LWT - Food Science and Technology 91 (2018) 353–360

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LWT - Food Science and Technology

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Isolation and application of bacteriophages to reduce Salmonella T

contamination in raw chicken meat
Hoang Minh Duc1, Hoang Minh Son1, Ken-ichi Honjoh, Takahisa Miyamoto∗
Laboratory of Food Hygienic Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka,
812-8581, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Chicken meats are considered as main sources associated with Salmonella infections in humans. In this study,
Bacteriophages lytic phages against Salmonella were isolated and examined for their efficacy to control Salmonella. Eighteen lytic
Bio-control phages were isolated from raw chicken skin and gizzard. Five phages belonging to Myoviridae and Siphoviridae
Salmonella families were characterized and selected for bacterial challenge tests. The treatment of raw chicken breast
Food safety
samples contaminated with S. Enteritidis and S. Typhimurium at 8 °C by the cocktail of five phages significantly
reduced (P < 0.05) viable counts by 1.41 and 1.86 log CFU/piece, respectively. When incubated at 25 °C, the
highest reductions of viable counts of S. Enteritidis and S. Typhimurium in the phage-treated samples were 3.06
and 2.21 log CFU/piece, respectively (P < 0.05). These data suggested that the phages isolated from raw
chicken meats are potential agents for controlling Salmonella in raw meats.

1. Introduction contamination in foods. However, these methods still represent some

Salmonella spp. is one of the most important foodborne pathogens
worldwide. In the United States, more than 40,000 cases of salmo-
nellosis were annually reported (Finstad, O'Bryan, Marcy, Crandall, &
Ricke, 2012). In Europe, Salmonella is responsible for more than 94,625
cases in 2015 (Authority & Control, European Centre for Disease
Prevention and, 2016). In Japan, the second most common foodborne
pathogen is Salmonella (Kumagai et al., 2015), causing 2551 cases of
illnesses in 2008 (Ishihara et al., 2009; Kumagai et al., 2015). More
than 2500 serovars of Salmonella have been identified but some of these
serovars, especially S. enterica serovar Enteritidis (S. Enteritidis) and S.
enterica serovar Typhimurium (S. Typhimurium) are frequently asso-
ciated with human infection (Hendriksen et al., 2011). Salmonella is
found in intestinal tract of warm-blood animals, especially chicken,
which consequently allows them to be easily transferred to meat during
slaughtering and processing (Antunes, Mourao, Campos, & Peixe,
2016). Chicken meats have been considered as the main sources of
Salmonella infections in humans (Authority & Control, European Centre
for Disease Prevention and, 2016; Doi et al., 2003; Noda et al., 2015).
Thus, reducing Salmonella contamination in chicken meats is necessary
to prevent human salmonellosis.
Various methods either based on chemical, physical, or biological
agents have been developed for the treatment of bacterial
major disadvantages such as harmfulness or toxicity to the foods, al-
tering the quality, or high cost for practical applications (Aymerich,
Picouet, & Monfort, 2008; Chen et al., 2012; Grant & Parveen, 2017;
Troy, Ojha, Kerry, & Tiwari, 2016). Lytic phages or bacteriophages are
bacterial viruses that infect their hosts and were used increasingly as
alternative tools to disinfect pathogenic bacteria in foods (Guenther,
Huwyler, Richard, & Loessner, 2009; Pang, Lambertini, Buchanan,
Schaffner, & Pradhan, 2017; M.; Sharma, Patel, Conway, Ferguson, &
Sulakvelidze, 2009). Using phage-based methods for treatment of bac-
terial contamination has some obvious advantages over conventional
ones. Firstly, phages only kill their host bacteria even antibiotics re-
sistant ones thus they are harmless to others, especially beneficial mi-
croorganisms which also present in the foods and human intestinal tract
(Loc-Carrillo & Abedon, 2011). Secondly, the addition of the lytic
phages to the foods is unlikely to induce negative impacts to food
properties (Perera, Abuladze, Li, Woolston, & Sulakvelidze, 2015). In
addition, the phages are abundant living organisms in natural en-
vironment, self-replicating, and self-limiting, which show great chances
for their successful isolation, economic and environmental benefits
(Hagens & Loessner, 2010; Loc-Carrillo & Abedon, 2011). Some com-
mercial phage products including ListShield™, Listex P-100™, EcoSh-
ield™, SalmoFresh™, and Salmonelex™ have been officially approved by
the United States Food and Drug Administration (FDA) and the United

∗
Corresponding author.
E-mail addresses: hmduc@agr.kyushu-u.ac.jp (H.M. Duc), sonbiotechn@gmail.com (H.M. Son), tmiyamot@agr.kyushu-u.ac.jp (T. Miyamoto).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.lwt.2018.01.072
Received 10 November 2017; Received in revised form 22 January 2018; Accepted 24 January 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
H.M. Duc et al. LWT - Food Science and Technology 91 (2018) 353–360

States Department of Agriculture (USDA) for use in foods to control Evaluation (NITE), Biological Research Center (NBRC), Chiba, Japan
foodborne pathogens (Atterbury, 2009; Garcia, Martinez, Obeso, & were used as hosts for phage isolation. Other Salmonella serovars were
Rodriguez, 2008; Hagens & Loessner, 2010). isolated in our laboratory. To prepare bacterial inoculum, frozen stock
Various phages infecting different foodborne pathogens have been was streaked on Tryptic Soy Agar (TSA; Becton, Dickinson and Com-
isolated from environmental samples such as municipal waste water pany) and incubated at 37 °C for 24 h. A single colony was inoculated
(Higgins et al., 2005; Hong, Pan, & Ebner, 2014), sewage (Carey-Smith, into 5 ml Luria Bertani broth (LB; Becton, Dickinson and Company) and
Billington, Cornelius, Hudson, & Heinemann, 2006; Hooton, Atterbury, incubated overnight at 37 °C. The bacterial cultures were serially di-
& Connerton, 2011; Hudson et al., 2013; Pereira et al., 2016), human luted in Phosphate Buffered Saline (PBS) to obtain desired concentra-
and animal feces (Bao et al., 2015; Bigot et al., 2011; Bigwood, Hudson, tions for each experiment.
Billington, Carey-Smith, & Heinemann, 2008; Hungaro, Mendonça,
Gouvêa, Vanetti, & Pinto, 2013; O'Flynn, Ross, Fitzgerald, & Coffey, 2.2. Phage isolation, purification and propagation
2004; Tomat, Migliore, Aquili, Quiberoni, & Balague, 2013; Viazis,
Akhtar, Feirtag, Brabban, & Diez-Gonzalez, 2011a). In these reports, the Forty-one raw chicken meat samples (12 gizzard, 11 liver, and 18
isolated phages have also been demonstrated to be active against their skin samples) purchased from local supermarkets in Fukuoka, Japan
hosts either in the broth or food environment. However, the safety of were used as the primary sources for phage isolation. Each meat sample
them when used in foods for human consumption is a matter of con- (50 g) was inoculated with overnight culture (100 μl) of S. Enteritidis
cern. For this reason, the isolation of phages directly from meat samples and S. Typhimurium, homogenized with LB broth (100 ml) supple-
could be a good choice for ensuring the safety of phages because meats mented with 10 mM CaCl2 in a sterile stomacher bag, and incubated
carrying phages are daily consumed by humans without causing serious overnight at 37 °C. After incubation, the culture (10 ml) was centrifuged
health problems. Despite the usefulness of the isolation of phages from at 12,000 × g for 5 min at 4 °C, and the supernatant was filtrated
food samples, it has been rarely reported in literature (Atterbury, through a 0.45 μm-pore size membrane filter (Merck Millipore, Ireland)
Connerton, Dodd, Rees, & Connerton, 2003; Firlieyanti, Connerton, & to yield crude phage lysate.
Connerton, 2016; Hoang Minh, Hoang Minh, Honjoh, & Miyamoto, To isolate lytic phage, the crude phage lysate (100 μl) was serially
2016). The aim of this study was to isolate lytic phages against Sal- diluted in Saline Magnesium (SM) buffer (0.05 M Tris-HCl, pH 7.5,
monella from raw chicken meat products and examine their efficacy in 0.1 M NaCl, 0.008 M MgSO4, 0.01% gelatin), mixed with 100 μl of each
reducing S. Enteritidis and S. Typhimurium in vitro and in chicken host strains in 4 ml molten agar (LB broth, agar powder 0.4% [w/v]).
breast. The mixture was then poured on TSA. The double layer agar plates were
incubated overnight at 37 °C. Following incubation, a single plaque was
2. Materials and methods picked up and re-suspended in SM buffer to generate phage suspension.
The suspension was serially diluted with SM buffer, mixed with its re-
2.1. Bacterial strains spective host, poured on TSA plates and incubated overnight at 37 °C
for producing a single plaque and these processes were repeated at least
Twenty-two Salmonella serovars used in this study were listed in three times to ensure the purity of the phages.
Table 1. Salmonella Enteritidis NBRC3313 and Salmonella Typhimurium In order to produce high titer stocks for further experiments, the
NBRC12529 obtained from National Institute of Technology and isolated phages were propagated with their original hosts. Briefly,
phage suspension (1 ml) was mixed with host culture (30 ml) at ex-
Table 1 ponential phase and incubated overnight at 37 °C. After incubation, the
Lytic range of isolated phages. mixture was centrifuged at 12,000 × g for 5 min at 4 °C and the su-
pernatant was filtrated through 0.22 μm-pore size membrane filter
Serovar Serovar Phages
designation (Merck Millipore, Ireland) to obtain phage stock. The phage stock was
SEG5 SES8 STG2 STG5 STS9 then serially diluted in SM buffer. The dilution (100 μl) was mixed with
bacterial host (100 μl) in molten agar (4 ml). The mixture was poured
S78 S. Enteritidis NBRC + + + + +
on TSA plate and incubated at 37 °C overnight. The phage titer of the
33113
S79 S. Typhimurium + - + + +
stock was determined as the numbers of plaques per milliliter (PFU/
NBRC 12529 ml).
S96 S. Typhimurium - - - - +
S97 S. Arahus - - - + -
S98 S. Agona + - + - - 2.3. Characterization of isolated phages
S99 S. Anatum - - - - -
S100 S. Braenderup - - - - - 2.3.1. Phage host range
S101 S. Derby + - + - - The host range of each isolated phages was examined on 22
S103 S. Enteritidis + + + + +
S104 S. Hadar - - + - -
Salmonella serovars listed in Table 1. Aliquot of phage stock (10 μl) was
S106 S. Typhi ± - + - ± dropped on the surface of double layer agar inoculated with 100 μl of
S107 S. Heidelberg + - + - ± each bacterial culture followed by the incubation at 37 °C for 24 h. The
S108 S. Istanbul + - + + ± presence of a clear zone at the application points indicated strong lytic
S109 S. Infantis + - - - -
activity while the absence of this zone was regarded as no lytic ability.
S110 S. Litchfield + - + + -
S111 S. London + - - + -
S112 S. Montevideo + - - - - 2.3.2. Phage morphology
S113 S. Muenster + - - + -
For morphological observation, the purified stocks of five phages
S114 S. Schwarzengrund + - + + +
S115 S. Stanley ± - + + + (STG2, SEG5, STG5, SES8, and STS9) were dropped onto carbon-coated
S116 S. Sofia - - - - + grid, negatively stained with 2% uranyl acetate, and visualized by using
S117 S. Thompson + - - - - the transmission electron microscope (TEM) (Hitachi H-7650, Hitachi,
Japan) operating at a voltage of 80 kV. The phage pictures were taken
Total infected strains (%) 16/22 2/22 (9) 12/22 10/22 10/22
(73) (55) (46) (46) at a magnification of 150,000. The morphological characteristics were
used to classify phages into families according to the phage typing in-
(+), clear lysis; ( ± ), turbid; (−), no lysis. structions (Ackermann, 2009).

354
H.M. Duc et al.
2.3.3. One-step growth curve determination
One-step growth of phages STG2, STG5, and STS9 on host S.
Typhimurium and phages SEG5 and SES8 on host S. Enteritidis was
performed according to the protocol described by Hong et al. with some
modifications (Hong et al., 2014). Briefly, the diluted phage stock
containing 106 PFU/ml was mixed with bacterial host containing
108 CFU/ml to obtain multiplicity of infection (MOI) of 0.01. The
mixture was incubated at 37 °C in a water bath for 10 min to allow the
absorption of phages before centrifuging at 10,000 × g for 30 s at room
temperature. The pellet containing phages bound to bacterial host cells
were re-suspended in 10 ml LB broth and the suspension was then kept
in a water bath at 37 °C with constant shaking. A portion of sample
(100 μl) was withdrawn and subjected to plaque assay in duplicate at
5 min interval for 60 min. Latent period was determined as the time
period between phage absorption and the first release of the phage
progeny. Burst size was estimated as the ratio of the number of released
phage progenies to the infected bacterial host cells (Pelzek, Schuch,
Schmitz, & Fischetti, 2008).
2.4. Effect of phage cocktail on viability of S. Enteritidis and S.
Typhimurium in vitro
To examine efficacy of phage cocktail on viability of S. Enteritidis
and S. Typhimurium in vitro, the suspension of 5 phages (SEG5, SES8,
STG2, STG5, and STS9) were mixed at equal parts to produce a cocktail
of 1010 PFU/ml. The diluted cultures of S. Enteritidis or S.
Typhimurium containing of 105 CFU were inoculated in 5 ml of LB
broth. The inoculated broth was treated with 100 μl (109 PFU) of phage
cocktail. SM buffer (100 μl) was used instead of the phage cocktail in
the control. The treatment and control were incubated at 25 °C and 8 °C
with constant shaking. At 2, 4, 6, and 24 h of incubation, the samples
(100 μl) were collected and serially diluted in PBS buffer. The dilutions
were then spread on TSA plates and incubated at 37 °C for 24 h. Next
day, the viable counts of Salmonella were enumerated.
2.5. Effect of phage cocktail on viability of S. Enteritidis and S.
Typhimurium on chicken breast
Raw chicken breast samples were purchased from local super-
markets in Fukuoka, Japan. The samples were cut into small pieces
(2 cm × 2 cm) and divided into two parts, one for the experiments with
S. Enteritidis and the other for those with S. Typhimurium. The phage
cocktail was prepared the same as above. The meat pieces were in-
oculated with 30 μl (3 × 104 CFU) of bacterial suspension of S.
Enteritidis or S. Typhimurium (in the experiments with S.
Typhimurium) and kept at room temperature for 30 min to allow bac-
terial attachment to meat surface. To treat the bacterial hosts, 30 μl
(3 × 108 PFU) of phage suspension was added to the meat piece. For
non-treatment, SM buffer (30 μl) was used instead of the phage cocktail.
The meat samples were incubated at 8 °C and 25 °C. At 2, 4, 6, and 24 h
of incubation, the meat pieces were withdrawn and homogenized in
10 ml of PBS buffer in a stomacher for 2 min. A portion (1 ml) of
homogenate was collected and serially diluted in PBS buffer. The ap-
propriate dilutions were spread onto Deoxycholate Hydrogen sulfide
Lactose (DHL) agar plates. Viable counts of Salmonella were enumerated
after incubation of DHL plates at 37 °C for 24 h. The efficiency of the
phage treatment was also calculated as the differences of viable counts
in the treatment and control meat pieces.
2.6. Statistical analysis
All experiments were repeated at least three times. The viable
counts were calculated as mean values and standard deviation of the
mean. The differences between treatment and control groups were
determined by t-test (Microsoft Excel 2011 for Mac OS). A value of
P < 0.05 was considered a statistically significant difference.
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LWT - Food Science and Technology 91 (2018) 353–360
3. Results
3.1. Phage isolation, purification and propagation
A total of 18 lytic phages were isolated from either skin or gizzard
samples. None of them were isolated from liver samples. The names of
the isolated phages were designed according to their initial hosts (SE for
S. Enteritidis NBRC3313, ST for S. Typhimurium NBRC12529) and the
type of meat samples (G for gizzard and S for skin). Of 18 isolated
phages, five phages SEG5, SES8, STG2, STG5, and STS9 were found to
produce clear plaques and stocks of high titer (> 109 PFU/ml)
(Table 1). Three phages SEG5, STG2, and STG5 were isolated from
chicken gizzard while 2 phages SES8 and STS9 from skin. To minimize
the cost of analysis, only these 5 phages were selected for further ex-
periments.
3.2. Characterization of phage
The host range of five tested phages was shown in Table 1. Phage
SEG5 isolated from chicken gizzard and propagated in S. Enteritidis
exhibited the widest host range, capable of infecting 16 (73%) of 22
Salmonella serovars tested. Phage SES8 isolated from chicken skin had
the narrowest lytic spectrum, lysing only 2 (9%) of 22 Salmonella ser-
ovars tested. Among hosts used for lytic range analysis, S. Enteritidis
NBRC3313 was the most sensitive host strain, being susceptible to all 5
phages. The host S. Typhimurium NBRC12529 was susceptible to 3 of 5
tested phages.
Transmission electron microscopy revealed that five tested phages
belonged to the Caudovirales order (Fig. 1). Phages STG2 and STG5
were characterized as members of Siphoviridae family with long and
non-contractile tails. The tail length of STG2 was 200 nm, whereas that
of STG5 was 216 nm. Phages STG2 and STG5 had icosahedral heads of
78 nm and 84 nm in diameter, respectively. Three remaining phages
(SEG5, SES8, and STS9) possessed typical morphological features of
Myoviridae family (Fig. 1). Among these three phages, SES8 had the
longest tail (119 nm), followed by STS9 (105 nm), and SEG5 (101 nm).
Phages SES8 and STS9 also had icosahedral heads with diameter of
76 nm and 75 nm, respectively while phage SEG5 had different head's
shape with its diameter of 55 nm (Fig. 1).
Fig. 2 indicated that the latent period of phages STG2, SEG5, and
STS9 was 15 min while that of phages STG5 and SES8 was 25 and
20 min, respectively. Phage STS9 had the largest burst size of 209 PFU/
cell, followed by phage SEG5 (95 PFU/cell), SES8 (69 PFU/cell), STG2
(52 PFU/cell), and STG5 (49 PFU/cell).
3.3. Effect of phage cocktail on viability of S. Enteritidis and S.
Typhimurium in vitro
In the experiment at 25 °C, viable counts of S. Enteritidis and S.
Typhimurium in the controls were similar to those found in the growth
of other popular enterobacteria with their counts rising steady after
incubation and reaching to plateau at 24 h (Fig. 3a and b). In contrast,
viability of S. Enteritidis and S. Typhimurium in the phage-treated
samples was significantly decreased (P < 0.05). The viable counts of S.
Enteritidis in the treatment were 2.2 and 3.7 log lower (P < 0.05)
compared to those in the control at 2 h and 4 h, respectively and below
the detection limit (< 10 CFU/ml) at 24 h (Fig. 3a). Similarly, the re-
ductions (P < 0.05) by 2.8, 4.9, and 4.2 log were found in the treat-
ment of S. Typhimurium at 2, 4, and 24 h, respectively (Fig. 3b). In the
experiment at 8 °C, both S. Enteritidis and S. Typhimurium could not
grow in the controls as their viability at tested time-points were nearly
the same with the initial inoculum whereas these bacterial hosts were
significantly reduced (P < 0.05) in the treatments (Fig. 4a and b). The
viable counts of S. Enteritidis and S. Typhimurium at 2 h incubation
were decreased (P < 0.05) by 2.6 log and 2.2 log, respectively and
these levels of reduction were maintained until 24 h (Fig. 4a and b).
H.M. Duc et al.
Fig. 1. Transmission electron micrographs (TEM) of phage STG2 (a), SEG5 (b), STG5 (c), SES8 (d), and STS9 (e).
3.4. Effect of phage cocktail on viability of S. Enteritidis and S.
Typhimurium on chicken breast
The effects of phage cocktail on viability of S. Enteritidis and S.
Typhimurium on chicken breast were also examined at 25 °C and 8 °C.
In all experiments, the naturally occurring Salmonella was not detected
in the chicken breast samples. The addition of phage cocktail to the
meat samples contaminated with S. Enteritidis and S. Typhimurium
induced significant reductions (P < 0.05) of the bacterial hosts. At
25 °C, the phage cocktail in the treatment reduced (P < 0.05) the
counts of S. Enteritidis by 1.5, 1.7, and 2.7 log CFU/piece at 2, 4, and
6 h, respectively compared to non-treatment (Fig. 5a) and in the ex-
periment with S. Typhimurium, the phage cocktail also decreased
(P < 0.05) bacterial cells by 2.0, 1.9, and 2.2 log CFU/piece at 2, 4,
and 6 h, respectively (Fig. 5b). Compared to the inoculation level, vi-
able counts of S. Enteritidis and S. Typhimurium after 6 h treatment
were 1.8 log and 1.2 log lower (P < 0.05), respectively (Fig. 5a and b).
In the phage treated samples at 24 h, regrowth of S. Enteritidis and S.
Typhimurium was observed but their viabilities were still lower
(P < 0.05) than those found in the phage free control samples (Fig. 5a
and b). When meat samples incubated at 8 °C, viable counts of S. En-
teritidis and S. Typhimurium in the controls were remained stable and
close to the initial inoculum during incubation time. Instead, the via-
bility of S. Enteritidis and S. Typhimurium in the treated samples was
significantly reduced (P < 0.05) by 1.2 and 1.4 log CFU/piece after 2 h
of incubation, respectively (Fig. 6a and b). At the end of the experiment
(24 h), the counts of S. Enteritidis and S. Typhimurium were 1.4 and 1.9
log CFU/piece less than those in the control, respectively (P < 0.05).
4. Discussion
In this study, raw chicken meats including skin, gizzard, and liver
were used as sources for isolation of phages specific to Salmonella.
Overall, 18 lytic phages specific to Salmonella were isolated only from
skin and gizzard samples. Studies have previously shown the isolation
of lytic phages against pathogenic Salmonella contaminated in foods
from different sources such as feces (Hungaro et al., 2013), chicken
meat (Thung et al., 2017), or intestinal content of broiler (Augustine &
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LWT - Food Science and Technology 91 (2018) 353–360
Bhat, 2015). To our best knowledge, our study is the first description of
isolation of lytic phages for S. Enteritidis and S. Typhimurium from
chicken skin and gizzard samples.
To reduce cost of analysis, only 5 phages (STG2, SEG5, STG5, SES8,
and STS9) that produce clear and large plaques during the isolation
steps were selected for further experiments. Morphological character-
izations showed that 5 phages belong to Myoviridae and Siphoviridae
families and have different particle sizes implicating diversity in their
morphotypes. This finding is consistent with other studies showing that
phages specific to Salmonella hosts are members of Caudovirales order
(Bardina, Spricigo, Cortes, & Llagostera, 2012). It was proposed that the
phages having short latent period and large burst size are one of the
desired candidates suitable for applications (Gill & Hyman, 2010). Five
phages isolated in this study had relatively short latent periods
(15–25 min) and large burst sizes (49–209 PFU/cell) indicating their
applicability for a bacterial treatment scheme.
In order to study lytic abilities of the isolated phages, cocktail of 5
phages was formed and mixed with S. Enteritidis and S. Typhimurium
in LB broth. The mixture was incubated at both room temperature
(25 °C) that would support normal growth of Salmonella, and at cool
temperature (8 °C) that is commonly found in most household re-
frigerators (Laguerre, Derens, & Palagos, 2002). At both conditions, the
co-incubation of phage cocktail with S. Enteritidis or S. Typhimurium in
LB broth showed rapid and significant reductions in viable counts of
these Salmonella hosts demonstrating lysis of the bacteria occurring in
the phage treated samples. With the same levels of bacterial inoculum,
the treatment of S. Enteritidis reduced the bacterial counts to below
detection limit after 24 h of incubation at 25 °C while that of S. Ty-
phimurium failed to prevent the bacterial regrowth after 4 h. This result
indicated that susceptibility of S. Enteritidis and S. Typhimurium to the
phage cocktail used in this study was not the same. S. Enteritidis was
possibly more susceptible to the phages used than S. Typhimurium at
25 °C. Researches have also pointed out that different Salmonella ser-
ovars showed different degrees of susceptibilities to phages (Grant,
Parveen, Schwarz, Hashem, & Vimini, 2017; C. S.; Sharma, Dhakal, &
Nannapaneni, 2015).
Phages depend on the growth of bacterial hosts for their replication
and production of offspring. At an optimal temperature range, the
H.M. Duc et al.
Fig. 2. One-step growth curves of phages SEG5 (a), SES8 (b), STG2 (c), STG5 (e), and STS9 (d). L, Latent period; B, Burst size. Error bars show standard deviations.
bacterial hosts usually grow faster, which promotes the replication of
phage particles while at lower temperature, the speed of phage re-
plication is considerably decreased or halted due to lower growth rate
of their hosts (Galarce, Bravo, Robeson, & Borie, 2014; Hungaro et al.,
2013). Therefore, the temperature used for an analysis of phage-host
interaction is normally considered as one of the most common extrinsic
factors affecting the application's outcome. In our study, the treatments
of S. Enteritidis and S. Typhimurium inoculated in LB at 25 °C produced
higher reductions in viable counts compared to the treatments of these
hosts at 8 °C. This result is consistent with previous reports from phage
bio-control studies that found greater bacterial reductions in the ex-
periments performed at high temperature (Hungaro et al., 2013; Tomat,
Quiberoni, Mercanti, & Balagué, 2014; Tomat et al., 2013).
357
LWT - Food Science and Technology 91 (2018) 353–360
As expected, the lytic ability of phage cocktail was not only shown
in the LB broth but also in the raw chicken breast, which was indicated
by the reductions of Salmonella viable counts in the treated meat sam-
ples. These results suggested that phage cocktail could be applied on
raw chicken meat to reduce contamination of S. Enteritidis and S.
Typhimurium.
For assurance of microbial safety and quality of raw meats, low
temperature is usually required not only during slaughtering of ani-
mals, processing, and transportation but also throughout storage period
of raw meats at retail store or household. However, temperature abuse
may occur somewhere in these sequential steps, which consequently
facilitates the growth of spoilage and/or harmful bacteria if they pre-
sent in the meat products (Alonso-Hernando, Alonso-Calleja, & Capita,
H.M. Duc et al. LWT - Food Science and Technology 91 (2018) 353–360

Fig. 3. Effects of phage cocktail on viability of S. Enteritidis (a) and S. Typhimurium (b) in LB broth at 25°C. S. Enteritidis and S. Typhimurium were inoculated in 5 ml LB broth at
2 × 104 CFU/ml without phages (dashed line) and with phage cocktail at 2 × 108 PFU/ml (solid line). Error bars show standard deviations.

Fig. 4. Effects of phage cocktail on viability of S. Enteritidis (a) and S. Typhimurium (b) in LB broth at 8°C. S. Enteritidis and S. Typhimurium were inoculated in 5 ml LB broth at
2 × 104 CFU/ml without phages (dashed line) and with phage cocktail at 2 × 108 PFU/ml (solid line). Error bars show standard deviations.

Fig. 5. Effects of phage cocktail on viability of S. Enteritidis (a) and S. Typhimurium (b) spiked onto chicken breast at 25°C. S. Enteritidis and S. Typhimurium were inoculated
onto chicken breast at 3 × 104 CFU/piece without phage cocktail (dashed line) and with phage cocktail at 3 × 108 PFU/piece (solid line). Error bars show standard deviations.

2013; Likar & Jevšnik, 2006). Furthermore, it was recorded that the
failure in maintaining proper temperature during storage of poultry
meats was a main reason inducing growth of Salmonella leading to
salmonellosis in humans (Juneja et al., 2007). Therefore, we evaluated
the killing abilities of our phage cocktail at 25 °C that is inappropriate
temperature for long and safe preservation of raw meats. Although
Salmonella hosts in the treated samples were found to regrow to the
levels higher than the initial inoculum when the experiment was ended
at 24 h of incubation, this phenomenon was not unusual in phage bio-
control researches (Bigot et al., 2011; Hudson et al., 2013; Tomat et al.,
2013) and it was attributed to the emergence of phage resistant bacteria
as described in the study on bio-control of S. Typhimurium by
Guenther, Herzig, Fieseler, Klumpp, & Loessner, 2012 (Guenther et al.,
2012) or the complexity of food matrix interfering diffusion and
homogeneous distribution of the phages, which hence decreased the
likelihood of phage-host contact (Bigwood et al., 2008; Tomat et al.,
2013). One of possible solutions to overcome this obstacle is applying
phage during tumbling to enhance the uniform distribution of phage on
the meat surface (Yeh et al., 2017). Even if the viable cells of both
Salmonella hosts were found to be higher than initial inoculum at the
end of experiment, the phage treatment of Salmonella hosts in raw
chicken for first 6 h were still significant as it reduced viable counts of

358
H.M. Duc et al.
Fig. 6. Effects of phage cocktail on viability of S. Enteritidis (a) and S. Typhimurium (b) spiked onto chicken breast at 8°C. S. Enteritidis and S. Typhimurium were inoculated on
chicken breast at 3 × 104 CFU/piece without phage cocktail (dashed line) and with phage cocktail at 3 × 108 PFU/piece (solid line) and. Error bars show standard deviations.
S. Enteritidis and S. Typhimurium by 1.8 log and 1.2 log compared to
the initial inoculum (4.5 log), respectively (P < 0.05). It is important
to note that most raw meats used for human consumption are not
normally stored at 25 °C for long time. Raw meats are practically stored
at low temperature. When the phage cocktail was applied onto the
chicken meats contaminated with S. Enteritidis and S. Typhimurium
and held at 8 °C, compared to the inoculum level, at least 1 log re-
duction (P < 0.05) in viable counts of these hosts was observed
throughout the course of experiment. This represents its applicability in
controlling the growth of Salmonella at storage condition. Although the
antibacterial effects of phages at low temperature have been recorded
previously (Bao et al., 2015; C. S.; Sharma et al., 2015), the exact
mechanism of these effects has not been fully understood. It could be
related to the absorption of a large number of phages onto bacterial
cells resulting in the rapid damage of cell wall without a need of phage
replication, a phenomenon called lysis from without (Abedon, 2010;
Galarce et al., 2014) or bacterial lysis occurring during dilution and
plating process (Viazis, Akhtar, Feirtag, & Diez-Gonzalez, 2011b).
Even though the phage treatment was unable to completely elim-
inate artificially contaminated S. Enteritidis and S. Typhimurium in
chicken breast samples, the reductions of these hosts in the phage
treated meats implicated practical utilities. In natural setting, the level
of Salmonella contamination in foods is usually very low (Boughton
et al., 2004; Dufrenne, Ritmeester, Delfgou-van Asch, van Leusden, & de
Jonge, 2001). The cell numbers of S. Enteritidis and S. Typhimurium
(3 × 104 CFU/piece) used to inoculate into meat samples in our study
was much higher than the counts of Salmonella actually found in foods.
Assuming that the Salmonella inoculum used in our meat experiments
was as low as the level contaminated in real conditions, phage appli-
cation might be more effective. Previous study showed that viable cells
of Salmonella were completely diminished by phage treatment when
low bacterial inoculum was applied (Goode, Allen, & Barrow, 2003).
5. Conclusion
The results from this study demonstrated that lytic phages against
various Salmonella serovars were successfully isolated from raw chicken
skin and gizzard. The cocktail of phages significantly reduced viability
of S. Enteritidis and S. Typhimurium in vitro and on raw chicken breast
at room and cool temperature. The phages isolated in this study seem to
be a potential bactericide for bio-control of Salmonella in raw meats.
Conflicts of interest
The authors have no conflict to declare.
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LWT - Food Science and Technology 91 (2018) 353–360
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