAGAGAGTCCCCTTTTAATCGAACCTAAGTTTTGGCAAATG oK3 F-AAAGGGGACTCTCTATGTTTTGTCATAATTC oK4 R-TTTGAGCTCACAATCTTAACCAACATCCC

1340F F-TCGCCCAAAGCAACCCAA 1340R R-CCGTAGCTTCATTTTCCCTT

Figure S1. Examination of phage-host interactions (wild-type+KVP40, ΔvanT+KVP40, and ΔvanO+KVP40) in marine broth under phase-contrast microscopy after 24 h incubation. Images represent random fields obtained from microscopic slides prepared after 24 h incubation of the indicated V. anguillarum strain with phage KVP40.

Figure S2. Phage KVP40 production and bacterial growth upon exposure to synthetic AHLs. Shown are phage concentrations in the cell-free supernatant of cultures incubated with (closed symbols) or without (open symbols) 10 μm AHLs over the course of 7 h (squares). AHLs were added at time = -1h, and phage were added at an API of 1 at time = 0 h. To determine whether the AHLs affected bacterial growth in the absence of phage, the number of colony forming units (CFU) in parallel cultures +/- AHL but without phage addition were quantified (circles). Error bars represent standard deviations of experiments carried out in duplicate.

Figure S3. (A) Spot assay for testing host specificity of phage KVP40 against wild type, QS mutants (ΔvanT and ΔvanO) and ompK mutants (ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK) as well. Five μl serial 100-fold dilutions of phage KVP40 lysate (1, 109; 2, 107; 3, 105; 4; 103 PFU ml-1) were shown by spot titration onto top agar lawns of the indicated strains. (B) Plaque assay quantification by using wild type and QS-mutants (ΔvanT and ΔvanO) as host, respectively. Error bars represent standard deviations (duplicate samples).

Figure S4. (A) Biofilm formation in V. anguillarum wild-type and QS mutants (ΔvanT and ΔvanO) pre- treated with phage KVP40 (open symbols) and in controls without phage KVP40 addition (closed symbols) quantified by crystal violet (OD595nm) staining during 8 days incubation. (B) Viable phage KVP40 recovered from free-living phases of wt+KVP40, ΔvanT+KVP40 and ΔvanO+KVP40, respectively, were quantified by plaque assay for 7 days. Error bars represent standard deviations from all experiments carried out in duplicate. It should be noted that the quantification of KVP40 most likely represent an underestimation of the actual phage production as only detached phage particles were counted. It is likely therefore, that the actual phage concentration was higher in the wild-type and ΔvanT cultures than in the ΔvanO cultures as more phages were likely trapped in the more developed biofilm of those cultures. wild-type Δ ompk

C-source Bromosuccinic Acid + + L-Histidine + + D-Fructose + + β-Methyl- D-Glucoside + + Inosine + + Dextrin + + D-Psicose + + Citric Acid + + Uridine + + Thymidine + + Tween 40 + + Tween 80 + + α-D-Glucose + + D-Sorbitol + + D,L-Lactic Acid + + L-Proline + + Sucrose + + D-Gluconic Acid + + N-Acetyl-DGlucosamine + + α-D-Lactose + + D-Trehalose + + L-Asparagine + + L-Aspartic Acid + + L-Serine + + Maltose + + L-Glutamic Acid + + D-Mannitol + + D,L-α-Glycerol Phosphate + + D-Cellobiose + + D-Mannose + + Mono-Methyl-Succinate + + Succinic Acid + + Cis-Aconitic Acid + - Glucose-6-Phosphate + - L-Alaninaide + - L-Alanine + - Glucose-1-Phosphate + - D-Galactose + + Glycerol + + D,L-α-Glycerol Phosphate + + Hydroxy-L-Proline + + α-Keto Glutaric Acid + + D-Alanine + + L-Alanylglycine + + L-Threonine + + Propionic Acid + + Acetic Acid + + Urocanic Acid + +

Figure S5. Carbon substrate utilization (BIOLOG GN2) between wild-type strain and ΔompK mutant. +, positive reaction at 24 h; -, negative reaction after 48 h.

Figure S6. Competition assay between wild-type (balck) and ΔompK (grey) at a ratio of 1 was performed. Colonies were picked at intervals and subsequently identified by PCR. Each bar represents (percent plotted in y axis) the fraction of the mixture.