i An update to this article is included at the end

Current Research in Food Science 2 (2020) 25–32

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Current Research in Food Science

journal homepage: www.editorialmanager.com/crfs/

Research Paper

Inactivation of Salmonella enterica on post-harvest cantaloupe and lettuce by

a lytic bacteriophage cocktail
Catherine W.Y. Wong a, Pascal Delaquis b, Lawrence Goodridge c, Roger C. L
evesque d,
Karen Fong a, Siyun Wang a, *
a
Department of Food Science, University of British Columbia, 2205 East Mall, Vancouver, BC, V6R 1Z4, Canada
b
Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC, V0H 1Z0, Canada
c
Department of Food Science and Agricultural Chemistry, McGill University, Montr
eal, QC, Canada
d
Institute for Integrative and Systems Biology, Universit
e Laval, Qu
ebec City, QC, Canada

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

Keywords: Salmonella enterica (S. enterica) is a causative agent of multiple outbreaks of foodborne illness associated with fresh
Salmonella enterica produce, including pre-cut melon and leafy vegetables. Current industrial antimicrobial interventions have been
Bacteriophage shown to reduce microbial populations by <90%. Consequently, bacteriophages have been suggested as an
Post-harvest
alternative to chemical sanitizers. Seven S. enterica strains from four serovars (105 CFU/mL) were separately
Lettuce
Cantaloupe
inoculated onto excised pieces of Romaine lettuce leaf and cantaloupe flesh treated with a five-strain bacterio-
Biocontrol phage cocktail 24 h before S. enterica inoculation. S. enterica, total aerobic populations and water activity were
measured immediately after inoculation and after 1 and 2 days of incubation at 8
C. The efficacy of the
bacteriophage cocktail varied between strains. Populations of S. enterica Enteritidis strain S3, S. Javiana S203, S.
Javiana S200 were reduced by > 3 log CFU/g and S. Newport S2 by 1 log CFU/g on both lettuce and cantaloupe
tissues at all sampling times. In contrast, populations of strains S. Thompson S193 and S194 were reduced by 2 log
CFU/g on day 0 on lettuce, but were not significantly different (P > 0.05) from the controls thereafter, S. Newport
S195 populations were reduced on lettuce by 1 log CFU/g on day 0 and no reductions were found on cantaloupe
tissue. Both aerobic populations and water activity were higher on cantaloupe than on lettuce. The water activity
of lettuce decreased significantly (P < 0.05) from 0.845  0.027 on day 0–0.494  0.022 on day 1, but that of
cantaloupe remained between 0.977 and 0.993 from day 0–2. The results of this study showed that bacterio-
phages can reduce S. enterica populations on lettuce and cantaloupe tissues but that the magnitude of the effect
was strain-dependent.

1. Introduction Recalled foods included products containing pre-cut watermelon, hon-

eydew melon, cantaloupe and any combination of at least one of the
Non-typhoidal Salmonella enterica (S. enterica) is a major etiological above-mentioned fruits (Centers for Disease Control and Prevention,
agent of foodborne illness (Centers for Disease Control and Prevention, 2019b). Given the number of produce-related outbreaks, these incidents
2011; Scallan et al., 2011). In the 10-year period between 1998 and suggest that additional antimicrobial strategies may be required to
2008, leafy vegetables were the cause of 2.5% of all S. enterica outbreaks improve the safety of fresh produce.
associated with the consumption of fresh produce in the United States Leafy vegetables and many fresh-cut vegetable or fruit products
(US) (Jackson et al., 2013). In 2018 alone, S. enterica was responsible for are consumed without the application of a heat treatment to inacti-
16 known outbreaks in the US (Centers for Disease Control and Pre- vate human pathogens. Consequently, the safety of these foods relies
vention, 2019a). Four of the 16 outbreaks (25%) were associated with on the maintenance of appropriate sanitary measures during pro-
plant-based foods, including frozen shredded coconut, raw sprouts, dried duction and harvest, and application of non-thermal treatments to
coconut and pre-cut melon (Centers for Disease Control and Prevention, reduce the risk of contamination prior to distribution (Sapers, 2001).
2019a). An outbreak of salmonellosis was definitively linked to pre-cut Unfortunately, non-thermal treatments such as washing in
melon in 2019 (Centers for Disease Control and Prevention, 2019b). chlorinated water cannot fully eliminate enteric bacterial pathogens

* Corresponding author.
E-mail address: [email protected] (S. Wang).

https://doi.org/10.1016/j.crfs.2019.11.004
2665-9271/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C.W.Y. Wong et al. Current Research in Food Science 2 (2020) 25–32

due to biofilm formation, attachment site inaccessibility, strength of 2019). Bacteriophage propagation and purification were performed
attachment or internalization of the pathogen (Burnett et al., 2000; according to methods described by Fong et al. (2019) and Fong et al.
Carmichael et al., 1998; Costerton, 1995; Fett, 2000; Liao and Cooke, (2017) using S. enterica host strains shown in Table 1. Bacteriophage
2001; Sapers, 2001; Seo and Frank, 1999; Takeuchi et al., 2000; cocktail titers were measured by a plaque assay described below.
Takeuchi and Frank, 2000). Briefly, decimal dilutions were prepared in capped microcentrifuge
The use of bacteriophages as a means to inactivate bacteria was tubes (VWR International, PA, USA) containing 450 μl Tryptic Soy
proposed soon after their discovery in 1915 (Sillankorva et al., 2012). Broth (TSB, Difco, Becton Dickinson, NJ, USA) þ 1.0 mM CaCl2
Advantages of using bacteriophages as antimicrobial agents in foods (TSB-Ca). Fifty μl of S. enterica culture (see Table 2) prepared according
include high specificity, self-replication, lack of negative sensory effects to methods provided in section 2.3 were added to each tube and the
and general non-toxicity to humans (Moye et al., 2018; Sillankorva et al., contents were mixed by agitation. After 30 min incubation at 37
C the
2012). The high specificity of bacteriophages allows for the general contents were applied to the surface of Tryptic Soy Agar þ 1.0 mM
microbiota to remain untouched, thus avoiding undesirable effects due to CaCl2 (TSA-Ca) in 100 mm  15 mm petri plates. The plates were
alterations in spoilage patterns or the inactivation of desirable microor- incubated at 37
C for 24 h prior to counting the plaques. The con-
ganisms in the food system (Sillankorva et al., 2012). Moreover, bacte- centration of the bacteriophage cocktail was approximately
riophages are tasteless and their addition does not alter the sensory 2.5  108 PFU/mL.
characteristics or attributes of the food, which is of concern to many food
processors (Moye et al., 2018).
There have been a few attempts to use bacteriophages for the control 2.3. Salmonella strains and inoculum preparation
of S. enterica on leafy vegetables and fresh-cut melon (Leverentz et al.,
2001; Spricigo et al., 2013; Zhang et al., 2019). The results of the The S. enterica strains selected for the experiments (Table 2) were
aforementioned research have shown bacteriophages to reduce Salmo- previously shown to colonize lettuce seedlings (Wong et al., 2019). All
nella populations on sprouts and lettuce (Leverentz et al., 2001; Spricigo were obtained from the Syst-OMICS Database (SALFOS, Universit
e Laval,
et al., 2013; Ye et al., 2010; Zhang et al., 2019). The present work was QC, Canada, https://salfos.ibis.ulaval.ca/). All strains were maintained
carried out to determine the efficacy of a bacteriophage cocktail against in TSB supplemented with 20% glycerol (VWR International, PA, USA) at
several strains of S. enterica on Romaine lettuce leaf and cantaloupe 80
C. Inocula were prepared with overnight incubation at 37
C in
tissues. 10 mL TSB under agitation at 175 rpm. The overnight cultures were then
spun at 1811g for 10 min and the supernatant was decanted. The
2. Materials and methods resulting pellets were washed twice with 10 mL 0.5 mM potassium
phosphate buffer (PPB), pH 6.8–7.0 (Amresco, OH, USA). The density of
2.1. Romaine lettuce and cantaloupe sample preparation the final suspension was spectrophotometrically adjusted with UV-1800
UV/Vis Spectrometer (Shimadzu, MD, USA) to an OD600 of 0.47–0.52
Whole Romaine lettuce heads and pre-cut cantaloupe were purchased and further diluted with 0.5 mM PPB to obtain a cell density of
from a local grocery store in Coquitlam, British Columbia, Canada. 106 CFU/mL.
Romaine leaf sections (2  2 cm) (L x W) and cantaloupe flesh sections
(2  2  0.2 cm) (L x W x D) were excised with a sterile sharp knife and
placed in 60 mm  15 mm Petri plates at 8  1
C (VWR International, 2.4. Bacteriophage application and inoculation of Romaine lettuce and
PA, USA). cantaloupe tissue

The bacteriophage cocktail was applied 24 h prior to inoculation with

2.2. Bacteriophage cocktail preparation
S. enterica to examine their ability to inactivate contaminants transferred
to plant tissues during post-harvest handling. The bacteriophage cocktail
Five Salmonella bacteriophages isolated from a variety of sources
(100 μL) was applied to lettuce leaf and cantaloupe flesh sections with a
(Table 1) were used to formulate a cocktail. The isolates were selected
micro-pipettor and the liquids were gently spread over the surface using
on the basis of their ability to lyse at least 35 of 43 S. enterica strains
disposable sterile plate spreaders. As a control, the CaCl2 solution
from 31 different serotypes, including the strains used to inoculate
(1.0 mM) was applied to the flesh sections in parallel. The treated sec-
fresh produce in the experiments described in 2.3 and 2.4 (Fong et al.,
tions were then transferred to 60 mm  15 mm Petri plates and stored in
an incubator set at 8  1
C. A temperature of 8
C was chosen to reflect
the average expected in household refrigerators (James et al., 2017,
Table 1 2008; Koutsoumanis et al., 2010). After 24 h incubation, 100 μl of each
Bacteriophages used in this study. S. enterica inoculum was applied singly to the surface of each section in
the same manner as used for the pre-treatment to achieve an inoculation
Bacteriophage Source Location Salmonella Salmonella host
host strain for strain for level of 105 CFU/mL. Salmonella populations were measured immedi-
isolation propagation ately after inoculation (day 0) and after 1 and 2 days at 8  1
C described
Φ3 Pretreated Montreal, Enteritidis S7 Enteritidis S7
below in 2.5.
sludge QC
Φ6 Pretreated Montreal, Javiana Javiana S1297
sludge QC S1297
Felix01 Felix Quebec Typhi Paratyphi B Table 2
d’Herelle City, QC S. enterica strains used in this study.
virus
Strain Serotype Source
collection
HER20 Felix Quebec Newport Newport S3 Enteritidis Human
d’Herelle City, QC C487-69 C487-69 S200 Javiana Human
virus S203 Javiana Octopus
collection S195 Newport Alfalfa seed
SE13 Sewage after Vancouver, Newport Newport S195 S2 Newport Human
first BC S195 S193 Thompson Spinach
treatment S194 Thompson Feather meal

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C.W.Y. Wong et al. Current Research in Food Science 2 (2020) 25–32

2.5. Measurement of Salmonella populations on Romaine lettuce and with strains S3, S200, S203, S2 (Fig. 1A and B, C, E), however populations
cantaloupe tissues of strains S193 and S194 were not significantly different (P > 0.05) after 1
or 2 days at 8
C (Fig. 1F and G). Populations of strains S3, S200, S203, S2
Tissue sections were placed in capped test tubes with 9 mL of 0.1% and S193 also remained lower on treated cantaloupe tissue than on con-
(w/v) peptone (Difco, Becton Dickinson, NJ, USA) solution and agitated trols (Fig. 2A and B, C, E, F), but populations of strain S194 increased from
on a vortex mixer set on high speed for 1 min. Decimal dilutions were to 2.97  0.24 to 4.80  0.23 log CFU/cm2 between day 1 and 2 (Fig. 2 G).
then prepared in 0.1% peptone and spread on Xylose Lysine Deoxy- The highest reductions in S. enterica measured in the present work
cholate (XLD) Agar (Oxoid, ThermoFisher Scientific, MA, USA). Pop- occurred on day 0 against strains S. Enteritidis S3, S. Javiana S200 and
ulations per surface area (CFU/cm2) of tissue were calculated from S203 and S. Thompson S194 (Fig. 1 A – C, G). However, populations
colony counts recorded after 24 h incubation at 37
C. declined on lettuce after day 1 and 2 irrespective of treatment applied,
likely in response to declining water activity. The water activity of lettuce
2.6. Measurement of aerobic mesophilic populations on Romaine lettuce and cantaloupe tissues during incubation is provided in Table 3. While
and cantaloupe tissues measurements obtained with cantaloupe tissue remained within the
range of 0.977–0.993 across all sampling times, the water activity of
Total aerobic mesophilic populations were measured on tissue sections lettuce tissue declined significantly (P < 0.05) from 0.845  0.027 to
that received no treatment prior to incubation. Dilutions prepared in 0.1% 0.494  0.022 during the first 24 h of incubation but remained un-
peptone as described above were applied to the surface of Tryptic Soy Agar changed (0.492  0.022) on day 2. An association between high water
(TSA) supplemented with yeast extract (1:200) (Oxoid, ThermoFisher Sci- activity and total aerobic populations can be discerned. Total aerobic
entific, MA, USA). Aerobic populations were measured on day 0, 1 and 2 at populations on untreated lettuce and cantaloupe tissues incubated at 8
C
8  1
C. Populations per surface area (CFU/cm2) of tissue were calculated for two days are provided in Fig. 3. Lettuce tissues harbored very low
from colony counts recorded after 24 h incubation at 37
C. aerobic populations that remained between 1.67  0.42 to 1.92  0.50
log CFU/cm2 between day 0 and 2. Total aerobic populations on canta-
2.7. Measurement of water activity loupe tissue were considerably higher at the outset, and increased
significantly (P < 0.05) from 5.35  0.98 log CFU/cm2 to 6.38  0.45 log
Water activity after 0, 1 and 2 days incubation at 8  1
C was CFU/cm2 after two days of incubation at 8
C.
measured on tissue sections that received no treatment prior to incuba- A cocktail formulated from five broad-host range lytic bacteriophages
tion. Individual sections were placed in 35 mm  10 mm Petri plates applied prior to a challenge with S. enterica reduced the populations of
(Corning, NY, USA) and were warmed to 25
C before water activity several strains inoculated onto Romaine lettuce leaf and cantaloupe tis-
measurement with an AquaLab Series 3 water activity meter (Decagon sues. Inactivation of enteric bacterial pathogens on plant tissues treated
Devices, Inc, WA, USA). with bacteriophage has been reported previously. A three-strain bacte-
riophage cocktail applied at a density of 7.5  107 PFU/cm2 after inocu-
2.8. Statistical analyses lation reduced populations of Escherichia coli O157:H7 on cantaloupe and
lettuce by 2.87 log CFU/g and 1.92 log CFU/g, respectively, after 2 days of
Five trials each were conducted on lettuce and cantaloupe tissue storage at 4
C (Sharma et al., 2009). S. enterica populations were reduced
sections using independently grown bacterial cultures. Five sections were by 1 log CFU/g on lettuce and 0.90 log CFU/g on mung bean sprouts stored
analyzed at each sampling time interval for each treatment: (1) S. enterica at 2
C and 10
C for 72 h after treatment with a commercial cocktail
alone, (2) S. enterica þ 1.0 mM CaCl2 and (3) S. enterica þ bacteriophage (SalmoFresh™) containing 108 PFU/mL of six bacteriophages (Zhang
cocktail in 1.0 mM CaCl2. Three to five water activity and total aerobic et al., 2019). Leverentz et al. (2001) found that a mixture of four bacte-
population measurements were obtained on day 0, 1 and 2. S. enterica riophage strains (SCPLX-1, Intralytix, Inc., 108 PFU/mL) could reduce S.
and aerobic populations were analyzed on log10-transformed data by a Enteritidis populations by 3.5 log CFU/g on honeydew melon stored at
two-way analysis of variance (ANOVA) and Tukey's honestly significant 5
C and 10
C. In the present work, the application of a five-phage cocktail
difference (HSD) for means separation. Measurements for water activity at a density of 108 PFU/cm2 resulted in Salmonella populations reductions
were analyzed by one-way ANOVA and Tukey's HSD for means separa- 4 log CFU/cm2, a seemingly high degree of inactivation given that
tion. All statistical analyses were performed using RStudio, version bacteriophage-based reductions of bacteria in solid foods generally range
1.1.453 (Rstudio, Inc, MA, US). between 1 and 3 log CFU/g and that complete elimination is rare (Higgins
et al., 2005; Leverentz et al., 2001; Moye et al., 2018; Whichard et al.,
3. Results and discussion 2003; Ye et al., 2010). However, comparisons of outcomes achieved from
individual studies on bacteriophage-based control of enteric bacterial
Seven S. enterica strains were singly inoculated onto the surface of pathogens in foods is difficult due to differences in experimental design,
Romaine lettuce leaf and cantaloupe tissues treated with a cocktail con- composition of cocktails, dosage and timing of bacteriophage application
sisting of equal proportions of five broad-host range lytic bacteriophage. and storage of target food products after treatment, among others.
The cocktail was applied at a density of approximately 2.5  108 PFU/cm2 While application of the bacteriophage cocktail could clearly inacti-
24 h prior to inoculation. Figs. 1 and 2 show that populations of six vate several strains of S. enterica, the efficacy was both strain and plant
S. enterica strains (S3, S200, S203, S2, S193, S194) were significantly species-dependent. Variable efficacy against different S. enterica strains
(P < 0.05) affected by the phage treatment. Populations of S3, S200 and has been reported. For example, Spricigo et al. (2013) showed that a
S203 were reduced by up to 4 log CFU/cm2 and neither S200 or S203 were bacteriophage cocktail could reduce S. Typhimurium populations by 3.9
recovered from lettuce tissue at a level of detection ¼ 0.36 log CFU/cm2 log CFU/g on fresh-cut lettuce, but only 2.2 log CFU/g of S. Enteritidis.
(Fig. 1, A – C). Population reductions of strains S2, S193 and S194 were Bacteriophage cocktails for the control of foodborne bacterial pathogens
lower, ranging between ~1 and 2 log CFU/cm2 (Fig. 1, E  G). On are generally formulated from broad-host range bacteriophage strains to
cantaloupe tissues, populations of strains S3, S200 and S203 were reduced ensure activity against all potential strains of the target species (Chan
by ~2–3 log CFU/cm2 (Fig. 2, A – C) and those of S2, S193 and S194 by et al., 2013). Despite formulation on this basis, the cocktail used in the
~0.5–1.5 log CFU/cm2 (Fig. 2, E  G). In contrast, there was no significant present study was ineffective against one of the seven strains (S. Newport
(P > 0.05) change in populations of strain S. Newport S195 measured S195) on both plant tissues, an unexpected outcome as the strain was
immediately after inoculation of either lettuce or cantaloupe tissues. susceptible to lysis by each of the bacteriophage in the cocktail when
Differences between S. enterica populations on control and treated samples tested by the plaque assay (Fong et al., 2017). Reasons for the lack of
were sustained during subsequent incubation of lettuce tissue inoculated activity against this strain on plant tissue surfaces are unclear. One of the

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C.W.Y. Wong et al. Current Research in Food Science 2 (2020) 25–32

Fig. 1. Populations (log CFU/cm2) of seven S. enterica strains on Romaine lettuce leaf tissue immediately after inoculation (Day 0) and after 1 (Day 1) and 2 days (Day 2)
incubation at 8  1
C. Treatments: : untreated tissue (controls); : 1.0 mM CaCl2 solution applied 24 h before inoculation; : 1.0 mM CaCl2 þ five-bacteriophage
cocktail applied 24 h before inoculation. (A) S. Enteritidis S3. (B) S. Javiana S200. (C) S. Javiana S203. (D) S. Newport S195. (E) S. Newport S2. (F) S. Thompson S193. (G)
S. Thompson S194. Different superscripts (a–d) denote significant differences (P < 0.05) between treatments on Day 0 between strains. Different superscripts (A–F)
denote significant differences (P < 0.05) between treatments on Day 1 between strains. Different superscripts (a–e) denote significant differences (P < 0.05) between
treatments on Day 2 between strains. Means and standard deviations were calculated using data from five biological replicates. Limit of detection is > 0.36 log CFU/cm2.

first initials steps in bacteriophage-host interactions is mediated by re- be the case with strain S. Newport S195 on lettuce or cantaloupe tissues.
ceptors on the Salmonella cell surface, for example vitamin B12 uptake Moreover, mutations acquired during replication can alter host range
outer membrane protein, flagellar or lipopolysaccharide-related O-anti- (Drake, 1991; Oechslin, 2018; Rokyta et al., 2009). These observations
gen proteins (Shin et al., 2012). Expression of receptors may not occur reinforce the need to assess the performance of bacteriophage-based
under all environmental conditions thereby precluding binding to and approaches to the control of enteric bacterial pathogens in fresh pro-
subsequent infection of the bacterial cell (Bull et al., 2014), which could duce against a wide range of strains.

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C.W.Y. Wong et al. Current Research in Food Science 2 (2020) 25–32

Fig. 2. Populations (log CFU/cm2) of seven S. enterica strains on cantaloupe tissue immediately after inoculation (Day 0) and after 1 (Day 1) and 2 days (Day 2) in-
cubation at 8  1
C. Treatments: : untreated tissue (controls); : 1.0 mM CaCl2 solution applied 24 h before inoculation; : 1.0 mM CaCl2 þ five-bacteriophage cocktail
applied 24 h before inoculation. (A) S. Enteritidis S3. (B) S. Javiana S200. (C) S. Javiana S203. (D) S. Newport S195. (E) S. Newport S2. (F) S. Thompson S193. (G) S.
Thompson S194. Different superscripts (a–d) denote significant differences (P < 0.05) between treatments on Day 0 between strains. Different superscripts (A–F) denote
significant differences (P < 0.05) between treatments on Day 1 between strains. Different superscripts (a–e) denote significant differences (P < 0.05) between treatments
on Day 2 between strains. Means and standard deviations were calculated using data from five biological replicates. Limit of detection is > 0.36 log CFU/cm2.

Overall, the cocktail examined here was more effective against A common observation derived from studies on bacteriophage usage in
S. enterica inoculated onto Romaine lettuce than cantaloupe tissues. foods is that the decrease in targeted bacterial populations is not
Variance in the extent of control achieved in plant-based foods has been consistent in different food matrices due to differences in water activity
ascribed to numerous factors, including differences in chemical compo- (Hudson et al., 2010; Guenther et al., 2012; Kang et al., 2013; Moye et al.,
sition and the microtopography of surfaces colonized by target bacteria 2018; Spricigo et al., 2013). The abundance of free water in moist food
(Abuladze et al., 2008; Leverentz et al., 2004, 2001; Sharma et al., 2009). matrices such as beverages or sliced melons is believed to facilitate the

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C.W.Y. Wong et al. Current Research in Food Science 2 (2020) 25–32

Table 3 However, a few studies have suggested that repetitive lytic bacteriophage
Water activity (aw)a of Romaine lettuce and cantaloupe tissues after 0, 1 replication cycles does not always occur under conditions used in some
and 2 days of incubation at 8  1
C. experimental food systems (Chibeu et al., 2013; Moye et al., 2018; Oli-
Time (days) Romaine Lettuceb Cantaloupec veira et al., 2014; Soni et al., 2012). Several factors influence host
0 0.845  0.027 a
0.983  0.002B
infection rates, notably temperature. In the present work, samples of
1 0.494  0.022b 0.993  0.006A inoculated plant tissues were stored at 8
C, a temperature at the higher
2 0.492  0.022b 0.977  0.002B end of the spectrum of domestic refrigerator temperatures (James et al.,
a
Means and standard deviations were calculated using data from
2017, 2008; Ziegler et al., 2018) but at the lower end of growth
n ¼ 3 to 5 replicates. Lettuce leaf and cantaloupe flesh sections permissive temperatures for Salmonella spp. (D'aoust, 1993; Matches and
(2  2 cm) were sampled at time points corresponding to day 0, 1 and 2 Liston, 1968). S. enterica has been reported to grow at 8
C on leafy greens
post inoculation. and fresh-cut cantaloupe (Huang et al., 2015; Posada-Izquierdo et al.,
b
Different letters (a to b) in the lettuce column indicate significant 2015), and to survive well at 4–5
C on leafy greens and fruits (Abd-Elall
differences (P < 0.05) among water activity from lettuce leaf sections on and Awadallah, 2015; Delbeke et al., 2015; Golden et al., 1993; Huang
day 0, 1 and 2. et al., 2015). Bacteriophage infection and lysis of enteric bacteria has
c
Different letters (A to B) in the cantaloupe column indicate signifi- been shown to occur at temperatures as low as 4
C (Jurczak-Kurek et al.,
cant differences (P < 0.05) among water activities from cantaloupe leaf 2016). Inactivation of S. enterica at 5 and 10
C and reports of higher
sections on day 0, 1 and 2.
activity at chill temperatures than at 20
C provides evidence that
bacteriophage retain the ability to infect Salmonella at low temperatures
(Leverentz et al., 2001; Zhang et al., 2019).
Previous research has shown that the infectivity of bacteriophages as
well as their stability during storage depends on the composition of so-
lutions used for stabilization and preservation. Adams (1949) found that
calcium ion solutions provide the highest infectivity and stability for
bacteriophage T5. Other solutions were less effective for the preservation
of T5, which lost its activity when stored in phosphate buffer and was
inactivated when stored in citrate solution (Adams, 1949). Moreover, the
infectivity of bacteriophage is known to decrease during time in storage
due to interactions with solution components (Mylon et al., 2010). For
example, Mylon et al. (2010) found that the MS2 bacteriophage aggre-
gated and lost infectivity when it was stored in increasing concentrations
of calcium chloride due to the neutralization of the negatively charged
moieties on the surface of the bacteriophage. Given this risk, bacterio-
phage suspensions for use in food systems are prepared in solutions
containing millimolar concentrations of calcium chloride and are used
soon after preparation.

4. Conclusions

In summary, the results of this work have indicated that the efficacy
of a bacteriophage cocktail is dependent on several factors, including the
host strain and commodity. Differences in susceptibility are not limited to
a specific bacteriophage but may extend to cocktails prepared from
Fig. 3. Total aerobic populations (log CFU/cm2) on : Romaine lettuce leaf and several bacteriophages. Consequently, differences in population re-
: cantaloupe tissues after 0, 1 and 2 days incubation at 8  1
C. Different ductions obtained with the seven strains of S. enterica inoculated onto
superscripts (a–c) denote significant differences (P < 0.05) between aerobic lettuce leaves and cantaloupe flesh may have been a consequence of
populations on lettuce leaves and cantaloupe flesh across day 0, 1 and 2. Means
variable susceptibility to infection dictated by the host range afforded by
and standard deviations were calculated using data from fourteen biological
the bacteriophage cocktail, and the concentration of the bacteriophage
replicates. Limit of detection is > 0.36 log CFU/cm2.
cocktail applied to the plant tissues.
The efficacy of the bacteriophage cocktail was strain-dependent but
reduced populations by 1–4 log CFU/cm2 on lettuce and cantaloupe flesh
transport of bacteriophages and to favor collision with target bacteria sections. The cocktail was not effective against S. Newport S195 on both
(Hudson et al., 2010; Moye et al., 2018). Given these assumptions, fresh produce commodities. Therefore, industrial applications may still
greater efficacy was anticipated against S. enterica on cantaloupe rather require additional treatments or measures to further reduce S. enterica
than the comparatively dry lettuce tissue surface. However, cut canta- populations.
loupe tissues can release an abundance of nutrients including readily
assimilated sugars, which are present at higher concentration Funding sources
(0.079 g/g) than in Romaine lettuce tissues (0.011 g/g) (L
opez et al.,
2014; Perkins-Veazie et al., 2012). Microbiological analysis showed that This work was supported by grants from Genome Canada (grant
aerobic microbial populations were several orders of magnitude greater number 8505); the National Sciences and Engineering Research Council
on cantaloupe than on lettuce tissues. Hence, the presence of large of Canada NSERC Discovery Grant (RGPIN-2015-04871); and the British
background microflorae on cut cantaloupe tissues may have negatively Columbia Ministry of Agriculture (URACP19-211).
affected the efficacy of the bacteriophage, possibly by providing other
non-target attachment sites. Acknowledgements
One of the anticipated benefits of bacteriophage-based food preser-
vation is the ongoing release of infectious virion progeny to ensure Special thanks to Dr. Sylvain Moineau from Universit
e Laval for
continuous inhibition of target bacteria (Howard-Varona et al., 2017). providing phages Felix01 and HER20.

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C.W.Y. Wong et al. Current Research in Food Science 2 (2020) 25–32

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 Update
Current Research in Food Science
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