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Article Type: Original Article
Running head: Characterization of phage Phda1
Characterization of a novel bacteriophage, Phda1, infecting the histamine-producing Photobacterium damselae subsp. damselae
Shogo Yamaki, Yuji Kawai, and Koji Yamazaki* Laboratory of Marine Food Science and Technology, Faculty of Fisheries Sciences, Hokkaido University 3-1-1, Minato, Hakodate 041-8611, Japan
Correspondence: Koji Yamazaki, Laboratory of Marine Food Science and Technology, Faculty of Fisheries Sciences, Hokkaido University 3-1-1, Minato, Hakodate 041-8611, Japan. Tel and fax: +81 138 40 5574; e-mail: [email protected]
Abstract Aims: Photobacterium damselae subsp. damselae is a potent histamine-producing microorganism. The aim of this study was to isolate and characterize a bacteriophage Phda1 that infected to P. damselae subsp. damselae to inhibit its growth and histamine accumulation. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12809 This article is protected by copyright. All rights reserved.
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Methods and Results: Phda1 was isolated from a raw oyster, and the host range, morphology, and the bacteriophage genome size were analyzed. Phda1 formed a clear plaque only against P. damselae subsp. damselae JCM8969 among 5 Gram-positive and 32 Gram-negative bacterial strains tested. Phda1 belongs to the family Myoviridae, and its genome size was estimated as 35.2–39.5 kb. According to the one-step growth curve analysis, the latent period, rise period, and burst size of Phda1 were 60 min, 50 min, and 19 PFU infected cell-1, respectively. Divalent cations, especially Ca2+ and Mg2+, strongly improved Phda1 adsorption to the host cells and its propagation. Phda1 treatment delayed the growth and histamine production of P. damselae subsp. damselae in an in vitro challenge test. Conclusions: The bacteriophage Phda1 might serve as a potential antimicrobial agent to inhibit the histamine poisoning caused by P. damselae subsp. damselae. Significance and Impact of the Study: This is the first description of a bacteriophage specifically infecting P. damselae subsp. damselae and its potential applications. Bacteriophage therapy could prove useful in the prevention of histamine poisoning.
Keywords: Bacteriophage, Photobacterium damselae subsp. damselae, phage therapy, histamine poisoning
Introduction Photobacterium damselae is a halophilic marine bacterium belonging to the family Vibrionaceae. This species is further categorized into P. damselae subsp. damselae (formerly Vibrio damsela) and P. damselae subsp. piscicida (formerly Pasteurella piscicida), both of which are known fish pathogens This article is protected by copyright. All rights reserved.
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Phage propagation and purification Phda1 was propagated and purified by the polyethylene glycol precipitation method and CsCl density gradient centrifugation. P. damselae subsp. damselae JCM8969 was incubated with TSBS at 25°C. Phda1 was added to the host strain culture in the exponential phase at a multiplicity of infection (MOI) of 0.1, and incubated at 25°C. Following Phda1 growth, the culture was centrifuged (10,000 × g, 30 min, 4°C) and filtered through 0.45-µm polyvinylidene difluoride filters (Merck Millipore; Billerica, MA). DNase I and RNase A (Sigma-Aldrich; St. Louis, MO) were added at 1 µg ml-1 to degrade bacterial DNA and RNA, and the lysate was incubated at room temperature for 1 h. NaCl was added to a final concentration of 1 mol l-1 and the lysate was incubated for 1 h on ice. The mixture was treated with 10% polyethylene glycol 6000 (Wako pure chemical industries; Osaka, Japan) at 4°C for 12 h, then the Phda1 lysate was centrifuged (10,000 × g, 20 min, 4°C), and the pellet was suspended in SM buffer. An equal volume of chloroform was added to the phage suspension, followed by another centrifugation step (10,000 × g, 10 min, 4°C) to remove polyethylene glycol. Propagated Phda1 was purified by CsCl density gradient centrifugation (100,000 × g, 2 h, 4°C, d = 1.3, 1.5, and 1.6), and purified Phda1 was stored at 4°C for further investigations.
Host range analysis Bacterial lawns listed in Table 1 were generated by the double agar overlay method. Two hundred microliters of each strain culture were added to 3.5 ml of 0.5% molten agar, and the mixture was overlaid on plate medium. After solidification of the soft agar, 10 µl of Phda1 solution was spotted onto each bacterial lawn. Plates were incubated and examined for clear zone development on the bacterial This article is protected by copyright. All rights reserved.
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Bacteriophages (phages) are viruses that infect bacteria, and have lately been considered as one of the most important antimicrobial agents. Although the therapeutic potential of phages has not been focused on since the discovery of antibiotics, the emergence of antibiotic-resistant bacteria makes phages an attractive agent to counteract these bacteria (Kutateladze and Adamia, 2010). The application of phages as an antimicrobial agent in phage therapy has recently been investigated in various areas such as medicine, veterinary science, agriculture, aquaculture, and food science. In fact, phages infecting a variety of pathogens, including Campylobacter coli, Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, and Staphylococcus aureus, were investigated for their potential use as effective antimicrobial agents (Carvalho et al., 2010a; Fu et al., 2010; Gupta and Prasad, 2011; Park et al., 2012, Chibeu et al., 2013). At present, the United States Department of Agriculture and the Food and Drug Administration have approved LISTEXTMP100 and SALMONELEXTM (Micreos Food Safety, Wageningen, The Netherlands), which are a L. monocytogenes phage and Salmonella phages, respectively, as ‘Generally Recognized As Safe’ food processing aids to control food pathogens. Nonetheless, not many studies have been performed to date on the evaluation of phage utilization to suppress histamine poisoning. Therefore, we have investigated the isolation of phages infecting histamine-producing bacteria and characterized the M. morganii-infecting phage FSP1 in a previous study (Yamaki et al., 2014). In the present study, we demonstrate the isolation and characterization of a specific phage infecting P. damselae subsp. damselae, one of the most potent histamine producers in seafood, for its use as an antimicrobial agent.
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Materials and Methods
Bacterial strains and growth conditions Bacterial strains used in this study are listed Table 1. Halophilic bacteria were incubated with tryptic soy broth (TSB, BD; Franklin Lakes, NJ, USA) supplemented with 1.5% NaCl (TSBS); all other bacteria were incubated with TSB or TSB supplemented with 0.6% yeast extract (BD).
Isolation of P. damselae subsp. damselae-infecting phages Phages infecting P. damselae subsp. damselae were isolated by the method of Carvalho et al. (2010b) with minor modifications. Raw oysters were mixed with nine times their weight of TSBS containing 400 µg ml-1 CaCl2 and 400 µg ml-1 MgSO4. P. damselae subsp. damselae JCM8969 cells in the exponential growth phase were added to the mixture and incubated at 20°C for 24 h. Ten milliliters of the culture were then treated with 5 % (v/v) chloroform to lyse the bacterial cells and centrifuged (10,000 × g, 30 min, 4°C). The collected supernatant was used for phage detection. The supernatants were spotted on a lawn of P. damselae subsp. damselae JCM8969 made with the double agar overlay method using tryptic soy agar (TSA, BD) plates supplemented with 1.5% NaCl and 0.5% soft agar supplemented with 2.0% NaCl. The plates were incubated at 25°C for 24 h. Plaques were picked up and suspended in 1 ml of SM buffer (100 mmol l-1 CaCl, 8 mmol l-1 MgSO4·7H2O, 50 mmol l-1 Tris-HCl, and 0.01% gelatin, pH 7.5), and single plaques were formed using the plaque assay method. This procedure was repeated more than three times and the isolated phage was termed Phda1.
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Phage propagation and purification Phda1 was propagated and purified by the polyethylene glycol precipitation method and CsCl density gradient centrifugation. P. damselae subsp. damselae JCM8969 was incubated with TSBS at 25°C. Phda1 was added to the host strain culture in the exponential phase at a multiplicity of infection (MOI) of 0.1, and incubated at 25°C. Following Phda1 growth, the culture was centrifuged (10,000 × g, 30 min, 4°C) and filtered through 0.45-µm polyvinylidene difluoride filters (Merck Millipore; Billerica, MA). DNase I and RNase A (Sigma-Aldrich; St. Louis, MO) were added at 1 µg ml-1 to degrade bacterial DNA and RNA, and the lysate was incubated at room temperature for 1 h. NaCl was added to a final concentration of 1 mol l-1 and the lysate was incubated for 1 h on ice. The mixture was treated with 10% polyethylene glycol 6000 (Wako pure chemical industries; Osaka, Japan) at 4°C for 12 h, then the Phda1 lysate was centrifuged (10,000 × g, 20 min, 4°C), and the pellet was suspended in SM buffer. An equal volume of chloroform was added to the phage suspension, followed by another centrifugation step (10,000 × g, 10 min, 4°C) to remove polyethylene glycol. Propagated Phda1 was purified by CsCl density gradient centrifugation (100,000 × g, 2 h, 4°C, d = 1.3, 1.5, and 1.6), and purified Phda1 was stored at 4°C for further investigations.
Host range analysis Bacterial lawns listed in Table 1 were generated by the double agar overlay method. Two hundred microliters of each strain culture were added to 3.5 ml of 0.5% molten agar, and the mixture was overlaid on plate medium. After solidification of the soft agar, 10 µl of Phda1 solution was spotted onto each bacterial lawn. Plates were incubated and examined for clear zone development on the bacterial This article is protected by copyright. All rights reserved.
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lawns.
Transmission electron microscopy The morphology of Phda1 was analyzed with a transmission electron microscope. Phda1 solution (11 log plaque-forming units [PFU] ml-1) was dropped onto a 200-mesh Cu grid (Nisshin EM; Tokyo, Japan) for 5 min, and 2.5% samarium triacetate, an appropriate negative staining reagent reported by Nakakoshi et al. (2011), was dropped onto the grid. Phda1 was identified by observation with a transmission electron microscope (JEM-1011, JEOL; Tokyo, Japan).
Phage DNA analysis Phda1 was suspended in TENS buffer (50 mmol l-1 Tris-HCl, 100 mmol l-1 EDTA, 100 mmol l-1 NaCl, and 0.3% sodium dodecyl sulfate; pH 8.0; Stenholm et al., 2008). Phda1 was treated with 200 µg ml-1 proteinase K at 65°C for 10 min to break Phda1 virions, and purification of Phda1 DNA was performed with the Phage DNA Isolation Kit (Norgen Biotek; Thorold, Ontario, Canada) according to the manufacturer’s instructions. Purified DNA was digested with EcoRV, PstI, and PvuII (Nippon Gene; Tokyo, Japan) for genome size estimation according to the manufacturer’s instructions. Digested DNA patterns were analyzed with agarose gel electrophoresis using 0.5% SeaKem® gold agarose (Takara Bio; Shiga, Japan) and SYBR® green nucleic acid stain (Sigma-Aldrich). Gene ladder wide I (Nippon Gene) and 2.5-kb DNA ladder (Takara Bio) were used as DNA size markers. The total genome size of Phda1 was estimated from the sum of the sizes of all digested fragments.
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Analysis of adsorption kinetics P. damselae subsp. damselae JCM8969 was incubated at 25°C with TSBS. When the OD600 nm of the culture reached 0.2, Phda1 was inoculated at MOI = 0.1 and incubated at 25°C. Samples were collected chronologically and centrifuged (10,000 × g, 1 min, 4°C), followed by filtration with a 0.45-µm polyvinylidene difluoride filter to remove phages adsorbed to bacteria. The PFU of free phage in the supernatant was determined by a plaque assay technique using a TSA plate supplemented with 1.5% NaCl and 25 mmol l-1 MgSO4 as the bottom agar and 0.5% soft agar supplemented with 2.0% NaCl and 25 mmol l-1 MgSO4 as the top agar. The measurement of PFU of Phda1 was carried out by this plaque assay technique throughout this study unless otherwise noted.
One-step growth curve analysis P. damselae subsp. damselae was incubated at 25°C with TSBS. When the OD600 nm of the culture reached 0.2, Phda1 was added at MOI = 0.1 and incubated at 25°C for 5 min for attachment of Phda1 to the host cells. The culture was centrifuged (10,000 × g, 2 min, 4°C) to remove free phages, and the pellet was suspended in fresh TSBS. The suspension was incubated at 25°C, and samples were collected every 10 min. The PFU of Phda1 in the sample was measured by serial dilution and the plaque assay technique. The latent period, rise period, and burst size of Phda1 were determined by monitoring the changes in PFU.
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Evaluation of heat and pH stability The heat and pH stability of Phda1 was evaluated by observing the changes in PFU after various treatments. In order to examine the thermal stability of Phda1, the phages (107 PFU ml-1) were suspended in TSBS and incubated at 60–85°C for 30 min. Samples was collected every 5 min and the PFU of surviving Phda1 phages was determined by serial dilution and the plaque assay technique. For the assessment of pH stability of Phda1, the phages (107 PFU ml-1) were added to TSBS (pH 1.0–12.0, adjusted with 6 mol l-1 HCl or 10 mol l-1 NaOH), and incubated at 30°C for 24 h. The PFU of surviving Phda1 phages was determined by serial dilution and the plaque assay technique.
Effects of divalent cations on Phda1 growth The adsorption kinetics and the one-step growth curve of Phda1 were examined in the presence of divalent cations (Ca2+, Mg2+, Fe2+, Mn2+, and Zn2+). These experiments were performed with cation broth (Bacto tryptone 17 g l-1, BBL PhytoneTM peptone 3 g l-1, glucose 2.5 g l-1, NaCl 20 g l-1; pH 7.3) supplemented with 1–25 mmol l-1 CaCl2, MgSO4, FeCl2, MnCl2, or ZnCl2, and the effects of divalent cations on the growth of Phda1 were evaluated.
Challenge test Phda1 (MOI = 100) and P. damselae subsp. damselae JCM8969 (3 log CFU ml-1) were inoculated into cation broth supplemented with 25 mmol l-1 CaCl2 and 1% histidine hydrochloride (pH 6.5). The culture was incubated at 8°C, 12°C, or 20°C. Viable cell counts of P. damselae subsp. damselae JCM8969 were investigated by spreading the culture onto a TSA plate supplemented with 1.5% NaCl This article is protected by copyright. All rights reserved.
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and incubation at 25°C for 24 h. The histamine concentration in the broth was also determined simultaneously with high-performance liquid chromatography (Mett and Sturgeon, 1982). Samples collected from the culture broth were mixed with an equal volume of 10% trichloroacetic acid and filtered through 0.2-µm polytetrafluoroethylene filters (Millipore). Filtered samples were subjected to high-performance liquid chromatography.
Results
Isolation of the P. damselae subsp. damselae-infecting phage Phda1 and its host range We isolated a P. damselae subsp. damselae-infecting phage from a raw oyster and termed it Phda1. The host range of Phda1 was analyzed for 32 strains of gram-negative bacteria and 5 strains of gram-positive bacteria. Phda1 formed a plaque only against P. damselae subsp. damselae JCM8969. Eight other strains of P. damselae subsp. damselae did not form plaques (Table 1), demonstrating a very narrow host range of Phda1.
Morphology and genome size of Phda1 The morphology of Phda1 was analyzed using a negative staining method. Transmission electron microscope observation determined the head length of Phda1 as 62 ± 4.6 nm (mean ± SD) and the contractile tail length as 110 ± 4.9 nm (mean ± SD) (n = 32). Moreover, Phda1 featured contracted tails attaching to the host bacteria P. damselae subsp. damselae JCM8969 (Fig. 1a). Based on the morphological analysis, Phda1 belongs to the order Caudovirales and the family Myoviridae, which This article is protected by copyright. All rights reserved.
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comprises tailed phages with a neck and contractile tail. For the estimation of the Phda1 genome size, the phage genome was digested with the restriction endonucleases EcoRV, PstI, and PvuII. The digestion patterns of each enzyme showed that the extracted nucleic acid of Phda1 was linear, double-stranded DNA, and the Phda1 DNA featured many restriction sites for each of the tested enzymes. The genome size of Phda1 was estimated by the sum of the bands resulting from the digestion, and was determined as 35.2–39.5 kb (Fig. 1b).
Adsorption kinetics and one-step growth curve of Phda1 The adsorption analysis revealed that 13 ± 4 % (mean ± SD) of Phda1 adsorbed to host cells at 10 min, and 66 ± 1 % (mean ± SD) adsorbed at 1 h in TSBS (data not shown). Moreover, the one-step growth curve analysis showed that the latent period of Phda1 was 60 min, the rise period was 50 min, and the burst size was 19 PFU infected cell-1 (Table 3).
Thermal and pH stability of Phda1 The stability of phages under various environmental conditions is an important factor for their evaluation as potential antimicrobial agents. Phda1 was stable at 60°C, but the PFU of Phda1 decreased to 4.7 log PFU ml-1 after heating at 70°C for 30 min. In addition, Phda1 was not detected after heating at 75°C and 80°C for 20 min, and at 85°C for 5 min (Fig. 2a). Based on the pH stability analysis, Phda1 was stable between pH 4–10, but strong acidic (pH ≤ 3) and alkaline (pH ≥ 11) conditions caused a loss of infectivity of Phda1 within 24 h (Fig. 2b). In addition, Phda1 was stable after freezing at −20°C, −40°C, and −80°C for 4 weeks (data not shown) in TSBS. These results suggest that Phda1 could be a This article is protected by copyright. All rights reserved.
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suitable antimicrobial agent due to its favorable stability under various environmental conditions.
Growth enhancement of Phda1 with divalent cations In order to elucidate the effect of divalent cations on the adsorption of Phda1, the adsorption rate was determined in cation broth supplemented with 1 mmol l-1 CaCl2, MgSO4, FeCl2, MnCl2, or ZnCl2. Compared with the control (without divalent cations), the adsorption of Phda1 was greatly improved in the presence of 1 mmol l-1 Ca2+, Mg2+, Fe2+, and Mn2+, while Zn2+ had no enhancing effect (Fig. 3). The one-step growth curves were analyzed next in the presence of 1 mmol l-1 CaCl2, MgSO4, and FeCl2. This analysis showed that Fe2+ had no effect on the growth of Phda1, while it was strongly enhanced by Ca2+ and Mg2+ (Fig. 4). Furthermore, the effects of Ca2+ and Mg2+ on the adsorption and growth of Phda1 were concentration-dependent. Adsorption rates of Phda1 after incubation at 25°C for 5 min increased to 79% and 74% in the presence of 25 mmol l-1 Ca2+ and Mg2+, respectively (Table 2). Based on the one-step growth analyses at various Ca2+ and Mg2+ concentrations, these divalent cations shortened the latent and rise periods to 30 min and increased the burst size 3–3.5-fold (Table 3). These results suggest that Ca2+ and Mg2+ play essential roles in the lytic cycles of Phda1.
Effects of Phda1 on the growth and histamine production of P. damselae subsp. damselae In order to evaluate the antimicrobial potential of Phda1, changes in the viable cell counts and histamine production by P. damselae subsp. damselae at different temperatures (8°C, 12°C, and 20°C) were investigated in the presence of Phda1. At 8°C, the viable cell counts of P. damselae subsp. damselae JCM8969 did not change, and the histamine level was below the detection limit (20 mg l-1) This article is protected by copyright. All rights reserved.
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during the incubation period of 120 h (Fig. 5a). At 12°C, the growth of P. damselae subsp. damselae was diminished by the presence of Phda1, and histamine production was also suppressed during the incubation period of 96 h compared with the control sample (Fig. 5b). Larger prevention effects were observed under 20°C incubation conditions. The population of P. damselae subsp. damselae increased to 8 log CFU ml-1 after 18 h in the control sample, while samples treated with Phda1 reached this point only after 27 h incubation. Similar to the viable cell counts, the histamine production in the presence of Phda1 was lagged by about 12 h in comparison with the control (Fig. 5c). These results suggest that phage treatment might be effective in the prevention of histamine accumulation.
Discussion Phage therapy is a highly anticipated alternative antimicrobial therapy due to the recent propagation of antibiotic-resistant bacterial strains (O’Flaherty et al., 2009). Many of the common pathogens, including Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Salmonella enterica, are target bacteria for the development of agents to improve their control (Carvalho et al., 2010a; Fu et al., 2010; Gupta and Prasad, 2011; Park et al., 2012, Chibeu et al., 2013). Phage applications are particularly attractive research fields for food science. Nevertheless, phages infecting bacteria that cause histamine poisoning, one of the major seafood-borne diseases in the world, have been hardly studied to date, with the exception of one previous study characterizing the M. morganii-infecting phage FSP1 (Yamaki et al., 2014). The aim of this study was to isolate and characterize a phage inhibiting P. damselae subsp. damselae, a potent histamine-producer, and to investigate the potential of phage therapy to prevent This article is protected by copyright. All rights reserved.
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histamine poisoning. The phage Phda1 infecting P. damselae subsp. damselae was isolated from a raw oyster and was subsequently identified as a member of the family Myoviridae by transmission electron microscope observation. Genome size of Phda1 was estimated with agarose gel electrophoresis using restriction enzymes, and this analysis showed that the genomic DNA of Phda1 has many restriction sites of restriction enzymes used in this study. However, precise genome sequence is needed in future because phages might have pathogenicity island, and these phages can’t be used for phage treatments due to risk of spread of pathogenic genes. We next examined the host range of Phda1 against 32 strains of gram-negative bacteria and 5 strains of gram-positive bacteria. Phda1 formed plaques only against P. damselae subsp. damselae JCM8969. This result suggests an extremely specific host range of Phda1. Because phages used for phage therapy often preferable exhibit a broad host range, Phda1 should be used as a supporting component in phage cocktails. These cocktails have been employed to supplement an individual phage’s host range and to suppress the development of phage-resistant bacteria (Lu and Koeris, 2011). Therefore, the isolation and characterization of a variety of phages is imperative for the generation of a phage cocktail against P. damselae subsp. damselae. Phage-resistant bacteria might eventually become a public concern similarly to antibiotic-resistant bacteria following a revival of phage therapy. Resistance acquisition is due to four main mechanisms: prevention of phage adsorption by changing or masking phage receptors, blockage of phage DNA injection, cutting of phage DNA by restriction-modification systems and the CRISPR-Cas system, and abortive infection systems (Labrie et al., 2010). The use of phage cocktails offers an effective strategy to prevent the development of phage-resistant bacteria, because bacterial resistance against one phage can This article is protected by copyright. All rights reserved.
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be prevented by other phages with different infection mechanisms. In fact, Tanji et al. (2004) reported that phage cocktails delayed the development of phage-resistant E. coli O157:H7, and O’Flynn et al. (2004) reported that the frequency of resistance development was 1.1 × 10-6 CFU in triple phage treatment compared with 1.2 × 10-6–3.3 × 10-4 CFU in single phage treatments. The stability of phages is one of the most important factors for the evaluation of their potential as antimicrobial agents. Thermal and pH stability of Phda1 were almost same to E. coli and S. enterica phage SFP10 belonging family Myoviridae (Park et al., 2012). Phda1 was suitable for use of the phage treatment because its wide range of stabilities covered growing temperature and pH for P. damselae subsp. damselae. Based on comparison with Vibrio parahaemolyticus phages Vp15P and Vp25P (Hidaka and Tokushige, 1978), latent period, and burst size of Phda1 were superior to these Myoviridae phages. Moreover, Phda1 was able to activate its lytic cycle, adsorption kinetics, and one-step growth curve in the presence of divalent cations, especially Ca2+ and Mg2+. These results suggest that Ca2+ and Mg2+ plays an essential role for Phda1 adsorption as well as in post-adsorption processes such as DNA injection, phage assembly, and host cell lysis. The activation of lytic cycles by addition of Ca2+ has been reported for many phages (Rountree, 1955; Landry and Zsigray, 1980; Bandara et al., 2012; Chhibber et al., 2014). Rountree (1951) suggested that staphylococcal phages have a requirement for Ca2+ with differences among individual strains. Moreover, Puck et al. (1951) hypothesized that cations in the environment are involved in the first adsorption to host cells by neutralizing the electronic repulsion that arises between the phages and the host cells. Potter and Nelson (1953) and Rountree (1955) further reported that Ca2+ supported the DNA penetration of phages into the host cells following adsorption. The antimicrobial effects of phages This article is protected by copyright. All rights reserved.
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might therefore be highly influenced by the specific divalent cation required for the steps of the infection process, such as adsorption, penetration, and others. Bandara et al. (2012) reported that the Bacillus cereus-infecting phages BCP1-1 and BCP8-2 could decrease the viable counts of B. cereus in fermented food to below detectable levels only in the presence of divalent cations. In the present study, the antimicrobial effects of Phda1 in the presence of divalent cations were greater than those without them (data not shown). Finally, we investigated the antimicrobial effects of Phda1 in order to evaluate the potential of phage application for inhibition of histamine poisoning. Phda1 treatment delayed both the growth and histamine accumulation in broth inoculated with P. damselae subsp. damselae. In the 12°C incubation condition, the histamine concentrations of the control samples after 96 h and 120 h incubation time were 94.7 mg l-1 and 1138 mg l-1, while those for phage-treated samples were 20.4 mg l-1 and 458 mg l-1, respectively. In the 20°C incubation condition, these values were 161 mg l-1 and 907 mg l-1 at 24 h and 30 h, respectively, in the control samples, while those of the phage-treated samples were below the detectable limit (24 h) and 33.2 mg l-1 (30 h). The environmental conditions, especially the pH, are important for histamine production of P. damselae subsp. damselae. Kimura et al. (2009) reported that transcripts of hdc genes associated with histamine production of P. damselae increased under acidic pH conditions and in the presence of extracellular histidine. In our experiments, no major induction of histidine decarboxylase expression was expected, because the broth pH hardly changed during the storage period (data not shown). Based on the histamine concentration standards for histidine-rich fishery products specified in the EU, Codex (100 mg kg-1), and the United States (50 mg kg-1), the phage treatment in this study might have potential as an effective approach for preventing histamine poisoning. This article is protected by copyright. All rights reserved.
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Although the phage treatment extended the growth of P. damselae subsp. damselae and accumulation of histamine, Phda1 has drawbacks of its poor host range and antimicrobial activity. Determination of host ranges of phages is strongly dependent on conditions of its receptor on bacterial surface, and host bacteria can hinder adsorption of phages by masking with cell surface components such as protein, exopolysaccharide (Labrie et al., 2010). For example, although phage T7, which recognized lipopolysaccharide as the receptor, couldn’t adsorb E. coli K1 due to the presence of capsule, enzymatic removal of K1 antigen enabled phage T7 to adsorb and infect (Scholl et al., 2005). Moreover, Erwinia amylovora phage L1 had a tail-associated depolymerase of extracellar polysaccharide, and its enzymatic capsule removal could enhance infection by other E. amylovora phage (Born et al., 2014). These report suggest that adequate chemical treatments or phage combinations can extend host ranges and antimicrobial activities of phages. Therefore, in order to fulfill the inhibition of histamine poisoning more definitely, isolation of many phages (phages having a broad host range and a stronger activity are preferable) using many strains of P. damselae subsp. damselae and performance of precise analysis of the Phda1 receptor to determine that the receptor is really strain-specific or masked by some components on other P. damselae subsp. damselae strains are needed. Practically, we should make the phage cocktail containing not only P. damselae phages but also other phages infecting histamine-producing bacteria. And the best way to carry out phage treatments to whole and processed fishes is to spray or dip as a post harvest treatment, because the contamination of histamine-producing bacteria is also occurred throughout various environments such as fish body, seawater, and processing plant. Moreover, the interaction of phages with food ingredients (e.g. carbohydrates, lipids, proteins) poses an important issue to consider, because the phage attachment to the This article is protected by copyright. All rights reserved.
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host cell is often inhibited by these interactions. Therefore, a challenge test incorporating food is one of the next steps in our research. In this study, we isolated and characterized the phage Phda1 that infects P. damselae subsp. damselae. Phda1 belongs to the family Myoviridae, and showed a suitable pH and thermal stability for its use as an antimicrobial agent. Moreover, the adsorption, lytic cycles, and antimicrobial effects of Phda1 were strongly improved in the presence of divalent cations. Finally, the challenge test showed that phage treatments might be potentially effective in the inhibition of histamine poisoning by P. damselae. To our knowledge, this is the first report of a phage preventing the growth and histamine production of P. damselae subsp. damselae.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26450278. Conflict of interest None declared.
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Born, Y., Fieseler, L., Klumpp, J., Eugster, M.R., Zurfluh, K., Duffy, B. and Loessner, M.J. (2014) The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage. Environ Microbiol 16, 2168-2180. Carvalho, C.M., Gannon, B.W., Halfhide, D.E., Santos, S.B., Hayes, C.M., Roe, J.M. and Azeredo, J. (2010a) The in vivo efficacy of two administration routes of a phage cocktail to reduce numbers of Campylobacter coli and Campylobacter jejuni in chickens. BMC Microbiol 10, 232. Carvalho, C., Susano, M., Fernandes, E., Santos, S., Gannon, B., Nicolau, A., Gibbs, P., Teixeira, P. and Azeredo, J. (2010b) Method for bacteriophage isolation against target Campylobacter strains. Lett Appl Microbiol 50, 192-197. Chhibber, S., Kaur, T. and Kaur, S. (2014) Essential role of calcium in the infection process of broad-spectrum methicillin-resistant Staphylococcus aureus bacteriophage. J Basic Microbiol 54, 775-780. Chibeu, A., Agius, L., Gao, A., Sabour, P.M., Kropinski, A.M. and Balamurugan, S. (2013) Efficacy of bacteriophage LISTEXTMP100 combined with chemical antimicrobials in reducing Listeria monocytogenes in cooked turkey and roast beef. Int J Food Microbiol 167, 208-214. D’Aloia, A., Vizzardi, E., Pina, P.D., Bugatti, S., Magro, F.D., Raddino, R., Curnis, A. and Cas, L.D. (2011) A scombroid poisoning causing a life-threatening acute pulmonary edema and coronary syndrome in a young healthy patient. Cardiovasc Toxicol 11, 280-283. Ferrario, C., Pegollo, C., Ricci, G., Borgo, F. and Fortina, M.G. (2012) PCR detection and identification of histamine-forming bacteria in filleted tuna fish samples. J Food Sci 77, 115-120. This article is protected by copyright. All rights reserved.
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Fu, W., Forster, T., Mayer, O., Curtin, J.J., Lehman, S.M. and Donlan, R.M. (2010) Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system. Antimicrob Agents Chemother 54, 397-404. Gupta, R. and Prasad, Y. (2011) Efficacy of polyvalent bacteriophage P-27/HP to control multidrug resistant Staphylococcus aureus associated with human infections. Curr Microbiol 62, 255-260. Hidaka, T. and Tokushige, A. (1978) Isolation and characterization of Vibrio parahaemolyticus bacteriophages in sea water. Mem Fac Fish, Kagoshima Univ 27, 79-90. Kanki, M., Yoda, T., Ishibashi, M. and Tsukamoto, T. (2004) Photobacterium phosphoreum caused a histamine fish poisoning incident. Int J Food Microbiol 92, 79-87. Kanki, M., Yoda, T., Tsukamoto, T. and Baba, E. (2007) Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury. Appl Environ Microbiol 73, 1467-1473. Kimura, B., Takahashi, H., Hokimoto, S., Tanaka, Y. and Fujii, T. (2009) Induction of the histidine decarboxylase genes of Photobacterium damselae subsp. damselae (formally P. histaminum) at low pH. J Appl Microbiol 107, 485-497. Kutateladze, M. and Adamia, R. (2010) Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol 28, 591-595. Labrie, S.J., Samson, J.E. and Moineau, S. (2010) Bacteriophage resistance mechanisms. Nat Rev Microbiol 8, 317-327. Landry, E.F. and Zsigray R.M. (1980) Effects of calcium on the lytic cycle of Bacillus subtilis Phage 41c. J Gen Virol 51, 125-135. Love, M., Teebken-Fisher, D., Hose, J.E., Farmer, J.J., Hickman, F.W. and Fanning, G.R. (1981) Vibrio This article is protected by copyright. All rights reserved.
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damsela, a marine bacterium, causes skin ulcers on the damselfish chromis punctipinnis. Science 214, 1139-1140. Lu, T.K. and Koeris, M.S. (2011) The next generation of bacteriophage therapy. Curr Opin Micoribol 14, 524-531. Magariños, B., Bonet, R., Romalde, J.L., Martínez, M.J., Congregado, F. and Toranzo, A.E. (1996) Influence of the capsular layer on the virulence of Pasteurella piscicida for fish. Microb Pathog 21, 289-297. Mett, C.L. and Sturgeon, R.J. (1982) Cation-exchange chromatography of histamine in the presence of ethylammonium chloride. J Chromatogr A 235, 536-538. Nakakoshi, M., Nishioka, H. and Katayama, E. (2011) New versatile staining reagents for biological transmission electron microscopy that substitute for uranyl acetate. J Electron Microsc 60, 401-407. O’Flaherty, S., Ross, R.P. and Coffey, A. (2009) Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol Rev 33, 801-819. O’Flynn, G., Ross, R.P., Fitzgerald, G.F. and Coffey, A. (2004) Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. Appl Environ Microbiol 70, 3417-3424. Park, M., Lee, J-H., Shin, H., Kim, M., Choi, J., Kang, D-H., Heu, S. and Ryu, S. (2012) Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7. Appl Environ Micobiol 78, 58-69. Pedersen, K., Dalsgaard, I. and Larsen, J.L. (1997) Vibrio damsela associated with diseased fish in Denmark. Appl Environ Microbiol 63, 3711-3715. This article is protected by copyright. All rights reserved.
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Potter, N.N. and Nelson, F.E. (1953) Role of calcium and related ions in proliferation of lactic streptococcus bacteriophage. J Bacteriol 66, 508-516. Puck, T.T., Garen, A. and Cline, J. (1951) The mechanism of virus attachment to host cells: I. The role of ions in the primary reaction. J Exp Med 93, 65-88. Rodtong, S., Nawong, S., Yongsawatdigul, J. (2005) Histamine accumulation and histamine-forming bacteria in Indian anchovy (Stolephorus indicus). Food Microbiol 22, 475-482. Rountree, P.M. (1951) The role of certain electrolytes in the adsorption of staphylococcal bacteriophages. J Gen Microbiol 5, 673-680. Rountree, P.M. (1955) The role of divalent cations in the multiplication of staphylococcal bacteriophage. J Gen Microbiol 12, 275-287. Sánchez-Guerrero, I.M., Vidal, J.B. and Escudero, A.I. (1997) Scombroid fish poisoning: A potentially life-threatening allergic-like reaction. J Allergy Clin immunol 100, 433-434. Scholl, D., Adhya, S. and Merril, C. (2005) Escherichia coli K1’s capsule is a barrier to bacteriophage T7. Appl Environ Microbiol 71, 4872-4874. Stenholm, A.R., Dalsgaard, I. and Middelboe, M. (2008) Isolation and characterization of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum. Appl Environ Microbiol 74, 4070-4078. Tanji, Y., Shimada, T., Yoichi, M., Miyanaga, K., Hori, K. and Unno, H. (2004) Toward rational control of Escherichia coli O157:H7 by a phage cocktail. Appl Microbiol Biotechnol 64, 270-274. Yamaki, S., Omachi, T., Kawai, Y. and Yamazaki, K. (2014) Characterization of a novel Morganella morganii bacteriophage FSP1 isolated from river water. FEMS Microbiol Lett 359, 166-172. This article is protected by copyright. All rights reserved.
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Figure legends Fig. 1. Morphology and genome size analysis of Phda1. (a) Transmission electron micrograph images of Phda1. Phda1 was negatively stained with 2.5% samarium triacetate. Arrows indicate that Phda1 adsorbed to a host cell. The scale bar indicates 200 nm. (b) Agarose gel electrophoresis analysis of Phda1. Lane 1: Gene ladder wide I (Nippon Gene; Tokyo, Japan); lane 2: Phda1 DNA; lanes 3, 4, and 5: Phda1 DNA digested with EcoRV, PstI, and PvuII (Nippon Gene); lane 6: 2.5-kb DNA ladder (Takara Bio; Tokyo, Japan).
Fig. 2. Thermal (a) and pH (b) stability of Phda1 in TSBS. Closed diamonds: heated at 60°C, open squares: heated at 70°C, closed triangles: heated at 75°C, open circles: heated at 80°C, closed squares: heated at 85°C. The results are shown as the mean ± standard deviation from three independent experiments.
Fig. 3. Effect of divalent cations on the adsorption rate of Phda1. Adsorption assays were conducted in cation broth without divalent cations or containing 1 mmol l-1 CaCl2, MgSO4, FeCl2, MnCl2, or ZnCl2. The results are shown as the mean ± standard deviation from three independent experiments.
Fig. 4. Effect of divalent cations on the one-step growth of Phda1 (PFU infected cell-1). One-step growth analyses were conducted in cation broth without divalent cations (diamond) or containing 1 mmol l-1 CaCl2 (circle), MgSO4 (square), or FeCl2 (triangle). The results are shown as the mean ± standard deviation from three independent experiments. This article is protected by copyright. All rights reserved.
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Fig. 5. Effect of Phda1 on the growth and histamine production of P. damselae subsp. damselae at 8°C (a), 12°C (b), and 20°C (c). Each symbol and column represents the viable cell count of P. damselae subsp. damselae with (closed circles) or without (closed diamonds) Phda1, and the histamine concentration with (closed columns) or without (open columns) Phda1. An asterisk (*) indicates a histamine concentration below the detectable limit (20 mg l-1).
Table 1. Bacterial strains used in this study and host range of Phda1. Bacterial species Gram-negative bacteria Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. piscicida Photobacterium phosphoreum Photobacterium phosphoreum
Strains
Plaque formation
JCM8967 JCM8968 JCM8969 MA1 MA2 MA3 OY1 OY2 SW1 Dpp-1 JCM20393 NBRC13896
+ -
Photobacterium phosphoreum Photobacterium ganghwense Photobacterium lutimaris Cronobacter muytjensii Escherichia coli
NBRC103031T JCM12487 JCM13586 ATCC51329 RIMD0509939
-
Morganella morganii subsp. morganii Proteus vulgaris Salmonella enterica serovar Enteritidis Salmonella enterica serovar Typhimurium
NBRC3848T ATCC33420 NBRC3313 IID1000
-
Yersinia enterocolitica
ATCC9610T
-
Vibrio alginolyticus
T
LMG4409
-
Vibrio anguillarum
LMG4437T
-
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Vibrio cincinnatiensis
LMG7891T
-
Vibrio fluvialis
LMG7894T
-
Vibrio harveyi
T
-
LMG4044
Vibrio metschnikovii
T
LMG11664
-
Vibrio natriegens
LMG10935T
-
T
Vibrio furnissii
-
LMG7910
T
Vibrio parahaemolyticus Vibrio vulnificus Gram-positive bacteria Bacillus cereus Listeria grayi Listeria innocua Listeria monocytogenes Staphylococcus aureus +, clear plaque; -, no plaque.
NBRC12711
-
LMG13545T
-
JCM2152T ATCC25401
-
ATCC33090T ATCC7644 NBRC14462
-
Table 2. Effect of calcium and magnesium ions on the adsorption rate (%) of Phda1 after incubation at 25°C for 5 min. 0 mmol l-1
1.0 mmol l-1
10 mmol l-1
25 mmol l-1
Ca2+
3.1 ± 4.32
17.7 ± 4.89
59.5 ± 11.5
79.3 ± 2.29
Mg2+
3.1 ± 4.32
18.9 ± 7.56
42.1 ± 1.83
74.1 ± 2.27
Table 3. Effect of calcium and magnesium ions on the latent period, rise period, and burst size of Phda1.
Control -1
1.0 mmol l
10 mmol l 25 mmol l
Burst size
(min)
(min)
(PFU infected cell-1)
60
50
19.1 ± 3.62
40
30
58.1 ± 7.30
2+
50
40
52.4 ± 14.6
2+
30
30
72.8 ± 4.61
2+
40
40
67.0 ± 13.4
2+
30
30
83.9 ± 21.5
2+
30
30
46.0 ± 17.3
Ca Ca
Mg -1
Rise period
2+
Mg -1
Latent period
Ca
Mg
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Article Type: Original Article
Running head: Characterization of phage Phda1
Characterization of a novel bacteriophage, Phda1, infecting the histamine-producing Photobacterium damselae subsp. damselae
Shogo Yamaki, Yuji Kawai, and Koji Yamazaki* Laboratory of Marine Food Science and Technology, Faculty of Fisheries Sciences, Hokkaido University 3-1-1, Minato, Hakodate 041-8611, Japan
Correspondence: Koji Yamazaki, Laboratory of Marine Food Science and Technology, Faculty of Fisheries Sciences, Hokkaido University 3-1-1, Minato, Hakodate 041-8611, Japan. Tel and fax: +81 138 40 5574; e-mail: [email protected]
Abstract Aims: Photobacterium damselae subsp. damselae is a potent histamine-producing microorganism. The aim of this study was to isolate and characterize a bacteriophage Phda1 that infected to P. damselae subsp. damselae to inhibit its growth and histamine accumulation. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12809 This article is protected by copyright. All rights reserved.
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Methods and Results: Phda1 was isolated from a raw oyster, and the host range, morphology, and the bacteriophage genome size were analyzed. Phda1 formed a clear plaque only against P. damselae subsp. damselae JCM8969 among 5 Gram-positive and 32 Gram-negative bacterial strains tested. Phda1 belongs to the family Myoviridae, and its genome size was estimated as 35.2–39.5 kb. According to the one-step growth curve analysis, the latent period, rise period, and burst size of Phda1 were 60 min, 50 min, and 19 PFU infected cell-1, respectively. Divalent cations, especially Ca2+ and Mg2+, strongly improved Phda1 adsorption to the host cells and its propagation. Phda1 treatment delayed the growth and histamine production of P. damselae subsp. damselae in an in vitro challenge test. Conclusions: The bacteriophage Phda1 might serve as a potential antimicrobial agent to inhibit the histamine poisoning caused by P. damselae subsp. damselae. Significance and Impact of the Study: This is the first description of a bacteriophage specifically infecting P. damselae subsp. damselae and its potential applications. Bacteriophage therapy could prove useful in the prevention of histamine poisoning.
Keywords: Bacteriophage, Photobacterium damselae subsp. damselae, phage therapy, histamine poisoning
Introduction Photobacterium damselae is a halophilic marine bacterium belonging to the family Vibrionaceae. This species is further categorized into P. damselae subsp. damselae (formerly Vibrio damsela) and P. damselae subsp. piscicida (formerly Pasteurella piscicida), both of which are known fish pathogens This article is protected by copyright. All rights reserved.
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Phage propagation and purification Phda1 was propagated and purified by the polyethylene glycol precipitation method and CsCl density gradient centrifugation. P. damselae subsp. damselae JCM8969 was incubated with TSBS at 25°C. Phda1 was added to the host strain culture in the exponential phase at a multiplicity of infection (MOI) of 0.1, and incubated at 25°C. Following Phda1 growth, the culture was centrifuged (10,000 × g, 30 min, 4°C) and filtered through 0.45-µm polyvinylidene difluoride filters (Merck Millipore; Billerica, MA). DNase I and RNase A (Sigma-Aldrich; St. Louis, MO) were added at 1 µg ml-1 to degrade bacterial DNA and RNA, and the lysate was incubated at room temperature for 1 h. NaCl was added to a final concentration of 1 mol l-1 and the lysate was incubated for 1 h on ice. The mixture was treated with 10% polyethylene glycol 6000 (Wako pure chemical industries; Osaka, Japan) at 4°C for 12 h, then the Phda1 lysate was centrifuged (10,000 × g, 20 min, 4°C), and the pellet was suspended in SM buffer. An equal volume of chloroform was added to the phage suspension, followed by another centrifugation step (10,000 × g, 10 min, 4°C) to remove polyethylene glycol. Propagated Phda1 was purified by CsCl density gradient centrifugation (100,000 × g, 2 h, 4°C, d = 1.3, 1.5, and 1.6), and purified Phda1 was stored at 4°C for further investigations.
Host range analysis Bacterial lawns listed in Table 1 were generated by the double agar overlay method. Two hundred microliters of each strain culture were added to 3.5 ml of 0.5% molten agar, and the mixture was overlaid on plate medium. After solidification of the soft agar, 10 µl of Phda1 solution was spotted onto each bacterial lawn. Plates were incubated and examined for clear zone development on the bacterial This article is protected by copyright. All rights reserved.
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Bacteriophages (phages) are viruses that infect bacteria, and have lately been considered as one of the most important antimicrobial agents. Although the therapeutic potential of phages has not been focused on since the discovery of antibiotics, the emergence of antibiotic-resistant bacteria makes phages an attractive agent to counteract these bacteria (Kutateladze and Adamia, 2010). The application of phages as an antimicrobial agent in phage therapy has recently been investigated in various areas such as medicine, veterinary science, agriculture, aquaculture, and food science. In fact, phages infecting a variety of pathogens, including Campylobacter coli, Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, and Staphylococcus aureus, were investigated for their potential use as effective antimicrobial agents (Carvalho et al., 2010a; Fu et al., 2010; Gupta and Prasad, 2011; Park et al., 2012, Chibeu et al., 2013). At present, the United States Department of Agriculture and the Food and Drug Administration have approved LISTEXTMP100 and SALMONELEXTM (Micreos Food Safety, Wageningen, The Netherlands), which are a L. monocytogenes phage and Salmonella phages, respectively, as ‘Generally Recognized As Safe’ food processing aids to control food pathogens. Nonetheless, not many studies have been performed to date on the evaluation of phage utilization to suppress histamine poisoning. Therefore, we have investigated the isolation of phages infecting histamine-producing bacteria and characterized the M. morganii-infecting phage FSP1 in a previous study (Yamaki et al., 2014). In the present study, we demonstrate the isolation and characterization of a specific phage infecting P. damselae subsp. damselae, one of the most potent histamine producers in seafood, for its use as an antimicrobial agent.
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Materials and Methods
Bacterial strains and growth conditions Bacterial strains used in this study are listed Table 1. Halophilic bacteria were incubated with tryptic soy broth (TSB, BD; Franklin Lakes, NJ, USA) supplemented with 1.5% NaCl (TSBS); all other bacteria were incubated with TSB or TSB supplemented with 0.6% yeast extract (BD).
Isolation of P. damselae subsp. damselae-infecting phages Phages infecting P. damselae subsp. damselae were isolated by the method of Carvalho et al. (2010b) with minor modifications. Raw oysters were mixed with nine times their weight of TSBS containing 400 µg ml-1 CaCl2 and 400 µg ml-1 MgSO4. P. damselae subsp. damselae JCM8969 cells in the exponential growth phase were added to the mixture and incubated at 20°C for 24 h. Ten milliliters of the culture were then treated with 5 % (v/v) chloroform to lyse the bacterial cells and centrifuged (10,000 × g, 30 min, 4°C). The collected supernatant was used for phage detection. The supernatants were spotted on a lawn of P. damselae subsp. damselae JCM8969 made with the double agar overlay method using tryptic soy agar (TSA, BD) plates supplemented with 1.5% NaCl and 0.5% soft agar supplemented with 2.0% NaCl. The plates were incubated at 25°C for 24 h. Plaques were picked up and suspended in 1 ml of SM buffer (100 mmol l-1 CaCl, 8 mmol l-1 MgSO4·7H2O, 50 mmol l-1 Tris-HCl, and 0.01% gelatin, pH 7.5), and single plaques were formed using the plaque assay method. This procedure was repeated more than three times and the isolated phage was termed Phda1.
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Phage propagation and purification Phda1 was propagated and purified by the polyethylene glycol precipitation method and CsCl density gradient centrifugation. P. damselae subsp. damselae JCM8969 was incubated with TSBS at 25°C. Phda1 was added to the host strain culture in the exponential phase at a multiplicity of infection (MOI) of 0.1, and incubated at 25°C. Following Phda1 growth, the culture was centrifuged (10,000 × g, 30 min, 4°C) and filtered through 0.45-µm polyvinylidene difluoride filters (Merck Millipore; Billerica, MA). DNase I and RNase A (Sigma-Aldrich; St. Louis, MO) were added at 1 µg ml-1 to degrade bacterial DNA and RNA, and the lysate was incubated at room temperature for 1 h. NaCl was added to a final concentration of 1 mol l-1 and the lysate was incubated for 1 h on ice. The mixture was treated with 10% polyethylene glycol 6000 (Wako pure chemical industries; Osaka, Japan) at 4°C for 12 h, then the Phda1 lysate was centrifuged (10,000 × g, 20 min, 4°C), and the pellet was suspended in SM buffer. An equal volume of chloroform was added to the phage suspension, followed by another centrifugation step (10,000 × g, 10 min, 4°C) to remove polyethylene glycol. Propagated Phda1 was purified by CsCl density gradient centrifugation (100,000 × g, 2 h, 4°C, d = 1.3, 1.5, and 1.6), and purified Phda1 was stored at 4°C for further investigations.
Host range analysis Bacterial lawns listed in Table 1 were generated by the double agar overlay method. Two hundred microliters of each strain culture were added to 3.5 ml of 0.5% molten agar, and the mixture was overlaid on plate medium. After solidification of the soft agar, 10 µl of Phda1 solution was spotted onto each bacterial lawn. Plates were incubated and examined for clear zone development on the bacterial This article is protected by copyright. All rights reserved.
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lawns.
Transmission electron microscopy The morphology of Phda1 was analyzed with a transmission electron microscope. Phda1 solution (11 log plaque-forming units [PFU] ml-1) was dropped onto a 200-mesh Cu grid (Nisshin EM; Tokyo, Japan) for 5 min, and 2.5% samarium triacetate, an appropriate negative staining reagent reported by Nakakoshi et al. (2011), was dropped onto the grid. Phda1 was identified by observation with a transmission electron microscope (JEM-1011, JEOL; Tokyo, Japan).
Phage DNA analysis Phda1 was suspended in TENS buffer (50 mmol l-1 Tris-HCl, 100 mmol l-1 EDTA, 100 mmol l-1 NaCl, and 0.3% sodium dodecyl sulfate; pH 8.0; Stenholm et al., 2008). Phda1 was treated with 200 µg ml-1 proteinase K at 65°C for 10 min to break Phda1 virions, and purification of Phda1 DNA was performed with the Phage DNA Isolation Kit (Norgen Biotek; Thorold, Ontario, Canada) according to the manufacturer’s instructions. Purified DNA was digested with EcoRV, PstI, and PvuII (Nippon Gene; Tokyo, Japan) for genome size estimation according to the manufacturer’s instructions. Digested DNA patterns were analyzed with agarose gel electrophoresis using 0.5% SeaKem® gold agarose (Takara Bio; Shiga, Japan) and SYBR® green nucleic acid stain (Sigma-Aldrich). Gene ladder wide I (Nippon Gene) and 2.5-kb DNA ladder (Takara Bio) were used as DNA size markers. The total genome size of Phda1 was estimated from the sum of the sizes of all digested fragments.
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Analysis of adsorption kinetics P. damselae subsp. damselae JCM8969 was incubated at 25°C with TSBS. When the OD600 nm of the culture reached 0.2, Phda1 was inoculated at MOI = 0.1 and incubated at 25°C. Samples were collected chronologically and centrifuged (10,000 × g, 1 min, 4°C), followed by filtration with a 0.45-µm polyvinylidene difluoride filter to remove phages adsorbed to bacteria. The PFU of free phage in the supernatant was determined by a plaque assay technique using a TSA plate supplemented with 1.5% NaCl and 25 mmol l-1 MgSO4 as the bottom agar and 0.5% soft agar supplemented with 2.0% NaCl and 25 mmol l-1 MgSO4 as the top agar. The measurement of PFU of Phda1 was carried out by this plaque assay technique throughout this study unless otherwise noted.
One-step growth curve analysis P. damselae subsp. damselae was incubated at 25°C with TSBS. When the OD600 nm of the culture reached 0.2, Phda1 was added at MOI = 0.1 and incubated at 25°C for 5 min for attachment of Phda1 to the host cells. The culture was centrifuged (10,000 × g, 2 min, 4°C) to remove free phages, and the pellet was suspended in fresh TSBS. The suspension was incubated at 25°C, and samples were collected every 10 min. The PFU of Phda1 in the sample was measured by serial dilution and the plaque assay technique. The latent period, rise period, and burst size of Phda1 were determined by monitoring the changes in PFU.
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Evaluation of heat and pH stability The heat and pH stability of Phda1 was evaluated by observing the changes in PFU after various treatments. In order to examine the thermal stability of Phda1, the phages (107 PFU ml-1) were suspended in TSBS and incubated at 60–85°C for 30 min. Samples was collected every 5 min and the PFU of surviving Phda1 phages was determined by serial dilution and the plaque assay technique. For the assessment of pH stability of Phda1, the phages (107 PFU ml-1) were added to TSBS (pH 1.0–12.0, adjusted with 6 mol l-1 HCl or 10 mol l-1 NaOH), and incubated at 30°C for 24 h. The PFU of surviving Phda1 phages was determined by serial dilution and the plaque assay technique.
Effects of divalent cations on Phda1 growth The adsorption kinetics and the one-step growth curve of Phda1 were examined in the presence of divalent cations (Ca2+, Mg2+, Fe2+, Mn2+, and Zn2+). These experiments were performed with cation broth (Bacto tryptone 17 g l-1, BBL PhytoneTM peptone 3 g l-1, glucose 2.5 g l-1, NaCl 20 g l-1; pH 7.3) supplemented with 1–25 mmol l-1 CaCl2, MgSO4, FeCl2, MnCl2, or ZnCl2, and the effects of divalent cations on the growth of Phda1 were evaluated.
Challenge test Phda1 (MOI = 100) and P. damselae subsp. damselae JCM8969 (3 log CFU ml-1) were inoculated into cation broth supplemented with 25 mmol l-1 CaCl2 and 1% histidine hydrochloride (pH 6.5). The culture was incubated at 8°C, 12°C, or 20°C. Viable cell counts of P. damselae subsp. damselae JCM8969 were investigated by spreading the culture onto a TSA plate supplemented with 1.5% NaCl This article is protected by copyright. All rights reserved.
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and incubation at 25°C for 24 h. The histamine concentration in the broth was also determined simultaneously with high-performance liquid chromatography (Mett and Sturgeon, 1982). Samples collected from the culture broth were mixed with an equal volume of 10% trichloroacetic acid and filtered through 0.2-µm polytetrafluoroethylene filters (Millipore). Filtered samples were subjected to high-performance liquid chromatography.
Results
Isolation of the P. damselae subsp. damselae-infecting phage Phda1 and its host range We isolated a P. damselae subsp. damselae-infecting phage from a raw oyster and termed it Phda1. The host range of Phda1 was analyzed for 32 strains of gram-negative bacteria and 5 strains of gram-positive bacteria. Phda1 formed a plaque only against P. damselae subsp. damselae JCM8969. Eight other strains of P. damselae subsp. damselae did not form plaques (Table 1), demonstrating a very narrow host range of Phda1.
Morphology and genome size of Phda1 The morphology of Phda1 was analyzed using a negative staining method. Transmission electron microscope observation determined the head length of Phda1 as 62 ± 4.6 nm (mean ± SD) and the contractile tail length as 110 ± 4.9 nm (mean ± SD) (n = 32). Moreover, Phda1 featured contracted tails attaching to the host bacteria P. damselae subsp. damselae JCM8969 (Fig. 1a). Based on the morphological analysis, Phda1 belongs to the order Caudovirales and the family Myoviridae, which This article is protected by copyright. All rights reserved.
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comprises tailed phages with a neck and contractile tail. For the estimation of the Phda1 genome size, the phage genome was digested with the restriction endonucleases EcoRV, PstI, and PvuII. The digestion patterns of each enzyme showed that the extracted nucleic acid of Phda1 was linear, double-stranded DNA, and the Phda1 DNA featured many restriction sites for each of the tested enzymes. The genome size of Phda1 was estimated by the sum of the bands resulting from the digestion, and was determined as 35.2–39.5 kb (Fig. 1b).
Adsorption kinetics and one-step growth curve of Phda1 The adsorption analysis revealed that 13 ± 4 % (mean ± SD) of Phda1 adsorbed to host cells at 10 min, and 66 ± 1 % (mean ± SD) adsorbed at 1 h in TSBS (data not shown). Moreover, the one-step growth curve analysis showed that the latent period of Phda1 was 60 min, the rise period was 50 min, and the burst size was 19 PFU infected cell-1 (Table 3).
Thermal and pH stability of Phda1 The stability of phages under various environmental conditions is an important factor for their evaluation as potential antimicrobial agents. Phda1 was stable at 60°C, but the PFU of Phda1 decreased to 4.7 log PFU ml-1 after heating at 70°C for 30 min. In addition, Phda1 was not detected after heating at 75°C and 80°C for 20 min, and at 85°C for 5 min (Fig. 2a). Based on the pH stability analysis, Phda1 was stable between pH 4–10, but strong acidic (pH ≤ 3) and alkaline (pH ≥ 11) conditions caused a loss of infectivity of Phda1 within 24 h (Fig. 2b). In addition, Phda1 was stable after freezing at −20°C, −40°C, and −80°C for 4 weeks (data not shown) in TSBS. These results suggest that Phda1 could be a This article is protected by copyright. All rights reserved.
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suitable antimicrobial agent due to its favorable stability under various environmental conditions.
Growth enhancement of Phda1 with divalent cations In order to elucidate the effect of divalent cations on the adsorption of Phda1, the adsorption rate was determined in cation broth supplemented with 1 mmol l-1 CaCl2, MgSO4, FeCl2, MnCl2, or ZnCl2. Compared with the control (without divalent cations), the adsorption of Phda1 was greatly improved in the presence of 1 mmol l-1 Ca2+, Mg2+, Fe2+, and Mn2+, while Zn2+ had no enhancing effect (Fig. 3). The one-step growth curves were analyzed next in the presence of 1 mmol l-1 CaCl2, MgSO4, and FeCl2. This analysis showed that Fe2+ had no effect on the growth of Phda1, while it was strongly enhanced by Ca2+ and Mg2+ (Fig. 4). Furthermore, the effects of Ca2+ and Mg2+ on the adsorption and growth of Phda1 were concentration-dependent. Adsorption rates of Phda1 after incubation at 25°C for 5 min increased to 79% and 74% in the presence of 25 mmol l-1 Ca2+ and Mg2+, respectively (Table 2). Based on the one-step growth analyses at various Ca2+ and Mg2+ concentrations, these divalent cations shortened the latent and rise periods to 30 min and increased the burst size 3–3.5-fold (Table 3). These results suggest that Ca2+ and Mg2+ play essential roles in the lytic cycles of Phda1.
Effects of Phda1 on the growth and histamine production of P. damselae subsp. damselae In order to evaluate the antimicrobial potential of Phda1, changes in the viable cell counts and histamine production by P. damselae subsp. damselae at different temperatures (8°C, 12°C, and 20°C) were investigated in the presence of Phda1. At 8°C, the viable cell counts of P. damselae subsp. damselae JCM8969 did not change, and the histamine level was below the detection limit (20 mg l-1) This article is protected by copyright. All rights reserved.
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during the incubation period of 120 h (Fig. 5a). At 12°C, the growth of P. damselae subsp. damselae was diminished by the presence of Phda1, and histamine production was also suppressed during the incubation period of 96 h compared with the control sample (Fig. 5b). Larger prevention effects were observed under 20°C incubation conditions. The population of P. damselae subsp. damselae increased to 8 log CFU ml-1 after 18 h in the control sample, while samples treated with Phda1 reached this point only after 27 h incubation. Similar to the viable cell counts, the histamine production in the presence of Phda1 was lagged by about 12 h in comparison with the control (Fig. 5c). These results suggest that phage treatment might be effective in the prevention of histamine accumulation.
Discussion Phage therapy is a highly anticipated alternative antimicrobial therapy due to the recent propagation of antibiotic-resistant bacterial strains (O’Flaherty et al., 2009). Many of the common pathogens, including Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Salmonella enterica, are target bacteria for the development of agents to improve their control (Carvalho et al., 2010a; Fu et al., 2010; Gupta and Prasad, 2011; Park et al., 2012, Chibeu et al., 2013). Phage applications are particularly attractive research fields for food science. Nevertheless, phages infecting bacteria that cause histamine poisoning, one of the major seafood-borne diseases in the world, have been hardly studied to date, with the exception of one previous study characterizing the M. morganii-infecting phage FSP1 (Yamaki et al., 2014). The aim of this study was to isolate and characterize a phage inhibiting P. damselae subsp. damselae, a potent histamine-producer, and to investigate the potential of phage therapy to prevent This article is protected by copyright. All rights reserved.
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histamine poisoning. The phage Phda1 infecting P. damselae subsp. damselae was isolated from a raw oyster and was subsequently identified as a member of the family Myoviridae by transmission electron microscope observation. Genome size of Phda1 was estimated with agarose gel electrophoresis using restriction enzymes, and this analysis showed that the genomic DNA of Phda1 has many restriction sites of restriction enzymes used in this study. However, precise genome sequence is needed in future because phages might have pathogenicity island, and these phages can’t be used for phage treatments due to risk of spread of pathogenic genes. We next examined the host range of Phda1 against 32 strains of gram-negative bacteria and 5 strains of gram-positive bacteria. Phda1 formed plaques only against P. damselae subsp. damselae JCM8969. This result suggests an extremely specific host range of Phda1. Because phages used for phage therapy often preferable exhibit a broad host range, Phda1 should be used as a supporting component in phage cocktails. These cocktails have been employed to supplement an individual phage’s host range and to suppress the development of phage-resistant bacteria (Lu and Koeris, 2011). Therefore, the isolation and characterization of a variety of phages is imperative for the generation of a phage cocktail against P. damselae subsp. damselae. Phage-resistant bacteria might eventually become a public concern similarly to antibiotic-resistant bacteria following a revival of phage therapy. Resistance acquisition is due to four main mechanisms: prevention of phage adsorption by changing or masking phage receptors, blockage of phage DNA injection, cutting of phage DNA by restriction-modification systems and the CRISPR-Cas system, and abortive infection systems (Labrie et al., 2010). The use of phage cocktails offers an effective strategy to prevent the development of phage-resistant bacteria, because bacterial resistance against one phage can This article is protected by copyright. All rights reserved.
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be prevented by other phages with different infection mechanisms. In fact, Tanji et al. (2004) reported that phage cocktails delayed the development of phage-resistant E. coli O157:H7, and O’Flynn et al. (2004) reported that the frequency of resistance development was 1.1 × 10-6 CFU in triple phage treatment compared with 1.2 × 10-6–3.3 × 10-4 CFU in single phage treatments. The stability of phages is one of the most important factors for the evaluation of their potential as antimicrobial agents. Thermal and pH stability of Phda1 were almost same to E. coli and S. enterica phage SFP10 belonging family Myoviridae (Park et al., 2012). Phda1 was suitable for use of the phage treatment because its wide range of stabilities covered growing temperature and pH for P. damselae subsp. damselae. Based on comparison with Vibrio parahaemolyticus phages Vp15P and Vp25P (Hidaka and Tokushige, 1978), latent period, and burst size of Phda1 were superior to these Myoviridae phages. Moreover, Phda1 was able to activate its lytic cycle, adsorption kinetics, and one-step growth curve in the presence of divalent cations, especially Ca2+ and Mg2+. These results suggest that Ca2+ and Mg2+ plays an essential role for Phda1 adsorption as well as in post-adsorption processes such as DNA injection, phage assembly, and host cell lysis. The activation of lytic cycles by addition of Ca2+ has been reported for many phages (Rountree, 1955; Landry and Zsigray, 1980; Bandara et al., 2012; Chhibber et al., 2014). Rountree (1951) suggested that staphylococcal phages have a requirement for Ca2+ with differences among individual strains. Moreover, Puck et al. (1951) hypothesized that cations in the environment are involved in the first adsorption to host cells by neutralizing the electronic repulsion that arises between the phages and the host cells. Potter and Nelson (1953) and Rountree (1955) further reported that Ca2+ supported the DNA penetration of phages into the host cells following adsorption. The antimicrobial effects of phages This article is protected by copyright. All rights reserved.
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might therefore be highly influenced by the specific divalent cation required for the steps of the infection process, such as adsorption, penetration, and others. Bandara et al. (2012) reported that the Bacillus cereus-infecting phages BCP1-1 and BCP8-2 could decrease the viable counts of B. cereus in fermented food to below detectable levels only in the presence of divalent cations. In the present study, the antimicrobial effects of Phda1 in the presence of divalent cations were greater than those without them (data not shown). Finally, we investigated the antimicrobial effects of Phda1 in order to evaluate the potential of phage application for inhibition of histamine poisoning. Phda1 treatment delayed both the growth and histamine accumulation in broth inoculated with P. damselae subsp. damselae. In the 12°C incubation condition, the histamine concentrations of the control samples after 96 h and 120 h incubation time were 94.7 mg l-1 and 1138 mg l-1, while those for phage-treated samples were 20.4 mg l-1 and 458 mg l-1, respectively. In the 20°C incubation condition, these values were 161 mg l-1 and 907 mg l-1 at 24 h and 30 h, respectively, in the control samples, while those of the phage-treated samples were below the detectable limit (24 h) and 33.2 mg l-1 (30 h). The environmental conditions, especially the pH, are important for histamine production of P. damselae subsp. damselae. Kimura et al. (2009) reported that transcripts of hdc genes associated with histamine production of P. damselae increased under acidic pH conditions and in the presence of extracellular histidine. In our experiments, no major induction of histidine decarboxylase expression was expected, because the broth pH hardly changed during the storage period (data not shown). Based on the histamine concentration standards for histidine-rich fishery products specified in the EU, Codex (100 mg kg-1), and the United States (50 mg kg-1), the phage treatment in this study might have potential as an effective approach for preventing histamine poisoning. This article is protected by copyright. All rights reserved.
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Although the phage treatment extended the growth of P. damselae subsp. damselae and accumulation of histamine, Phda1 has drawbacks of its poor host range and antimicrobial activity. Determination of host ranges of phages is strongly dependent on conditions of its receptor on bacterial surface, and host bacteria can hinder adsorption of phages by masking with cell surface components such as protein, exopolysaccharide (Labrie et al., 2010). For example, although phage T7, which recognized lipopolysaccharide as the receptor, couldn’t adsorb E. coli K1 due to the presence of capsule, enzymatic removal of K1 antigen enabled phage T7 to adsorb and infect (Scholl et al., 2005). Moreover, Erwinia amylovora phage L1 had a tail-associated depolymerase of extracellar polysaccharide, and its enzymatic capsule removal could enhance infection by other E. amylovora phage (Born et al., 2014). These report suggest that adequate chemical treatments or phage combinations can extend host ranges and antimicrobial activities of phages. Therefore, in order to fulfill the inhibition of histamine poisoning more definitely, isolation of many phages (phages having a broad host range and a stronger activity are preferable) using many strains of P. damselae subsp. damselae and performance of precise analysis of the Phda1 receptor to determine that the receptor is really strain-specific or masked by some components on other P. damselae subsp. damselae strains are needed. Practically, we should make the phage cocktail containing not only P. damselae phages but also other phages infecting histamine-producing bacteria. And the best way to carry out phage treatments to whole and processed fishes is to spray or dip as a post harvest treatment, because the contamination of histamine-producing bacteria is also occurred throughout various environments such as fish body, seawater, and processing plant. Moreover, the interaction of phages with food ingredients (e.g. carbohydrates, lipids, proteins) poses an important issue to consider, because the phage attachment to the This article is protected by copyright. All rights reserved.
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host cell is often inhibited by these interactions. Therefore, a challenge test incorporating food is one of the next steps in our research. In this study, we isolated and characterized the phage Phda1 that infects P. damselae subsp. damselae. Phda1 belongs to the family Myoviridae, and showed a suitable pH and thermal stability for its use as an antimicrobial agent. Moreover, the adsorption, lytic cycles, and antimicrobial effects of Phda1 were strongly improved in the presence of divalent cations. Finally, the challenge test showed that phage treatments might be potentially effective in the inhibition of histamine poisoning by P. damselae. To our knowledge, this is the first report of a phage preventing the growth and histamine production of P. damselae subsp. damselae.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26450278. Conflict of interest None declared.
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damsela, a marine bacterium, causes skin ulcers on the damselfish chromis punctipinnis. Science 214, 1139-1140. Lu, T.K. and Koeris, M.S. (2011) The next generation of bacteriophage therapy. Curr Opin Micoribol 14, 524-531. Magariños, B., Bonet, R., Romalde, J.L., Martínez, M.J., Congregado, F. and Toranzo, A.E. (1996) Influence of the capsular layer on the virulence of Pasteurella piscicida for fish. Microb Pathog 21, 289-297. Mett, C.L. and Sturgeon, R.J. (1982) Cation-exchange chromatography of histamine in the presence of ethylammonium chloride. J Chromatogr A 235, 536-538. Nakakoshi, M., Nishioka, H. and Katayama, E. (2011) New versatile staining reagents for biological transmission electron microscopy that substitute for uranyl acetate. J Electron Microsc 60, 401-407. O’Flaherty, S., Ross, R.P. and Coffey, A. (2009) Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol Rev 33, 801-819. O’Flynn, G., Ross, R.P., Fitzgerald, G.F. and Coffey, A. (2004) Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. Appl Environ Microbiol 70, 3417-3424. Park, M., Lee, J-H., Shin, H., Kim, M., Choi, J., Kang, D-H., Heu, S. and Ryu, S. (2012) Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7. Appl Environ Micobiol 78, 58-69. Pedersen, K., Dalsgaard, I. and Larsen, J.L. (1997) Vibrio damsela associated with diseased fish in Denmark. Appl Environ Microbiol 63, 3711-3715. This article is protected by copyright. All rights reserved.
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Potter, N.N. and Nelson, F.E. (1953) Role of calcium and related ions in proliferation of lactic streptococcus bacteriophage. J Bacteriol 66, 508-516. Puck, T.T., Garen, A. and Cline, J. (1951) The mechanism of virus attachment to host cells: I. The role of ions in the primary reaction. J Exp Med 93, 65-88. Rodtong, S., Nawong, S., Yongsawatdigul, J. (2005) Histamine accumulation and histamine-forming bacteria in Indian anchovy (Stolephorus indicus). Food Microbiol 22, 475-482. Rountree, P.M. (1951) The role of certain electrolytes in the adsorption of staphylococcal bacteriophages. J Gen Microbiol 5, 673-680. Rountree, P.M. (1955) The role of divalent cations in the multiplication of staphylococcal bacteriophage. J Gen Microbiol 12, 275-287. Sánchez-Guerrero, I.M., Vidal, J.B. and Escudero, A.I. (1997) Scombroid fish poisoning: A potentially life-threatening allergic-like reaction. J Allergy Clin immunol 100, 433-434. Scholl, D., Adhya, S. and Merril, C. (2005) Escherichia coli K1’s capsule is a barrier to bacteriophage T7. Appl Environ Microbiol 71, 4872-4874. Stenholm, A.R., Dalsgaard, I. and Middelboe, M. (2008) Isolation and characterization of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum. Appl Environ Microbiol 74, 4070-4078. Tanji, Y., Shimada, T., Yoichi, M., Miyanaga, K., Hori, K. and Unno, H. (2004) Toward rational control of Escherichia coli O157:H7 by a phage cocktail. Appl Microbiol Biotechnol 64, 270-274. Yamaki, S., Omachi, T., Kawai, Y. and Yamazaki, K. (2014) Characterization of a novel Morganella morganii bacteriophage FSP1 isolated from river water. FEMS Microbiol Lett 359, 166-172. This article is protected by copyright. All rights reserved.
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Figure legends Fig. 1. Morphology and genome size analysis of Phda1. (a) Transmission electron micrograph images of Phda1. Phda1 was negatively stained with 2.5% samarium triacetate. Arrows indicate that Phda1 adsorbed to a host cell. The scale bar indicates 200 nm. (b) Agarose gel electrophoresis analysis of Phda1. Lane 1: Gene ladder wide I (Nippon Gene; Tokyo, Japan); lane 2: Phda1 DNA; lanes 3, 4, and 5: Phda1 DNA digested with EcoRV, PstI, and PvuII (Nippon Gene); lane 6: 2.5-kb DNA ladder (Takara Bio; Tokyo, Japan).
Fig. 2. Thermal (a) and pH (b) stability of Phda1 in TSBS. Closed diamonds: heated at 60°C, open squares: heated at 70°C, closed triangles: heated at 75°C, open circles: heated at 80°C, closed squares: heated at 85°C. The results are shown as the mean ± standard deviation from three independent experiments.
Fig. 3. Effect of divalent cations on the adsorption rate of Phda1. Adsorption assays were conducted in cation broth without divalent cations or containing 1 mmol l-1 CaCl2, MgSO4, FeCl2, MnCl2, or ZnCl2. The results are shown as the mean ± standard deviation from three independent experiments.
Fig. 4. Effect of divalent cations on the one-step growth of Phda1 (PFU infected cell-1). One-step growth analyses were conducted in cation broth without divalent cations (diamond) or containing 1 mmol l-1 CaCl2 (circle), MgSO4 (square), or FeCl2 (triangle). The results are shown as the mean ± standard deviation from three independent experiments. This article is protected by copyright. All rights reserved.
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Fig. 5. Effect of Phda1 on the growth and histamine production of P. damselae subsp. damselae at 8°C (a), 12°C (b), and 20°C (c). Each symbol and column represents the viable cell count of P. damselae subsp. damselae with (closed circles) or without (closed diamonds) Phda1, and the histamine concentration with (closed columns) or without (open columns) Phda1. An asterisk (*) indicates a histamine concentration below the detectable limit (20 mg l-1).
Table 1. Bacterial strains used in this study and host range of Phda1. Bacterial species Gram-negative bacteria Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. damselae Photobacterium damselae subsp. piscicida Photobacterium phosphoreum Photobacterium phosphoreum
Strains
Plaque formation
JCM8967 JCM8968 JCM8969 MA1 MA2 MA3 OY1 OY2 SW1 Dpp-1 JCM20393 NBRC13896
+ -
Photobacterium phosphoreum Photobacterium ganghwense Photobacterium lutimaris Cronobacter muytjensii Escherichia coli
NBRC103031T JCM12487 JCM13586 ATCC51329 RIMD0509939
-
Morganella morganii subsp. morganii Proteus vulgaris Salmonella enterica serovar Enteritidis Salmonella enterica serovar Typhimurium
NBRC3848T ATCC33420 NBRC3313 IID1000
-
Yersinia enterocolitica
ATCC9610T
-
Vibrio alginolyticus
T
LMG4409
-
Vibrio anguillarum
LMG4437T
-
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Vibrio cincinnatiensis
LMG7891T
-
Vibrio fluvialis
LMG7894T
-
Vibrio harveyi
T
-
LMG4044
Vibrio metschnikovii
T
LMG11664
-
Vibrio natriegens
LMG10935T
-
T
Vibrio furnissii
-
LMG7910
T
Vibrio parahaemolyticus Vibrio vulnificus Gram-positive bacteria Bacillus cereus Listeria grayi Listeria innocua Listeria monocytogenes Staphylococcus aureus +, clear plaque; -, no plaque.
NBRC12711
-
LMG13545T
-
JCM2152T ATCC25401
-
ATCC33090T ATCC7644 NBRC14462
-
Table 2. Effect of calcium and magnesium ions on the adsorption rate (%) of Phda1 after incubation at 25°C for 5 min. 0 mmol l-1
1.0 mmol l-1
10 mmol l-1
25 mmol l-1
Ca2+
3.1 ± 4.32
17.7 ± 4.89
59.5 ± 11.5
79.3 ± 2.29
Mg2+
3.1 ± 4.32
18.9 ± 7.56
42.1 ± 1.83
74.1 ± 2.27
Table 3. Effect of calcium and magnesium ions on the latent period, rise period, and burst size of Phda1.
Control -1
1.0 mmol l
10 mmol l 25 mmol l
Burst size
(min)
(min)
(PFU infected cell-1)
60
50
19.1 ± 3.62
40
30
58.1 ± 7.30
2+
50
40
52.4 ± 14.6
2+
30
30
72.8 ± 4.61
2+
40
40
67.0 ± 13.4
2+
30
30
83.9 ± 21.5
2+
30
30
46.0 ± 17.3
Ca Ca
Mg -1
Rise period
2+
Mg -1
Latent period
Ca
Mg
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