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Inhibitory effect of a combination with novel jumbo

bacteriophages ΦMV-1 and ΦMV-4 on Morganella morganii
subsp. morganii growth and histamine accumulation

Shogo Yamaki, Soya Kuronuma, Yuji Kawai, Koji Yamazaki

PII: S0168-1605(19)30388-5
DOI: https://doi.org/10.1016/j.ijfoodmicro.2019.108457
Reference: FOOD 108457

To appear in: International Journal of Food Microbiology

Received date: 2 July 2019

Revised date: 19 November 2019
Accepted date: 21 November 2019

Please cite this article as: S. Yamaki, S. Kuronuma, Y. Kawai, et al., Inhibitory effect
of a combination with novel jumbo bacteriophages ΦMV-1 and ΦMV-4 on Morganella
morganii subsp. morganii growth and histamine accumulation, International Journal of
Food Microbiology (2019), https://doi.org/10.1016/j.ijfoodmicro.2019.108457

This is a PDF file of an article that has undergone enhancements after acceptance, such
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© 2019 Published by Elsevier.

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Running title: Inhibitory effect of M. morganii phages

Inhibitory effect of a combination with novel jumbo bacteriophages ΦMV-1 and

ΦMV-4 on Morganella morganii subsp. morganii growth and histamine accumulation

Shogo Yamaki1, Soya Kuronuma2, Yuji Kawai1 and Koji Yamazaki1

1
Laboratory of Marine Food Science and Technology, Faculty of Fisheries Sciences,

Hokkaido University, Hakodate, Japan

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Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan

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Correspondence: Shogo Yamaki, Laboratory of Marine Food Science and Technology, Faculty
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of Fisheries Sciences, Hokkaido University 3-1-1, Minato, Hakodate 041-8611, Japan. Tel:

+81 138 40 5567; e-mail: yamaki@fish.hokudai.ac.jp

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Abstract

Histamine (scombroid) poisoning is a foodborne illness caused by ingestion of

histamine-contaminated seafood; therefore, inhibition of the growth of histamine-producing

bacteria is key for it prevention. Infection of pathogenic bacteria by bacteriophages (phages)

is being developed to prevent multiple foodborne illnesses. Here, we describe the inhibitory

effect of a phage mixture on growth and histamine accumulation of Morganella morganii

subsp. morganii, the primary causative agent of histamine poisoning in fish meat. We isolated

novel two phages, ΦMV-1 and ΦMV-4, which infected M. morganii subsp. morganii strains

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tested in this study. ΦMV-1 and ΦMV-4 belong to family Myoviridae. Pulsed-field gel

electrophoresis revealed that these phages are jumbo bacteriophages with large genomes. The

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latent period, rise period and burst size of ΦMV-1 were 30 min, 60 min, and 224 PFU per
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infected cell, respectively, and those of ΦMV-4 were 60 min, 50 min, and 62 PFU per infected

cell, respectively. A mixture of ΦMV-1 and ΦMV-4 effectively prevented regrowth of M.

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morganii subsp. morganii after phage treatment, suggesting that the phage mixture treatment
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is more effective for inhibition of growth and histamine accumulation by M. morganii subsp.

morganii than single phage treatment. Treatment with phage mixture inhibited growth and
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histamine accumulation by M. morganii subsp. morganii in canned and fresh tuna. The phage

mixture might be an effective way to prevent growth of the histamine producer and
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accumulation of histamine in seafood.

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Keywords: Bacteriophage, phage utilization, Morganella morganii subsp. morganii,

histamine poisoning

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1. Introduction

Histamine poisoning, also known as scombroid poisoning, is a common type of

poisoning caused by seafood. Unlike most cases of food poisoning, the disease is not caused

by infection of the patient, but by ingesting the histamine secreted into fish meat by bacteria.

Histidine-rich fish meats, including tuna, mackerel, bonito, and sardine, are causative foods.

The symptoms of scombroid poisoning are mild, allergy-like symptoms including hives, rash,

flushing and facial swelling (Hungerford, 2010). However, there are some reports of severe

cases with potentially life-threatening events (D’Aloia et al., 2011; Sánchez-Guerrero et al.,

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1997). The elimination of histamine from foods is impractical because it is stable during

heating and freezing. The good hygienic practice is important for avoiding contamination of

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histamine-producing bacteria. However, some of the histamine-producing bacteria are natural
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contaminants of seafood. Therefore, to prevent poisoning, the growth of histamine-producing

bacteria must also be suppressed before they secrete high levels of histamine.
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Morganella morganii, a motile gram-negative bacterium, belongs to the family
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Enterobacteriaceae and is the causative agent of many diseases including diarrhea, urinary

tract infections, bacteremia, and sepsis (Chang et al., 2011; Müller, 1986). M. morganii has a
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strong ability to secrete histamine in decomposed fish meats and is a leading cause of

histamine poisoning in humans because of its high histidine decarboxylase activity

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(López-Sabater et al., 1996; Rodtong et al., 2005). M. morganii is widely distributed in fish
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and their environment. Kim et al. (2003) showed M. morganii in the gills, skin, and intestine

of fish, as well as on the surfaces of conveyor belts and plastic totes used in fish processing.
Hence, inhibiting the growth of M. morganii is critical during seafood processing and storage

to minimize histamine poisoning.

Bacteriophages (phages) are viruses that specifically infect and lyse bacterial cells.

They are now being considered as agents for controlling or reducing pathogens. Therapeutic

applications of phages infecting vancomycin-resistant Enterococcus faecium,

multidrug-resistant Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas

aeruginosa have been investigated in multiple studies (Biswas et al., 2002; Gupta and Prasad,

2011; McVay et al., 2007; Shen et al., 2012). Phages can also be used to control foodborne

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pathogens. Some phage products, such as LISTEXTM P100 against Listeria monocytogenes,

SalmonelexTM against Salmonella enterica (Micreos Food Safety, Wageningen, The

Netherlands), and EcoShieldTM against Escherichia coli (Intralytix, Baltimore, MD, USA),

have already been developed. The anti-Listeria activity of LISTEXTM P100 has been

demonstrated on cheese, melon, pear, cooked turkey, roast beef, and raw salmon fillets

(Chibeu et al., 2013; Oliveira et al., 2014; Soni et al., 2012; Soni and Nannapaneni, 2010).

The Department of Agriculture and the Food and Drug Administration of the United States

have approved LISTEXTM P100 as a “Generally Recognized As Safe” food processing aid for

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control of L. monocytogenes. For biogenic amine reduction, Ladero et al. (2016) reported the

efficacy of using Enterococcus faecalis phage Q69 to inhibit tyramine-producing E. faecalis

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in cheese production. In addition, our previous report indicated possibility of phage utilization
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for the prevention of histamine accumulation in tuna (Yamaki et al., 2018).

Practical uses of phages for food safety have some limitations, particularly limited
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host range as well as regrowth of the target bacteria. Our previous report showed regrowth of
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M. morganii on challenge testing in vitro after phage treatment, although phage treatment

caused a 6-log CFU/mL reduction of the viable cell count (Yamaki et al., 2014). Mixtures of
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multiple phages appear to be more effective in inhibiting regrowth and the emergence of

phage-resistant bacteria. Tanji et al. (2004) reported that a mixture of two phages could delay
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the emergence of phage-resistant E. coli O157:H7, and Kim et al. (2014) mentioned that the
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mixture of phage SSU 5 and SSU 14, which adsorb to the lipopolysaccharide core or

O-antigen repeats of S. Typhimurium, might delay the growth of S. Typhimurium in liquid

medium compared to single-phage treatment. Our previous reports have shown that phage

utilization has potential for inhibiting histamine poisoning (Yamaki et al., 2014; Yamaki et al.,

2015: Yamaki et al., 2018). However, in the past, few phages for the biological control of M.

morganii have been identified (Oliveira et al., 2017), and additional M. morganii phages are

needed for efficient control by treatment with phage mixture. In this study, we have isolated

and characterized two previously unknown phages infecting M. morganii subsp. morganii and

evaluated phage-mixture treatment for inhibiting the growth of M. morganii subsp. morganii

and accumulation of histamine.

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2. Materials and Methods

2.1. Bacterial strains and growth conditions

Table 1 lists bacterial strains used in this study, and Table S1 shows the capability of

histamine production of used strains. All strains except M. psychrotolerans and

Photobacterium were grown in tryptic soy broth (TSB; Becton, Dickinson and Company

(BD), Franklin Lakes, NJ, USA) at 30°C for 18 h. M. psychrotolerans was grown in TSB at

25°C for 24 h, and Photobacterium was grown in TSB supplemented with 1.5% NaCl for 24 h.

P. phosphoreum was incubated at 20°C, and P. damselae was incubated at 25°C. For bacterial

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challenge assays using raw tuna, chloramphenicol-resistant M. morganii subsp. morganii

NBRC3848cr was used for selective detection of inoculated M. morganii among contaminants

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in the raw tuna. This strain was grown in TSB supplemented with 1% histidine and 50 µg/mL
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chloramphenicol at 30°C for 18 h.
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2.2. Isolation of phages
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We isolated M. morganii phages using the method of Carvalho et al. (2010) with

minor modifications. Briefly, 400 mL of river water was added to 400 mL of 2× TSB
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containing 400 µg/mL CaCl2 and 400 µg/mL MgSO4. M. morganii subsp. morganii

NBRC3848T, NBRC3168, or ATCC25829 cells in log phase were inoculated into the resulting
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mixture (105 CFU/mL) and incubated at 30°C for 6 h. After incubation, 10 mL of each sample
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was centrifuged (6,000 × g, 20 min, 4°C), and the resulting supernatants were decanted and

filtered using 0.45-µm pore size polyvinylidene fluoride membrane filters (Merck Millipore,
Billerica, MA, USA). The filtrates were used for phage detection. Ten µL of filtrate was

spotted onto 0.5% top agar inoculated with M. morganii subsp. morganii overlaid, onto tryptic

soy agar (TSA; BD). Plates were incubated at 30°C for 18 h. Clear single plaques were picked

and suspended in SM buffer (100 mmol/L NaCl, 8 mmol/L MgSO4·7H2O, 50 mmol/L

Tris-HCl, and 0.01% gelatin, pH 7.5). These steps were repeated at least three times for phage

purification. To propagate the isolated phages, 800 µL of phage suspension were inoculated at

108 PFU/mL to 800 mL of a culture of M. morganii NBRC3848T (OD600 = 0.1) and incubated

at 30°C for 6 h. The culture was centrifuged (6,000 × g, 20 min, 4°C) and the supernatant was

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decanted and filtered as described above. Phages were concentrated by polyethylene glycol

precipitation method and purified on a CsCl density gradient. Purified phages were dialyzed

and stored in SM buffer at 4°C.

2.3. Host range determination

For each fresh bacterial broth culture listed in Table 1, 100 µL of each fresh liquid

culture was added to 3 mL of 0.5% soft agar and overlaid onto a TSA or TSA supplemented

with 1.5% NaCl plate. After solidification of the agar, 10 µL of phage solution was spotted

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onto the bacterial lawn. Plates were incubated, and plaque formation were checked. Host

ranges were evaluated by plaque formation and efficiency of plating (EOP). EOP was

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calculated by a comparison of phage titers on each bacterial strain with those of the strains
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obtained as maximum phage titers.
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2.4. Transmission electron microscopy (TEM)
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High-titer phage solutions (>109 PFU/mL) were dropped onto a 200-mesh Cu grid

(Nisshin EM, Tokyo, Japan) and negatively stained with 2.5% samarium triacetate, a good
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substitute for uranyl acetate for negative staining (Nakakoshi et al., 2011). The morphologies

of isolated phages were observed with a JEM-1011 (JEOL, Tokyo, Japan). Phages were
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classified by morphology.
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2.5. Pulsed field gel electrophoresis (PFGE)

PFGE was used for genome size estimation as described by Lingohr et al. (2009).

Purified phages were embedded in agarose plugs (SeaKem Gold Agarose, Lonza, Rockland,

ME, USA) and lysed by soaking in lysis buffer and Proteinase K. PFGE was performed on a

CHEF-DR II system (Bio-Rad Laboratories, Hercules, CA, USA).

2.6. Analysis of adsorption kinetics

Liquid culture of M. morganii NBRC3848T was inoculated at 105 CFU/mL into

TSB and grown at 30°C. When the OD600 of the culture reached 0.8, bacteria were pelleted

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(10,000 × g, 5 min, RT) and washed with fresh TSB. Five mL of the culture was

pre-incubated at 30°C for 5 min, then each phage was added at 106 PFU/mL. The mixture was

incubated at 30°C, and 100 µL of each sample was added to 900 µL of SM buffer. The sample

solution was filtered through a 0.45-µm membrane, then PFU were quantitated by a

double-agar overlay method. One hundred µL of each sample and broth culture of M.

morganii NBRC3848T were added to 4 mL of 0.5% molten soft agar and poured onto a TSA

bottom plate. After incubation at 30°C for 24 h, plaques were counted.

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2.7. One-step growth curves

M. morganii NBRC3848T was inoculated into TSB and incubated at 30°C. When

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the OD600 of the culture reached 0.1, phages were added at 105 PFU/mL and adsorbed at 30°C
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for 10 min. Following centrifugation (10,000 × g, 2 min, 4°C), the pellet containing infected

cells was resuspended in fresh TSB, incubated at 30°C, and samples for measurement were
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collected chronologically. Phage titers were measured as described above.
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2.8. Heat and pH tolerance

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To evaluate thermal stability, phages were added to preheated TSB (40–70°C) to a

final concentration of 107 PFU/mL. During heating (40–70°C) for intervals between 0 to 30
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min, samples were taken every 5 min and placed on ice. Phage titers were measured as
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described above. The pH stability of phages was examined by incubating them in TSB (pH 3–

12) at 30°C for 24 h before measurement.

2.9. Bacterial challenge assays in liquid medium

Equivalent mixture of M. morganii NBRC3848T and NBRC3168 was inoculated at

1×103 CFU/mL into TSB containing 1% histidine (pH 6.5). Phage ΦMV-1 (1 × 105 PFU/mL),

ΦMV-4 (1 × 105 PFU/mL), or an equivalent phage mixture of ΦMV-1 and ΦMV-4 (1 × 105

PFU/mL), were added to cultures. Cultures were incubated at 12°C for 12 days, and samples

were collected for determination of viable cell counts at 2, 4, 6, 8, 10 and 12 days. Collected

samples were diluted and spread onto TSA plates for determination of viable cell count.

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2.10. Analysis of regrowth rates after phage treatment

One-hundred microliter of equivalent mixtures of M. morganii NBRC3848T and

NBRC3168 inoculated in TSB were added to wells of 96-well microtiter plates. Thereafter,

ΦMV-1, ΦMV-4, or the equivalent mixture of ΦMV-1 and ΦMV-4 were diluted in TSB and

100 µL of dilution was inoculated. TSB inoculated M. morganii without phage was used as a

control. Final concentrations of bacteria and phage were 1 × 103 CFU/mL and 1 × 107

PFU/mL, respectively. Plates were incubated at 20°C, and OD595 absorbances were measured

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at 24-h intervals. Wells with OD595 > 0.05 were defined as regrown, and the regrowth rate (%)

was calculated as:

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(Number of wells with OD595 > 0.05 / Number of all wells) × 100
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2.11. Bacterial challenge assay using canned tuna
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Mixture of M. morganii NBRC3848T and NBRC3168 was inoculated at 3 × 103
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CFU/g to 75 g of sterilized canned tuna. After mixing, 750 µL of the phage mixture

(equivalent mixture of ΦMV-1 and ΦMV-4) was inoculated at 3 × 107 PFU/g. Inoculated
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canned tuna were mixed, stored at 12°C for 5 days or 20°C for 48 h, and 5 g of stored meats

were collected and homogenized with 45 mL of PBS at each sampling time during storage.
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Homogenized solution was diluted and spread onto TSA plates for viable cell counts of M.
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morganii. TSA plates were incubated at 30°C for 48 h, and CFU was determined.

For histamine assays, homogenized solutions were heated at 100°C for 30 min to
denature histidine decarboxylases and centrifuged. Histamine in supernatants was measured

with a Histamine Test Kit (Kikkoman Biochemifa, Tokyo, Japan) according to the

manufacturer’s instructions.

2.12. Bacterial challenge assay using raw tuna

Raw tuna purchased at a local supermarket was cut into 5-g pieces using a sterile

knife and each piece of meat was placed in a sterile petri dish. Fresh cultures of M. morganii

NBRC3848cr, which is chloramphenicol resistant, were washed and diluted. Diluent (50 µL)

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was added to each piece at 3 × 103 CFU/g. After incubation at room temperature for 30 min,

100 µL of the mixture of ΦMV-1 and ΦMV-4 diluted with PBS was added at 3 × 107 PFU/g.

Samples were incubated at 20°C, with samples taken to assay bacterial growth and histamine

concentration every 12 h.

For bacterial counts, pieces of tuna were homogenized with 45 mL of PBS and

spread onto agar plates. For counts of all bacteria, samples were spread onto TSA plates. For

M. morganii counts, samples were spread onto modified Niven’s agar (5.0 g/L tryptone, 5.0 g

/L yeast extract, 1.0 g/L CaCO3, 25.0 g/L L-histidine hydrochloride monohydrate, 5.0 g/L

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NaCl, 0.06 g/L bromocresol purple, and 20.0 g/L agar, pH 5.1; Niven et al., 1981)

supplemented with 50 µg/mL chloramphenicol. After spreading, TSA plates were incubated at

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30°C for 48 h, and Niven’s agar plates were incubated at 30°C for 24–48 h.
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2.13. Statistical analyses
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Bacterial and phage counts were performed in duplicate. Results are represented as
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mean values and standard deviation. Two-way ANOVA with Holm-Bonferroni method or

Welch’s test were conducted for comparisons of means. P values less than 0.05 were
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considered to be statistically significant. All statistical tests were conducted with R v3.3.1 (R

Core Team, 2016).

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3. Results

3.1. Isolation and host ranges of phages ΦMV-1 and ΦMV-4

In this study, we succeeded in isolating phages ΦMV-1 and ΦMV-4 infecting M.

morganii subsp. morganii from river water samples obtained in Hakodate, Hokkaido, Japan.

These phages formed plaques against M. morganii subsp. morganii. The host ranges of

ΦMV-1 and ΦMV-4 were tested against the 30 strains of gram-negative bacteria including 10

strains of genus Morganella (Table 1). M. morganii subsp. morganii strains were sensitive to

ΦMV-1 or ΦMV-4, and two strains (ATCC25829 and JNBP_03145) showed lysis only at high

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phage titer. M. psychrotolerans JCM16473T was sensitive to ΦMV-1. However, M. morganii

subsp. sibonii JCM16939T was not sensitive to both phages. Both phages did not show lysis

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against Proteus and Providencia that were related genera to Morganella. These observations
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suggest that ΦMV-1 and ΦMV-4 are useful phages for the control of M. morganii subsp.

morganii.
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3.2. Morphology and genome sizes of phages ΦMV-1 and ΦMV-4

The isolates ΦMV-1 and ΦMV-4 were further characterized by TEM. Based on
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morphology, ΦMV-1 and ΦMV-4 appeared to be members of the family Myoviridae, with

long, rigid, and contractile tails (Fig. 1A, B). The head diameter and tail length of ΦMV-1
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were 116 ± 8 nm and 307 ± 16 nm, respectively (n = 30), and those of ΦMV-4 were 121 ± 8
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nm and 122 ± 9 nm (n = 30), respectively. Genome sizes were analyzed by PFGE (Fig. 1C).

The genome sizes of ΦMV-1 and ΦMV-4 were approximately 230 and 360 kbp, respectively,
indicating that these phages were jumbo phages.

3.3. Adsorption kinetics and one-step growth curves of phages ΦMV-1 and ΦMV-4

Figure 2A show changes of rate of free phages during incubation. ΦMV-1 rapidly

adsorbed against M. morganii cells, and 88% of ΦMV-1 adsorbed at 2.5 min. However,

adsorption of ΦMV-4 was slow. After 10 min, only 50% of ΦMV-4 was adsorbed against M.

morganii cells. Adsorption constants of ΦMV-1 and ΦMV-4 were 1.11 × 10-9 mL/min and

9.05 × 10-11 mL/min. One-step growth curves (Fig. 2B) show that the latent period, rise period,

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and burst size of ΦMV-1 were 30 min, 60 min, and 224 PFU per infected cell, and those of

ΦMV-4 were 60 min, 50 min, and 62 PFU per infected cell, respectively.

3.4. Thermal and pH stability of ΦMV-1 and ΦMV-4

Phage stability is an extremely important consideration for industrial use as

biocontrol agents. Figures 3A and C show the viability of ΦMV-1 and ΦMV-4 as a function of

temperature. ΦMV-1 survived at 40°C and 50°C for 30 min, but viable ΦMV-1 was not

detected after heating at 60°C for 20 min or at 70°C (Fig. 3A), indicating that heating to 60°C

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is sufficient to inactivate ΦMV-1. The thermal stability of ΦMV-4 was superior to that of

ΦMV-1, as it was stable at 40–60°C for 30 min. Viable ΦMV-4 was not detected after heating

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to 70°C (Fig. 3C). The pH stability of the phages is shown in Figs. 3B and D. ΦMV-1 was
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stable at pH 5.0–8.0, for 24 h (Fig. 3B). ΦMV-4 had greater pH stability than ΦMV-1, as it

survived at pH 4.0–11.0, although a 1.5-log reduction was observed at pH 4.0. After pH

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treatment at pH 3.0 or 12.0 for 24 h, ΦMV-4 could not be detected (Fig. 3D). These results
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indicate that ΦMV-1 and ΦMV-4 have suitable stabilities for practical use in ready-to-eat

seafood or raw materials for processed seafood.

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3.5. Phage mixture suppresses growth and regrowth of M. morganii subsp. morganii
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Figure 4 shows changes of M. morganii growth in TSB containing 1% histidine at 12°C.

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Growth was suppressed by each phage or both together between 4 and 6 days of incubation (P

< 0.05). However, after 6 days, M. morganii showed regrowth and reached around 8 log
CFU/mL after 12 days of storage in samples treated with single phage; moreover, differences

from control cultures were not significant at 10 and 12 days (P > 0.05). Phage mixture

treatment using both ΦMV-1 and ΦMV-4 suppressed the growth of M. morganii throughout

the 12-day storage less than 5 log CFU/mL (P < 0.05). These results suggest that resistance

was developed in the samples treated with single phage, but both together were more effective

for the control of bacterial multiplication.

To confirm the inhibitory effect of the phage mixture on growth of M. morganii, we

then measured optical densities at 595 nm and calculated regrowth rates over 120 h in 96-well

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microtiter plate (Table 2). These results show that regrowth rates of single phage treatment

was 27.6% at 120 h post inoculation; however, phage mixture reduced the frequency of

positive wells more than 5-fold than single phage treatment.

3.6. Phage mixture controls growth of M. morganii subsp. morganii and prevents

histamine accumulation in canned tuna

Figure 5 shows changes in cell counts and histamine concentrations in canned tuna

treated with or without the phage mixture at 12°C or 20°C. At 12°C, in canned tuna without

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phage infection, cell counts exceeded 8 log CFU/g and histamine began to accumulate after 3

days storage. After 5 days, bacterial growth reached 9.0 log CFU/g and histamine

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concentration in canned tuna exceeded 4,000 mg/kg. However, infection with the phage
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mixture significantly decreased cell counts (P < 0.05). Histamine could not be detected during

the first 4 days. Only a slight amount of histamine was detected after 5 days (Fig. 5A and 5B).
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At 20°C, M. morganii rapidly grew and reached 8 log CFU/g after 24 h in
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uninfected control samples. Histamine concentrations were 625 mg/kg after 24 h and more

than 3,000 mg/kg after 48 h. In phage-infected canned tuna, growth was significantly
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inhibited. M. morganii decreased to 3.5 log CFU/g from 5.9 log CFU/g after 36 h, but it

increased to 5.3 log CFU/g 12 h later. Histamine accumulation in phage-treated canned tuna
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meats was significantly lower than that in control samples (P < 0.05), only 60 mg/kg at 48 h
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(Fig. 5C and 5D).

3.7. Phage mixture controls growth of M. morganii subsp. morganii and prevents

histamine accumulation in fresh tuna

Figure 6 shows changes of total viable counts, viable cell counts of M. morganii,

and histamine concentration during storage of fresh, raw tuna at 20°C. Initial total viable

counts were about 5 log CFU/g and reached about 9 log CFU/g during 36 h of storage.

Because of the host-specific antimicrobial effect of phages, there is no difference between

total viable count of control and phage treatment samples (Fig. 6A). Initial counts of M.

morganii in control tuna were 3.4 log CFU/g, and that in tuna treated with phage mixture was

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2.6 log CFU/g (P < 0.05), respectively. After that, phage treatment conditions showed

significant reductions in cell counts at all storage times (Fig. 6B).

Figure 6C shows changes in histamine concentrations of raw tuna during storage at

20°C. Histamine was detected at 24 h in both phage-treated and untreated tuna, but phage

treatment lowered the concentration almost 10-fold. Thereafter, histamine rapidly

accumulated in the control sample, but its accumulation in the phage-treated sample was

much slower than in the control sample (P < 0.05).

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4. Discussion

In this study, we isolated and characterized novel M. morganii subsp. morganii

phage ΦMV-1 and ΦMV-4. PFGE revealed that both isolated phages were jumbo phages with

genomes larger than 200 kbp (Yuan and Gao, 2017). Isolations of jumbo phages are rare

because their large virions tend to prevent visible plaque formation and passage through

membrane filters (Serwer et al., 2007). ΦMV-1 and ΦMV-4 may not be able to pass 0.22 µm

pore size membrane filters (Fig. 1A). In this study, we used the 0.45 µm pore size membrane

filters, allowing the successful isolation of jumbo M. morganii subsp. morganii phages. The

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biology of jumbo phages is not well understood, but they have been shown to differ from

smaller, classical phages (Yuan and Gao, 2017). To our knowledge, this is the first report of

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jumbo M. morganii subsp. morganii phages; further studies will reveal more about their
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biology.

Phage mixtures are an attractive tool for the prevention of phage resistance in
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bacteria. O’Flynn et al. (2004) observed frequencies of bacteriophage insensitive mutants for
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single-phage were between 1.2×10−6 and 3.3×10−4, but with double infections, mutant

frequencies were between 1.1×10−6 and 1.5×10−6. Moreover, Tanji et al. (2004) also showed
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that a phage mixture delayed the emergence of phage-resistant cells, but not all combinations

were effective. The mechanism of this prevention is hypothesized to be that bacteria resistant
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to one phage could still be killed by another phage with a different receptor-binding protein.
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When multiple phages are used to control pathogens, a combination of phages that recognize

different host receptors is recommended. Our results showed that the adsorption kinetics
between ΦMV-1 and ΦMV-4 were different (Fig. 2A). This result suggests that the

receptor-binding mechanisms of ΦMV-1 and ΦMV-4 are likely different from each other.

Our data suggest that the combination of phages ΦMV-1 and ΦMV-4 has potential

for control of M. morganii subsp. morganii. We confirmed the effect of the phage mixture

combining ΦMV-1 and ΦMV-4 on inhibition of bacterial growth and histamine accumulation.

Treatment with either single phage inhibited the growth of M. morganii subsp. morganii, but

regrowth of the bacteria eventually occurred. In contrast, the mixture of ΦMV-1 and ΦMV-4

suppressed the bacterial growth throughout the 12-day incubation period, although cell counts

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fluctuated between 3 and 5 log CFU/mL (Fig. 4). This fluctuation in cell counts might imply

generation of phage-resistant bacteria and mutant phage infecting phage-resistant bacteria.

Staphylococcal Twort-like phage ΦSA012 mutates its receptor-binding protein during

coincubation with the host, allowing mutant phages to infect ΦSA012-resistant strains

(Takeuchi et al., 2016).

Our data showed that single phages controlled bacterial growth. However,

Paez-Espino et al. (2015) reported that clustered regularly interspaced short palindromic

repeat (CRISPR) immunity drove phage genome evolution by single-nucleotide

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polymorphisms in sequences targeted by CRISPR, and the presence of more than one type of

phage increased the persistence of infection by the recombination-based creation of chimeric

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phage. Therefore, phage mixtures increase the potential for counterattack against
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phage-resistant bacteria by increased genetic or receptor-binding protein divergence. Indeed,

our results showed that the mixture of ΦMV-1 and ΦMV-4 significantly decreased the
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regrowth rates of M. morganii subsp. morganii during phage treatment (Table 2).
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After phage mixture treatment, we observed regrowth of M. morganii subsp.

morganii in tuna (Fig. 5 and 6). To confirm whether the regrowing M. morganii subsp.
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morganii were phage-resistant, we isolated 26 colonies and checked the sensitivity to

infection by ΦMV-1 and ΦMV-4 by spot testing. All 26 bacterial isolates were sensitive (data
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not shown). We hypothesize that this is because the phages were not diffusing effectively for
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immotility, so that bacteria were only subject to infection on the surface of the tuna. This is

consistent with high phage concentrations being important for control of foodborne pathogens
in foods (Hagens and Loessner, 2010); our previous report also showed that antimicrobial

effects were phage concentration-dependent (Yamaki et al., 2018).

Our results showed that phage mixture treatment is a promising method for

inhibition of histamine production by M. morganii subsp. morganii in tuna (Fig. 5 and 6).

However, we observed differences of histamine concentration between canned and raw tuna.

At 20°C, the histamine concentration of treated canned tuna was much lower than that of

treated raw tuna. This difference might result from bacteria present in raw tuna other than M.

morganii subsp. morganii. Other histamine producers, such as Photobacterium phosphoreum,

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P. damselae, and M. psychrotolerans, might be sources of histamine in raw tuna. However, as

shown in Figure 6C, the majority of accumulated histamine was produced by M. morganii

subsp. morganii and the phage mixture showed the significant reduction in histamine in raw

tuna. In the future, for more effective inhibition, it will likely be necessary to isolate and

characterize additional phages infecting histamine-producing bacteria, particularly M.

psychrotolerans, which is psychrotolerant (Emborg et al., 2006), and has been found to have

contaminated about 40% of retail seafood in Japan (Kato et al., 2017). We will attempt to

isolate phages infecting M. psychrotolerans, as we have done for other histamine-producing

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bacteria (Yamaki et al., 2014; Yamaki et al., 2015).

In this study, we isolated and characterized two jumbo M. morganii subsp. morganii

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phages, ΦMV-1 and ΦMV-4. The phage mixture combining ΦMV-1 with ΦMV-4 suppressed
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the growth of M. morganii subsp. morganii and significantly reduced histamine accumulation

level in canned and fresh tuna. These results suggest that application of the phage mixture
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might be an effective way to prevent histamine poisoning by M. morganii subsp. morganii
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caused by seafood.
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Acknowledgements

This work was supported in part by JSPS KAKENHI Grant Number JP26450278
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and a Grant-in-Aid for JSPS Research Fellow Grant Number JP16J03433. S. Yamaki is
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supported by a Research Fellowship for Young Scientists of the Japan Society for the

Promotion of Science (JSPS).

Conflict of Interest

None declared.

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Figure legends

Fig. 1. Transmission electron micrographs of MV-1 (A) and MV-4 (B). PFGE image (C)

for genome-size estimation. Bars, 200 nm. Lane 1, 2, 3, and 4 indicates Lambda PFG marker

(New England Biolabs, Ipswich, MA, USA), MV-1 genome, MV-4 genome, and Marker 7

GT (Nippon gene, Tokyo, Japan), respectively.

Fig. 2. Adsorption kinetics (A) and one-step growth curves (B) of MV-1 and MV-4.

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Symbols represent MV-1 (◆) and MV-4 (●). Rate of free phage was calculated by

dividing the phage titer at each time (P) by the phage titer at 0 min (P0). Results are shown as

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the means ± standard deviations from three independent experiments.
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Fig. 3. Thermal (A, C) and pH (B, D) stability of MV-1 (A, B) and MV-4 (C, D). Symbols
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represent samples heated at 40°C (◆), 50°C (■), 60°C (▲), and 70°C (●). Results are shown
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as means ± standard deviations from three independent experiments.

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Fig. 4. The growth of M. morganii not treated or treated with ΦMV-1, ΦMV-4, or the

equivalent mixture of ΦMV-1 and ΦMV-4 in TSB supplemented with 1% histidine (pH 6.5)
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at 12°C for 12 days. Bars represent cell counts of M. morganii treated with ΦMV-1 (dark
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gray), ΦMV-4 (light gray), ΦMV-1 and ΦMV-4 (white), or without phages (black). The

results are shown as means ± standard deviations from three independent experiments.

Fig. 5. The growth of M. morganii (A and C) and histamine accumulation (B and D) on

canned tuna during storage at 12°C (A and B) or 20°C (C and D). Symbols represent cell

counts with (●) or without (◆) phage mixture. Bars represent histamine concentrations with

(white) or without (black) the phage treatment. The results are shown as means ± standard

deviations from three independent experiments. †Below detection limits for cell counts (2 log

CFU/g) or histamine (8 mg/kg).

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Fig. 6. Growth of total aerobic bacteria (A), M. morganii (B), and histamine accumulation (C)

on fresh tuna during storage at 20°C. Symbols represent viable cell counts with (●) or without

(◆) the phage mixture. Bars represent histamine concentration with (white) or without (black)

the phage mixture. The results are shown as means ± standard deviations from three

independent experiments. †Below the detection limit for histamine (8 mg/kg).

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Table 1. Bacterial strains used in this study and host ranges of ΦMV-1 and ΦMV-4.
Host range†
Bacterial species Strain
ΦMV-1 ΦMV-4
Morganella morganii subsp. morganii NBRC3848T +++ ++
Morganella morganii subsp. morganii NBRC3168 +++ ++
Morganella morganii subsp. morganii ATCC25829 +/ +/
Morganella morganii subsp. morganii JNBP_03126‡  +++
Morganella morganii subsp. morganii JNBP_03128‡ +++ ++
Morganella morganii subsp. morganii JNBP_03133‡ +++ +
Morganella morganii subsp. morganii JNBP_03145‡ +++ +/

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Morganella morganii subsp. morganii JNBP_03147‡  +++
Morganella morganii subsp. sibonii JCM16939T  

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Morganella psychrotolerans JCM16473T + 
JCM1669T  
Proteus mirabillis
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Proteus vulgaris ATCC33420  
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Proteus hauseri ATCC13315  
Providencia rustigianii JCM3953T  
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Providencia rettgeri ATCC9250  

Providencia stuartii ATCC33672  
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Providencia alcalifaciens ATCC9886T  

Aeromonas hydrophilasubsp. hydrophila NBRC3820  
JCM1657T  
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Citrobacter freundii
Cronobactor sakazakii ATCC51329T  
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Escherichia coli JCM1649T  

Hafnia alvei JCM1666T  
Klebsiella pneumoniae subsp. pneumoniae ATCC13883T  
Photobacterium damselae subsp. damselae JCM8968  
Photobacterium phosphoreum NBRC13896  
Pseudomonas fluorescens JCM5963T  
Raoultella ornithinolytica ATCC31898T  
Salmonella bongori CIP8233T  
Salmonella Enteritidis NBRC3313  
Yersinia enterocolitica subsp. enterocolitica ATCC9610T  
†
+++, EOP 0.5–1.0; ++, EOP 0.2–0.5; +, EOP 0.1–0.2; +/−, weak lysis (lysis only at high
titer); −, no plaque formation. Maximum titers of ΦMV-1 and ΦMV-4 were obtained against

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strain JNBP_03128 and JNBP_03147, respectively.

‡
Subspecies of these strains were not determined in the culture collection. According to
Jensen et al. (1992), two subspecies differ in the ability of trehalose fermentation. These five
strains were trehalose-negative and identified as subsp. morganii.

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Table 2. Suppression of regrowth (OD595 > 0.05) by phage mixture during incubation in 96-well
microplates.
Percent of wells with growth (OD595 > 0.05)
Phage
0h 24 h 48 h 72 h 96 h 120 h

None (control) 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

MV-1 0a* 0a 4.2 ± 1.9a 20.8 ± 3.7a 23.4 ± 1.3a 27.6 ± 2.7a

MV-4 0a 0a 1.0 ± 1.5a 6.3 ± 3.4b 10.9 ± 10.0ab 27.6 ± 6.0a

MV-1 + MV-4 0a 0a 0a 1.6 ± 1.3b 2.6 ± 1.9b 4.7 ± 3.8b

*Different letters indicate significant differences (p < 0.05) for each time point between each
phage-treated condition.

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Highlights:

 Novel M. morganii phages ΦMV-1 and ΦMV-4 were isolated.

 PFGE analysis revealed ΦMV-1 and ΦMV-4 were jumbo phages.

 Combination of ΦMV-1 and ΦMV-4 inhibited the regrowth of M. morganii.

 Phage mixture could prevent histamine accumulation in canned and fresh tuna.

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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6