Food Microbiology 92 (2020) 103572

Contents lists available at ScienceDirect

Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Bacteriophage biocontrol of Shiga toxigenic Escherichia coli (STEC) O145 T

biofilms on stainless steel reduces the contamination of beef
Changbao Wanga,b, Hua Hanga, Shoubiao Zhoua,∗∗, Yan D. Niuc, Hechao Dub,c, Kim Stanfordd,
Tim A. McAllisterb,∗
a
College of Environmental Science and Engineering, Anhui Normal University, Wuhu, Anhui, 241002, PR China
b
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1, Canada
c
Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N 1N4, Canada
d
Alberta Agriculture and Forestry, Lethbridge, AB, T1J 4V6, Canada

ARTICLE INFO ABSTRACT:

Keywords: Shiga toxigenic Escherichia coli (STEC) can form biofilms and frequently cause serious foodborne illnesses. A
Shiga toxigenic Escherichia coli (STEC) strain of STEC O145:H25 (EC19990166) known to be a strong biofilm former was used to evaluate the efficacy of
Biofilm bacteriophage AZO145A against biofilms formed on stainless steel (SS) coupons. Exposure of STEC O145:H25 to
Bacteriophage phage AZO145A (1010 PFU/mL) for 2 h resulted in a 4.0 log10 reduction (P < 0.01) of planktonic cells grown in
Beef
M9 broth at 24 °C for 24 h, while reductions were 2.0 log10 CFU/mL if these cells were grown for 48 h or 72 h
Biocontrol
prior to phage treatment. STEC O145 biofilms formed on SS coupons for 24, 48 and 72 h were reduced
Stainless steel
(P < 0.01) 2.9, 1.9 and 1.9 log10 CFU/coupon by phages. STEC O145 cells in biofilms were readily transferred
from the surface of the SS coupon to beef (3.6 log10 CFU/coupon) even with as little as 10 s of contact with the
meat surface. However, transfer of STEC O145 cells from biofilms that formed on SS coupons for 48 h to beef was
reduced (P < 0.01) by 3.1 log10 CFU by phage (2 × 1010 PFU/mL) at 24 °C. Scanning electron microscopy
revealed that bacterial cells within indentations on the surface of SS coupons were reduced by phage. These
results suggest that bacteriophage AZO145A could be effective in reducing the viability of biofilm-adherent
STEC O145 on stainless steel in food industry environments.

1. Introduction to 2012 (Kühne et al., 2016). Contaminated beef products, in particular,

Shiga toxigenic Escherichia coli (STEC) are widespread foodborne
pathogens that can cause serious illness in humans, such as hemor-
rhagic colitis (HC), thrombotic thrombocytopenic purpura and hemo-
lytic uremic syndrome (HUS) and occasionally kidney failure (Singh
et al., 2019; Tarr et al., 2005). STEC O157:H7 is the predominant ser-
otype associated with foodborne outbreaks, but other STEC serogroups
(e.g., O26, O45, O91, O103, O111, O113, O121, O128, O145) have also
been linked to diarrhea and serious disease (Gould et al., 2013;
Calderon et al., 2010). Currently, more than 250 different STEC ser-
ogroups have been described, with more than 150 of these being as-
sociated with a variety of intestinal and extra-intestinal diseases (Carter
et al., 2016). The top six serogroups linked to disease include O26, O45,
O103, O111, O121, and O145, being responsible for 70% of non-O157
infections in the United States from 1983 to 2002 (Brooks et al., 2005)
and 81% of gastroenteritis and 32% HUS cases in Germany from 2008
are responsible for an estimated 37% of those illnesses in Canada
(Currie et al., 2019). Among the top six non-O157 serogroups, STEC
O145 is most commonly associated with human infections in the U.S
(Gould et al., 2013). STEC O145 carries the core virulence determinants
of STEC and shares a common evolutionary lineage with STEC O157
(Cooper et al., 2014).
Occasionally, STEC strains occur in beef processing plants, causing
serious contamination or cross contamination of beef products (Joseph
et al., 2018; Yang et al., 2017). Some STEC are capable of forming
strong biofilms on various food-contact surfaces (Adator et al., 2018;
Wang et al., 2016a), which are sessile bacterial communities that ad-
here to biotic and abiotic surfaces, and are embedded in a self-produced
extracellular polymeric matrix (Yin et al., 2018). Compared to plank-
tonic cells, cells within biofilms are more resistant to antimicrobials
including the sanitizers used in meat processing environments (Li et al.,
2012; Wang et al., 2012). Previous work in our laboratory identified a

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: zhoushoubiao@vip163.com (S. Zhou), tim.mcallister@canada.ca (T.A. McAllister).

https://doi.org/10.1016/j.fm.2020.103572
Received 27 January 2020; Received in revised form 1 June 2020; Accepted 13 June 2020
Available online 21 June 2020
0740-0020/ Crown Copyright © 2020 Published by Elsevier Ltd. All rights reserved.
C. Wang, et al. Food Microbiology 92 (2020) 103572

strain of STEC O145 that readily forms stable strong biofilms on poly- (Sigma-Aldrich) supplemented with 0.4% glucose, 0.02% MgSO4, and
styrene and stainless steel (Adator et al., 2018; Ma et al., 2019). 0.001% CaCl2 (w/v) and grown at 37 °C, on a rocker platform at
Numerous physical, chemical, and biological strategies such as ul- 180 rpm for 18 h.
trasound, UV-C irradiation, cold oxygen plasma, plant extracts, bac- Bacteriophage AZO145A was propagated to 1010 PFU/mL using
tericidal coatings, and quorum sensing inhibitors have been assessed as STEC O145:H25 as a host bacterium in LB broth. The host spectrum of
biofilm inhibitors and disruptors (Fan et al., 2018; Sadekuzzaman et al., this phage was spot tested as described by Kocharunchitt et al. (2009).
2017a). For example, ε-polylysine is a natural microbiological food The efficiency of plating (EOP) was tested according to the method of
preservative isolated originally from the fermentation broth of Strep- Kutter (2009) and phage titers were determined using the double layer
tomyces albulus. It is composed of 25–35 L-lysine units and certified as plate method. For this procedure, 100 μL of each phage was diluted
GRAS (Generally Recognized as Safe, No. GRN000135) by the US Food from 1010 PFU/mL to 102 PFU/mL and 100 μL of the respective bac-
and Drug Administration (FDA) and has been shown to have anti-bio- terial host culture were added to 3 mL of LB 0.3% as the top agarose
film activity (Zhang et al., 2019; Guillon et al., 2018). However, the layer and plated over a LB plate. The plates were incubated at 37 °C for
efficacy of some of these approaches has been shown to be limited and 12 h and the number of plaques counted. The results were expressed as
unsuitable for adoption by the food industry (Cui et al., 2018; plaque forming units per millilitre (PFU/mL).
Sadekuzzaman et al., 2017b). Bacteriophages are viruses capable of
infecting their bacterial host and have shown promise as anti-biofilm
agents (García-Anaya et al., 2020; Hungaro et al., 2013). Recently, 2.2. Determination of one-step growth curve, minimal inhibitory
there has been increased interest in the use of phage to eradicate bio- concentration (MIC) and minimal bactericidal concentration (MBC) of
films from food and food contact surfaces. It was reported that phage phage
BPECO 19 (Podoviridae) was capable of reducing the viability of E. coli
O157:H7 biofilm cells by 2–3 log CFU/cm2 on stainless steel, rubber, An one-step growth curve of phage AZO145A was performed as
and lettuce (Sadekuzzaman et al., 2017a). Furthermore, phages (5%, described by Wommack et al. (2009) with minor modifications. Briefly,
30 min) in combination with cold nitrogen plasma (CNP, 400 W, 2 min) 1 mL of mid-log phase STEC O145:H25 culture (2 × 108 CFU/mL) and
as a physical intervention, eliminated E. coli O157:H7 biofilms from the 1 mL of phage suspension (4 × 106 PFU/mL) were mixed to achieve a
surface of lettuce, cucumbers and carrots (Cui et al., 2018). Our la- multiplicity of infection (MOI) of ~0.02. The mixture was allowed to
boratory previously isolated a bacteriophage, AZO145A from beef adsorb for 10 min and then centrifuged at 10,000×g for 2 min, before
cattle feces that exhibited high activity against STEC. The T4-like phage the supernatant was discarded. The precipitate was then mixed with
was a member of the Myoviridae and exhibited high antibacterial ability 10 mL of LB broth pre-heated at 37 °C, and 0.1 mL was transferred to
against a number of STEC serogroups. The objective of this study was to 9.9 mL of pre-heated LB broth. The mixture was vortexed, and 0.1 mL
evaluate the efficacy of AZO145A against STEC O145 biofilms on was transferred to another 9.9 mL LB broth, vortexed and cultured at
stainless steel (SS) coupons. The extent to which phage treatment re- 37 °C for 2 h in a water bath. The titer of phage AZO145A was de-
duced the contamination of meat upon contact with the surface of SS termined using STEC O145:H25 after 0, 15, 20, 25, 30, 40, 50, 60, 70,
was also assessed. 80 and 100 min with this procedure repeated thrice. The latent period
was defined as the time interval between adsorption and the point at
with the phage titer began to increase. The burst size was calculated as
2. Materials and methods the ratio of the final number of the highest phage particles to the initial
number of infected host cells at the beginning of the test (phage titer at
2.1. Bacterial strains, phage isolates, media, and growth conditions time 0).
The minimal inhibitory concentration (MIC) and minimal bacter-
Twelve STEC strains and one Salmonella strain were used to evaluate icidal concentration (MBC) of AZO145A were determined using the 96-
host range of AZO145A (Table 1). Stock cultures of STEC O145:H25 well microplate (Cui et al., 2018). An overnight culture of STEC
EC19990166 were stored in Luria-Bertani (LB) broth (10 g/L Tryptone, O145:H25 in M9 broth was diluted with 10 mM phosphate buffered
10 g/L NaCl, 5 g/L Yeast Extract) (Sigma-Aldrich, Oakville, ON, CA) saline (PBS, pH 7.4) and prepared in tubes (105 CFU/mL) and then
supplemented with 15% (v/v) glycerol. The stock was streaked onto LB 180 μL was transferred to each microplate well. The phage suspension
broth agar (1.5% agar, Sigma-Aldrich) and incubated at 37 °C for 18 h. (2 × 1010 PFU/mL) was serially diluted and 20 μL added to each well.
A single colony was inoculated into 10 mL of minimal salt (M9) broth The microplate was then incubated at 24 °C for 48 h with agitation
(150 rpm). Absorbance was measured at 600 nm for all trials. Sus-
Table 1 pensions from the wells showing no growth were cultured on LB Agar.
Host spectrum of phage AZO145A by the spot test. The MIC was defined as the lowest concentration of phages that pre-
Serogroup Strain Ref. No. Lysis by phage AZO145A cluded visible growth of STEC O145:H25 on LB after incubation at 24 °C
for 24 h. The MBC was the lowest concentration of phages required to
O157:H7 EC2011007 +++ kill all of STEC O145:H25 on the LB after incubation at 24 °C for 48 h.
O145:H25 EC19990166 +++
O121:H19 EC19990161 +++
All experiments were performed in triplicate.
O111:NM EC20030053 +++ Bacterial inactivation was determined with the phage suspension
O103:H2 EC19970327 - (2 × 1010 PFU/mL) using STEC O145:H25 as a host. An overnight
O45:H2 EC19940040 ++ culture of STEC O145:H25 in M9 broth was diluted with 10 mM PBS to
O26:H11 EC19970119 +++
105 CFU/mL. Then, 100 μL of the bacterial culture and a 100 μL phage
O91:H21 EC20010076 +++
O113:H21 EC20020170 +++ suspension were mixed and allowed to adsorb for 10 min. Ten mL of M9
O128:NM EC19960949 +++ broth was then added to the mixture, which was incubated at 24 °C for
O154:H10 South Africa + 12 h. A bacterial culture incubated under the same conditions without
O129:H23 South Africa ++ phage served as a control. Aliquots of test samples and of the control
Salmonella typhimurium ATCC 14028 -
were collected after 0, 2, 4, 6 and 12 h of incubation. Bacterial con-
+++ complete clearing centration was determined in duplicate using LB agar plates after in-
++ clearing throughout but with faintly hazy background cubation for 24 h at 37 °C. All experiments were performed in triplicate.
+ substantial turbidity throughout the cleared zone
-no clearing.

2
C. Wang, et al.
2.3. Phage against STEC O145 planktonic cells of different ages in broth
STEC O145:H25 working stock was streaked onto LB broth agar
plates and incubated at 37 °C for 18 h. An isolated colony was then
inoculated into 10 mL of M9 broth supplemented with 0.4% glucose,
0.02% MgSO4, and 0.001% CaCl2 (w/v) and grown at 24 °C, on a rocker
platform for 24, 48, or 72 h, prior to a series of ten-fold dilutions with
M9. To evaluate the infectivity of phage to STEC O145:H25 cells at
different growth stages (24, 48 or 72 h), 100 μL of phage and 100 μL of
STEC O145:H25 (1.0 × 106 CFU/mL) were mixed and added to 9.8 mL
of M9 broth. This resulted in a final phage concentration of
1.0 × 109 PFU/mL and an STEC O145:H25 concentration of
1.0 × 104 CFU/mL. All treatments were allowed to adsorb for 10 min
prior to incubation at 24 °C. A bacterial culture containing 9.9 mL M9
broth without phage served as a control. The number of viable STEC
O145:H25 was determined after 2 h of incubation. All experiments
were performed in triplicate.
2.4. Biofilm removal on stainless steel coupon with phage
Stainless steel 304 (SS) coupons (2.5 cm × 7.6 cm × 0.08 cm) with
#4 finish were used to assess dry-surface biofilm formation and treat-
ment. Coupons were prepared as described by Ma et al. (2019). Briefly,
coupons were soaked in 10% bleach (0.5% hypochlorite) for 24 h prior
to use. Coupons were then rinsed three times with sterile distilled water
to remove residual hypochlorite and dried at 24 °C. Coupons were then
treated with 70% ethanol and air-dried for 5 min at 24 °C. Lastly, the
coupons were autoclaved at 121 °C for 15 min.
To assess the effect of AZO145A against biofilms of different ages,
overnight cultures of STEC O145:H25 (100 μL) were transferred into
50 mL Falcon tubes containing a sterile SS coupon and 20 mL of M9
broth. The tubes were loosely capped and incubated at 24 °C for 24, 48
and 72 h, at which point biofilm formation was assessed. Biofilms
formation was assessed as described by Ma et al. (2020). After in-
cubation, SS coupons were removed from tubes, rinsed in 25 ml of
sterilized deionized water through forty immersions, three times, so as
to remove loosely-attached bacteria. Coupons with adherent biofilms
were fixed with 25 ml absolute methanol (Sigma-Aldrich) for 15 min
and air-dried for 2 min. Coupons were then stained for 15 min with
0.5% (w/v) crystal violet solution (Sigma-Aldrich) on a shaking plat-
form at 40 rpm. Coupons were immersed three times in 25 ml sterile
water to remove excess crystal violet stain, and air-dried at 24 °C for
5 min. Coupons were then immersed in 25 ml of 33% glacial acetic acid
(Sigma-Aldrich) and left at room temperature for 15 min. Dissolved dye
was measured at 590 nm using a spectrophotometer (Genesys 20,
Thermo).
Phage suspension (5 mL, 2 × 1010 PFU/mL) or 5 mL-10 μg/mL ε-
polylysine (Bocscience, NY, USA) or M9 broth (w/v) (5 mL, control)
were dispensed directly onto the biofilms on SS coupons while being
held over a petri plate. Coupons were then placed on the plate and
contact was allowed for 1.5 h on each side of the coupon, resulting in
3 h exposure at 24 °C for each coupon. . After incubation, coupons were
rinsed as described above, deposited in 20 mL of sterile PBS and soni-
cated at 20 kHz for 10 min to promote the detachment of biofilms. After
sonication, the tubes containing coupons were vigorously vortexed for
1 min, and 1 mL of the bacterial suspension was serially diluted, plated
on LB agar, and incubated at 37 °C for 18 h. Colonies were enumerated
(CFU/mL) and all experiments were performed in triplicate.
2.5. STEC O145 transfer to beef from coupon before and after phage
treatment
Fresh beef fillets (eye of round) purchased at a local grocery store
served as the recipient surface and were cut into 2 × 2 × 0.5 cm, 4 g
pieces. E. coli contamination of the beef was assessed by scraping ~10 g
of tissue from the surface of each fillet, placing it in 90 mL of sterile
3
Food Microbiology 92 (2020) 103572
PBS, vortexing it for 2 min and plating 500 μL of the homogenate onto
MacConkey agar. All fillets that tested negative were subsequently used
in the experiment. Coupons with associated STEC O145:H25 biofilms
grown for 48 h were prepared and treated with phage, ε-polylysine or
left as a control as described in section 2.4. Coupons were then divided
into two groups of 9, with 3 coupons treated with phage, 3 coupons
with ε-polylysine treated, and 3 serving as controls. Two pieces of beef
(2 × 2 cm) for each coupon were allowed to touch each side of the SS
coupon thrice for 10 s (slight touch method), then the two pieces of beef
were transferred to a 50 mL Falcon tube containing 20 mL of sterile
PBS. For the second group, a SS coupon was placed on a prepared beef
fillet surface and completely covered with a second beef fillet in what
has been described as the “sandwich transfer method” (Wang et al.,
2015). Next, a sterilized 500 g glass bottle was placed on the upper
meat surface for 10 min, to apply a consistent pressure. Then the two
pieces of beef were moved into a 50 mL Falcon tube containing 20 mL
of sterile PBS (composite sample). Composite samples were sonicated at
20 kHz for 10 min, vigorously vortexed for 3 min with glass beads, and
a 100 μl aliquot was removed, subject to 10-fold serial dilution and
plated onto MacConkey agar to enumerate STEC O145:H25 colonies.
The plates were incubated at 37 °C for 24 h. Simultaneously, an aliquot
(1 ml) of tryptic soy broth was mixed with the initial homogeneous
solution and incubated at 24 °C for 24 h to recover STEC
O145:H25 cells from beef. The enriched suspension (100 μL) was spot-
plated onto MacConkey agar and plates were incubated at 37 °C for
24 h. Enrichments that were negative were incubated for another 24 h
and plated again as described above. Total recovery was estimated as
the sum of positive samples identified after 24 or 48 h. Positive rate [%
(n/N)] was calculated based on the number of samples testing positive
(n) divided by the total sample size (N). Recovery percentage [% (n*/
N*)] was calculated based on the number of samples testing positive
(n*) divided by the total sample size (N*) after enrichment. All ex-
periments were performed four times.
2.6. Scanning electron microscopy (SEM)
Biofilm formation on SS coupon by STEC O145:H25 was further
observed by scanning electron microscopy (SEM) as described by Ma
et al. (2019). Coupons with biofilms were rinsed three times as de-
scribed above, air-dried, and then fixed in 2.5% (v/v) glutaraldehyde
(Canemco INC., Quebec, CA) for 24 h. Subsequently, the samples were
dehydrated in an ethanol (Commercial alcohols, ON, CA) (v/v) (i.e.,
30%, 50%, 70%, 85%, 95% and 100%) and isobutyl alcohol series
(Sigma-Aldrich) (v/v) (i.e., 10%, 30%, 50%, 70%, 90%, and 100%).
The samples were then treated with 100% (v/v) hexamethyldisilazane
(Sigma-Aldrich) for 10 min, sputter coated with gold and visualized
using a SEM (HITACHI S-4800, Japan).
2.7. Statistical analysis
To assess the MIC of the phage, PFU/mL was included in the model.
To examine the formation of STEC O145:H25 biofilms on stainless steel
coupons, the model variables included treatment, biofilm age and
treatment ⅹ biofilm age in the model. To investigate the transfer of
STEC O145:H25 to beef, the model variables included treatment,
transfer method and treatment ⅹ transfer method in the model. Biofilm
transferring to beef data were compiled from the four independent
experiments. Significant differences were determined t using SAS (SAS
9.4, SAS Institute, Cary, NC, USA) with differences reported at P ˂ 0.05
using the LSMEANS function.
3. Results
3.1. Characteristics of phage AZO145A
Based on spot tests, the host spectrum of phage AZO145A included
C. Wang, et al.
Fig. 1. Characterization of phage AZO145A. A: Efficacy of plating of phage AZO145A on E. coli O145:H25 LB agar plate (phage concentrations from 102 - 1010 PFU/
mL); B: Plaques formed by phage AZO145A; C: One-step growth curve of phage.
11 E. coli O serogroups (Table 1), with clear plaques formed at both low
(103 PFU/mL) and high concentrations of phage on LB plates (Fig. 1A
and B). The burst size of AZO145A was approximately 98 PFU/cell,
after a latent period of 30 min (Fig. 1C). These results showed that
phage AZO145A exhibited a broad host spectrum against STEC ser-
ogroups.
3.2. Effect of phage AZO145A against STEC O145 in broth
The MIC value of phage against STEC O145:H25 was
1.2 × 109 PFU/mL (Fig. 2A) and the MBC was 1.0 × 1010 PFU/mL
(Table 2). AZO145A eliminated STEC O145 in broth cultures at 24 °C
with a multiplicity of infection (MOI) greater than 7200. Exposure to
2 × 1010 PFU/mL for 2 h reduced STEC O145:H25 cells in broth by 4.0
log10 CFU/mL (Fig. 2B). Viable counts were reduced below the limit of
detection (< 10 CFU/mL) after 2 h of phage treatment. However, after
12 h, STEC O145:H25 recovered by 0.4 log10 CFU/mL possibly as a
result of the development of resistance.
3.3. Effects of phage AZO145A against STEC O145 planktonic cells over
time
Phage AZO145A was evaluated for its lytic activity against STEC
O145:H25 strain grown for 24, 48 or 72 h in M9 broth and exposed to
phage for 2 h. Phage reduced (P < 0.01) STEC O145:H25 below the
limit of detection (< 10 CFU/mL) in 24 h (Fig. 3A). However, phage
4
Food Microbiology 92 (2020) 103572
reduced (P < 0.01) cell numbers by only 2.0 log10 CFU/mL after 48 h
or 72 h (Fig. 3B and C), but numbers were consistently lower than
controls.
3.4. Effects of phage AZO145A against STEC O145 biofilms on stainless
steel coupons
Biofilms of STEC O145:H25 on SS coupon grown in M9 broth for 24,
48 and 72 h were exposed to phage solutions for 3 h, and the cell
number were reduced (P < 0.01) by of 2.9, 1.9 and 1.9 log10 CFU/
coupon, respectively (Table 3).. ε-polylysine reduced attached cell
numbers in biofilms grown in M9 broth for 24, 48 and 72 h by 3.5, 1.8
and 1.7 log10 CFU/coupon, respectively. There was no difference
(P > 0.05) in efficacy between phage and ε-polylysine and neither
could completely eliminate the biofilm from the surface of SS coupons.
There were no significant changes in phage titer before and after
treatment (Table 3).
3.5. Biofilm reduction efficacy of phage at reducing the transfer of STEC
O145 from stainless steel coupons to beef
Cells associated with biofilms formed on SS coupon could readily
transfer to beef (Table 4). Even after 10 s of contact, 3.6 log10 CFU/
coupon STEC O145:H25 cells were transferred to the surface of beef.
Both phage and ε-polylysine equally reduced (P < 0.01) the transfer of
STEC O145:H25 cells from the surface of SS coupons to beef by 3.1
C. Wang, et al.
Fig. 2. Activity of phage against E. coli O145:H25 in M9 at 24 °C. A: MIC of
phage AZO145A against E. coli O145:H25 in M9 at 24 °C. Values represent
means ± SE (n = 4). Initial phage titer was 2.0 × 1010 PFU/mL. Initial E. coli
count was 6.9 × 105 CFU/mL. Letters differ at p < 0.05. B: Phage treatment of
E. coli O145:H25 grown in M9 at 24 °C for 12 h. Values represent means ± SE
(n = 3).
log10 CFU/coupon and 2.9 log10 CFU/coupon, respectively. Compared
to the slight touch method, the sandwich method transferred more
STEC O145:H25 cells from the surface of SS coupon to beef (4.3
log10 CFU/coupon transferred). Phage treatment and ε-polylysine re-
duced (P < 0.01) the transfer of STEC O145:H25 cells from the surface
of SS coupon to beef via the sandwich method by 3.8 log10 CFU/coupon
and 3.3 log10 CFU/coupon, respectively.
The highest positive rate of STEC O145:H25 after transfer to beef
was 75.0% for the ε-polylysine treatment by the sandwich method. In
contrast, the lowest positive rate of STEC O145:H25 without enrich-
ment was 25.0% for both the ε-polylysine and phage treatment by the
Table 2
The minimal bactericidal concentration (MBC) of phage AZO145A against E. coli O145:H25 in M9 at 24.
Phage concentration (PFU/mL) 0 2.0ⅹ1010 1.0ⅹ1010
E coli O145:H25 + - - +
+ growth, - no growth.
5
Food Microbiology 92 (2020) 103572
slight touch method (Table 5). In contrast, all controls resulted in the
transfer of STEC O145:H25 to beef. The lower positive rate suggests
that transfers of biofilms from SS coupons to beef was reduced
(P < 0.05) by phage treatment. However, STEC O145:H25 was re-
covered from all negative samples after 48 h of enrichment (Table 5).
After 24 h of enrichment, the recovery of STEC O145:H25 after phage
treatment was higher (P < 0.05) than for ε-polylysine. Extending the
enrichment period to 48 h increased the recovery of STEC from ε-
polylysine treatment. These results indicated that the attached cells on
the biofilm formed on SS coupon posed a high risk of contaminating
beef processing and phage or ε-polylysine could reduce, but not elim-
inate this risk.
3.6. Scanning electron microscope analysis
As observed by SEM, STEC O145:H25 formed compact and dense
biofilms on the surface of SS coupons (Fig. 4A, B and C). As shown in
Fig. 4D, E and F, there were attached cells with altered morphology
remaining on the surface of SS coupons after phage treatment, and
these were most oftern observed within the indentations on the surface
of coupons. Compared to control, the morphology of attached cells in
the cracks on the surface of SS coupons were different from those
treated with phage or ε-polylysine(Fig. 4).
4. Discussion
Currently, STEC strains frequently cause serious foodborne illnesses
and economic losses to the food industry, especially during a high-event
periods (HEP) (Wang et al., 2016b), when commercial beef processing
plants experience elevated levels of STEC. This study was conducted to
evaluate the anti-biofilm and antimicrobial activity of phage AZO145A
against STEC. This phage showed a broad host spectrum against STEC
strains, with most of these strains forming strong biofilms on poly-
styrene and stainless steel surfaces (Bumunang et al., 2019; Ma et al.,
2019; Wang et al., 2016a). Recently, phage have gained considerable
attention in the food industry as a potential means of controlling bio-
films on surfaces in food processing environments (Cha et al., 2019; Cui
et al., 2018; Sadekuzzaman et al., 2017a). In the present study, phage
AZO145A reduced the number of cells of STEC O145:H25, an ex-
ceptionally strong biofilm former on stainless steel coupons (Ma et al.,
2019). These results agree with some previous studies on phage bio-
control. Sharma et al., 2005 reported that treatment with bacteriophage
KH1 reduced populations of E. coli O157:H7 cells attached to SS coupon
surfaces. Cui et al. (2018) reported that 1010 PFU/mL E. coli O157:H7
phages combined with cold nitrogen plasma treatment resulted in ir-
reversible destruction of E. coli O157:H7 cells so as to reduce this pa-
thogen to undetectable levels on the surface of SS coupons.
Interestingly, the sensitivity of phage to STEC O145:H25 biofilm
was affected by the maturity of the biofilm (Table 3). These observa-
tions were consistent with other reports. For example, E. coli K-12 strain
AR3110 cells in 48 h biofilms were rapidly eradicated as a result of
exposure to T7 phages, while 60 h biofilms were resistant to phages
(Vidakovic et al., 2018). Listeria monocytogenes biofilms were more
sensitive to sanitizers (i.e., sodium hypochlorite, quaternary ammo-
nium compounds and peroxyacetic acid) on stainless steel coupons after
2 than after 7 days of biofilm formation (Stopforth et al., 2002). Cells of
S. enteritidis in biofilms grown for 48 h were more sensitive to trisodium
phosphate (pH 12.5), compared to cells grown for 72 h (Korber et al.,
5.0ⅹ109 2.5ⅹ109 1.2ⅹ109 6.0ⅹ108
+ + +
C. Wang, et al.
Fig. 3. E. coli O145:H25 planktonic cells before and after phage treatment.
Values represent means ± SE (n = 3). ∗: P < 0.05, compared to the control
group. (A) Planktonic cells grown at 24 °C for 24, (B) 48 or (C) 72 h and treated
with 1010 PFU/mL of phage after 2 h of incubation.
1997). These responses likely reflect that exponentially growing cells
are more sensitive to environmental stresses than stationary phase cells,
because of the high energy demand and intense regulation of growth
components associated with actively growing cells (Gauthier et al.,
1992). This may also explain why phages are less effective as bacteria
foten enter stasis at temperatures below 5 °C (Ma et al., 2019). Early
6
Food Microbiology 92 (2020) 103572
biofilm formation requires actively growing cells as microcolonies and
complex matrices are formed, rendering the cell more susceptible to
antimicrobials. This explanation is consistent with the results of phage
treatment of planktonic and biofilm cells in this study. Secondly, bio-
films are composed of a mixture of polymeric compounds such as
polysaccharides, proteins, nucleic acids, and lipids, with dense micro-
colonies separated by channels that distribute water, nutrients, oxygen,
enzymes, and cellular debris. This microenvironment protects bacteria
from antimicrobial agents, host immune defenses and other abiotic
factors (Flemming and Wingender, 2010). Despite these protective
properties, phages still significantly reduced the number of cells asso-
ciated with mature biofilms, indicating the potential of AZO145A as an
anti-biofilm agent.
Unfortunately, it is difficult for phage to completely remove all cells
attached and embedded within biofilms and the mechanisms whereby
phage attack biofilms are poorly understood. The presence of phage
resistant host cells within the biofilm could create ‘spatial refugees’
within the interior of biofilms (Tait et al., 2002). As bacterial commu-
nities in biofilms are surrounded by a polysaccharide matrix, phages
may not be able to gain access to bacterial cells that are deeply em-
bedded within biofilms (Briandet et al., 2008). This makes the re-
lationship between phage and host bacteria complex as phage need to
use host bacterial resources (i.e., proteins and nucleic acids) for re-
plication and thus rely on the presence of metabolically active bacteria
to maintain the balance known as “Red Queen” dynamics (Morgan
et al., 2010). Bacteria can harbor a number of phage resistance me-
chanisms, such as restriction modification systems, abortive infections
(i.e., dormancy or possible ‘suicidal’ responses upon phage infection in
bacteria), CRISPR-Cas systems and others (de Melo et al., 2018;
Vidakovic et al., 2018; Wang et al., 2019). Phages also developed dif-
ferent strategies to respond to these challenges (Watson et al., 2019)
such as the production of enzymes that degrade extracellular poly-
saccharide substances (EPS) (Azeredo and Sutherland, 2008).
Contamination of beef carcasses with E. coli may occur during the
dressing, chilling or cutting stages of processing via aerosols or direct
contact with the surface of equipment (Dourou et al., 2011). Some of
these procedures such as dressing and cutting may be performed at
room temperature in processing, retail and home environments. Pre-
vious studies have observed that E.coli cells could readily transfer from
biofilms formed on stainless steel to fresh lettuce, even from biofilms
that were desiccated over 30 d (Adator et al., 2018). E. coli O157:H7
attached to the surface of stainless steel were able to readily transfer to
meat, poultry, ready-to-eat deli foods, and produce. This pathogen
strongly adhered to the surface of cantaloupes, lettuce, carrots, and
spinach and was not removed by vigorous washing with water (Silagyi
et al., 2009). In the present study, STEC O145:H25 could readily
transfer from biofilms formed on SS coupon to the surface of beef even
after only 10 s of contact. If the contact time and contact force were
increased, it is likely that even more STEC O145:H25 cells would have
been transferred to beef.
In the present study, two transfer methods were used to simulate the
beef processing environments associated with SS surfaces. The slight
touch method simulated short-term contact between beef and the sur-
face of processing utensils, while the sandwich method simulated the
contact of beef with machinery during cutting and extrusion. The re-
sults showed that both methods readily resulted in the transfer of STEC
O145:H25 from biofilms to the surface of beef to a similar degree. The
sandwich method showed that the biofilm of STEC O145:H25 would
readily cause cross contamination in large-scale beef processing en-
vironments if not treated or control. These results are consistent with
observation that Salmonella cells within biofilms were readily trans-
ferred from stainless steel to meat products, such as salted sausage,
bacon, sliced ham, cantonese sausage and roast pork (Wang et al.,
2015). The transfer of L. monocytogenes cells from single species bio-
films on SS coupons to salmon fillets was high, posing a high risk of L.
monocytogenes contaminating salmon processing facilities (Pang and
C. Wang, et al. Food Microbiology 92 (2020) 103572

Table 3
Activity of bacteriophage AZO145A against biofilms of differing maturity formed on stainless steel coupons in M9 medium at 24 °C.
Biofilm age (h) STEC number (logCFU/coupon) Reduction (logCFU/coupon) Phage concentration (logPFU/mL)

CK phage ε-polylysine CK Phage ε-polylysine Before After

Aa Ab Ab
24 4.7 ± 0.2 1.8 ± 1.4 1.2 ± 1.3 - 2.9 3.5 10.35 ± 0.06 10.36 ± 0.04
48 5.4 ± 0.2Ba 3.5 ± 0.5Bb 3.6 ± 1.1Bb - 1.9 1.8 10.35 ± 0.06 10.37 ± 0.06
72 5.8 ± 0.1Ca 3.9 ± 0.7Bb 4.1 ± 0.8Bb - 1.9 1.7 10.35 ± 0.06 10.34 ± 0.05

Control: control without phage for 3 h; phage: treated with phage for 3 h; ε-polylysine: treated with 10 μg/mL ε-polylysine for 3 h.Values are means ± standard
deviations of three replicates experiments.Mean values in the same column with different capital letters differ (P > 0.05) and mean values in the same row with
different letters differ (P > 0.05).

Table 4
Transfer of E. coli strain O145 cells from biofilms on stainless steel coupons to beef after phage and ε-polylysine treatment.
Transfer method STEC number on beef (logCFU/coupon) Reduction (logCFU/coupon)

M9 Phage ε-polylysine M9 phage ε-polylysine

Slight method 3.6 ± 0.3Aa(UT1) 0.5 ± 0.9Ab(PT1) 0.7 ± 1.4Ab(ET1) - 3.1 2.9
Sandwich method 4.3 ± 0.3Ba(UT2) 0.5 ± 0.5Ab(PT2) 1.0 ± 0.8Ab(ET2) - 3.8 3.3
Reduction 0.7 0 0.3

M9: treated with M9 for 3 h serves as control; phage: treated with phage for 3 h; ε-polylysine: treated with 10 μg/mL ε-polylysine for 3 h. UT1: untreated coupon
using slight touch transfer method; PT1: phage treated coupon using slight touch transfer method; ET1: ε-polylysine treated coupon using slight touch transfer
method; UT2: untreated coupon using sandwich transfer method; PT2: phage treated coupon using sandwich transfer method; ET2: ε-Polylysine treated coupon using
sandwich transfer method.
Values are means ± standard deviations of four replicates experiments.
Mean values in the same column with different capital letters differ (P > 0.05) and mean values in the same row with different letters differ (P > 0.05).

Table 5
STEC recovery rate from the surfaces of beef after transferring from stainless steel coupon.
Treatment Positive rate after transfer, % Recovery percentage after enrichment, Recovery percentage after 24 h Recovery percentage after 48 hd
(n/N)a % (n1/N)b enrichment, % (n2/N1c) enrichment, % (n3/N1)

UT1 100.0(12/12) - - -
PT1 25.0(3/12) 75.0(9/12) 88.9(8/9) 11.1(1/9)
ET1 25.0(3/12) 75.0(9/12) 55.6(5/9) 44.4(4/9)
UT2 100.0(12/12) - - -
PT2 41.7(5/12) 58.3(7/12) 71.4(5/7) 28.6(2/7)
ET2 75.0(9/12) 25.0(3/12) 33.3(1/3) 66.7(2/3)

UT1: untreated coupon using slight touch method; PT1: phage treated coupon using slight touch method; ET1: ε-polylysine treated coupon using slight touch method;
UT2: untreated coupon using sandwich method; PT2: phage treated coupon using sandwich method; ET2: ε-polylysine treated coupon using sandwich method.
a
Positive rate [% (n/N)] was calculated based on the number of samples testing positive (n) divided by the total sample size (N).
b
Recovery percentage [% (n*/N*)] was calculated based on the number of samples testing positive (n*) divided by the total sample size (N*) after enrichment.
c
N1: The number of samples testing negative before enrichment.
d
If E. coli was not recovered on 24 h SS coupons, samples were further enriched for additional 24 h, resulting in a total enrichment period of 48 h.

Yuk, 2019). Based on these results, elimination of cross-contamination
of pathogenic STEC O145:H25 in food processing environment could
pose a great challenge, although it does appear that phage could reduce
the risk of contamination.
ε-polylysine was used as a positive control in the current experi-
ments, and its anti-biofilm activity was similar to that of AZO145A.
Although both phage and ε-polylysine reduced the biofilms formed on
the surface of SS coupons, there is evidence that their mechanisms were
quite different. After phage treatment (Fig. 4D, E, F) few bacterial cells
adhered to the surface of SS coupons. In contrast, after ε-polylysine
treatment remaining STEC O145:H25 cells remained intact and were
mostly distributed in the cracks of the surface of SS coupon (Fig. 4G, H,
I). Phage-borne enzymes have been shown to degrade exopoly-
saccharides, which are the major protective layer within biofilms (Chai
et al., 2014; Vidakovic et al., 2018). Additionally, it is also assumed that
phage can diffuse through the pores and channels of biofilms, thus
reaching cells that are embedded within the interior of biofilms
(Sadekuzzaman et al., 2017a). Consequently, phage could break down
the composition and structure of planktonic cells and biofilms. How-
ever, as an antimicrobial peptide, ε-polylysine kills bacteria by diffusing
through the cell membrane, resulting in osmotic pressure imbalances
and metabolic disorders (Li et al., 2014; Lin et al., 2018).
5. Conclusion
In conclusion, the current study found that phage AZO145A was
effective in inactivating STEC O145:H25 in solution and in biofilms.
Phage AZO145A reduced the number of STEC O145:H25 cells in bio-
films transferred from stainless steel to beef, possibly lowering the risk
of STEC contamination of beef in processing plants, retailers and the
home.. Food safety is an ever-changing topic, which requires vigilance
throughout the production chain to maintain the health of consumers
and reduce economic losses to the food industry. The present study only
investigated single species biofilms, but multi-species biofilms can also
form in food processing environments. Further studies are needed to
evaluate the effects of phage cocktails or the combination of phage and
other antimicrobial agents on mixed-species biofilms.

7
C. Wang, et al. Food Microbiology 92 (2020) 103572

Fig. 4. Representative SEM images of O145 biofilms before and after treatment after growing on stainless steel coupons in M9 medium at 24 °C for 24, 48, and 72 h.
A: untreated O145 biofilm grown for 24 h; B: untreated O145 biofilm grown for 48 h; C: untreated O145 biofilm for 72 h; D: phage treated O145 biofilm grown for
24 h; E: phage treated O145 biofilm grown for 48 h; F: phage treated O145 biofilm grown for 72 h; G: 10 μg/mL ε-polylysine treated O145 biofilm grown for 24 h; H:
10 μg/mL ε-polylysine treated O145 biofilm grown for 48 h; I: 10 μg/mL ε-polylysine treated O145 biofilm grown for 72 h. Bar is 20 μm. White arrow points towards
the abnormal shape of the attached cells remaining on the surface of SS coupons after phage treatment.

Declaration of competing interest biofilms. Curr. Pharmaceut. Biotechnol. 9, 261–266.

No conflict of interest is declared.
Acknowledgements
This work was financially supported by grants from Anhui
Provincial Natural Science Foundation (1808085MC73), and the
Natural Science Foundation of Anhui Higher Education Institutions
(KJ2018A0314), and the Doctor's Research Foundation of Anhui
Normal University (2018XJJ53) and the Agriculture and Agri-Food
Canada–Beef Cluster program STEC project FOS.07.17.We acknowledge
the technical assistance of Zhi Ma, Emmanuel W. Bumunang, Reuben
Ha, Cheyenne Sargeant, Wendi Smart, Rahat Zaheer, Krysty Thomas,
Amy Stratton, Grant Duke and Susanne Trapp during the study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fm.2020.103572.
References
Adator, E.H., Cheng, M., Holley, R., McAllister, T., Narvaez-Bravo, C., 2018. Ability of
Shiga toxigenic Escherichia coli to survive within dry-surface biofilms and transfer to
fresh lettuce. Int. J. Food Microbiol. 269, 52–59.
Azeredo, J., Sutherland, I.W., 2008. The use of phages for the removal of infectious
Briandet, R., Lacroix-Gueu, P., Renault, M., Lecart, S., Meylheuc, T., Bidnenko, E.,
Steenkeste, K., Bellon-Fontaine, M.-N., Fontaine-Aupart, M.-P., 2008. Fluorescence
correlation spectroscopy to study diffusion and reaction of bacteriophages inside
biofilms. Appl. Environ. Microbiol. 74, 2135–2143.
Brooks, J.T., Sowers, E.G., Wells, J.G., Greene, K.D., Griffin, P.M., Hoekstra, R.M.,
Strockbine, N.A.J.T., 2005. Non-O157 Shiga toxin–producing Escherichia coli infec-
tions in the United States, 1983–2002. J. Infect. Dis. 192, 1422–1429.
Bumunang, E.W., McAllister, T.A., Zaheer, R., Ortega Polo, R., Stanford, K., King, R., Niu,
Y.D., Ateba, C.N., 2019. Characterization of Non-O157 Escherichia coli from cattle
faecal samples in the North-West province of South Africa. Microorganisms 7.
Calderon, V.E., Chang, Q., McDermott, M., Lytle, M.B., McKee, G., Rodriguez, Rasko,
D.A., Sperandio, V., Torres, A.G., 2010. Outbreak caused by cad-negative Shiga toxin-
producing Escherichia coli O111, Oklahoma. Foodb. Pathog. Dis. 7, 107–109.
Carter, M.Q., Quinones, B., He, X., Zhong, W., Louie, J.W., Lee, B.G., Yambao, J.C.,
Mandrell, R.E., Cooley, M.B., 2016. An environmental Shiga Toxin-Producing
Escherichia coli O145 clonal population exhibits high-level phenotypic variation that
includes virulence traits. Appl. Environ. Microbiol. 82, 1090–1101.
Cha, Y., Son, B., Ryu, S., 2019. Effective removal of staphylococcal biofilms on various
food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. Food
Microbiol. 84.
Chai, Z., Wang, J., Tao, S., Mou, H., 2014. Application of bacteriophage-borne enzyme
combined with chlorine dioxide on controlling bacterial biofilm. LWT - Food Sci.
Technol. (Lebensmittel-Wissenschaft -Technol.) 59, 1159–1165.
Cooper, K.K., Mandrell, R.E., Louie, J.W., Korlach, J., Clark, T.A., Parker, C.T., Huynh, S.,
Chain, P.S., Ahmed, S., Carter, M.Q., 2014. Comparative genomics of enterohemor-
rhagic Escherichia coli O145: H28 demonstrates a common evolutionary lineage with
Escherichia coli O157: H7. BMC Genom. 15, 17.
Cui, H., Bai, M., Yuan, L., Surendhiran, D., Lin, L., 2018. Sequential effect of phages and
cold nitrogen plasma against Escherichia coli O157:H7 biofilms on different vege-
tables. Int. J. Food Microbiol. 268, 1–9.
Currie, A., Honish, L., Cutler, J., Locas, A., Lavoie, M.C., Gaulin, C., Galanis, E., Tschetter,
L., Chui, L., Taylor, M., Jamieson, F., Gilmour, M., Ng, C., Mutti, S., Mah, V., Hamel,
M., Martinez, A., Buenaventura, E., Hoang, L., Pacagnella, A., Ramsay, D., Bekal, S.,

8
C. Wang, et al.
Coetzee, K., Berry, C., Farber, J., Team, O., 2019. Outbreak of Escherichia coli
O157:H7 infections linked to mechanically tenderized beef and the largest beef recall
in Canada, 2012. J. Food Protect. 82, 1532–1538.
de Melo, A.G., Levesque, S., Moineau, S., 2018. Phages as friends and enemies in food
processing. Curr. Opin. Biotechnol. 49, 185–190.
Dourou, D., Beauchamp, C.S., Yoon, Y., Geornaras, I., Belk, K.E., Smith, G.C., Nychas,
G.J., Sofos, J.N., 2011. Attachment and biofilm formation by Escherichia coli O157:H7
at different temperatures, on various food-contact surfaces encountered in beef
processing. Int. J. Food Microbiol. 149, 262–268.
Fan, Q., Zhang, Y., Yang, H., Wu, Q., Shi, C., Zhang, C., Xia, X., Wang, X., 2018. Effect of
Coenzyme Q0 on biofilm formation and attachment-invasion efficiency of Listeria
monocytogenes. Food Contr. 90, 274–281.
Flemming, H.-C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623.
García-Anaya, M.C., Sepulveda, D.R., Sáenz-Mendoza, A.I., Rios-Velasco, C., Zamudio-
Flores, P.B., Acosta-Muñiz, C.H., 2020. Phages as biocontrol agents in dairy products.
Trends Food Sci. Technol. 95, 10–20.
Gauthier, M., Flatau, G., Clement, R., Munro, P., 1992. Sensitivity of Escherichia coli cells
to seawater closely depends on their growth stage. J. Appl. Bacteriol. 73, 257–262.
Gould, L.H., Mody, R.K., Ong, K.L., Clogher, P., Cronquist, A.B., Garman, K.N., Lathrop,
S., Medus, C., Spina, N.L., Webb, T.H., 2013. Increased recognition of non-O157
Shiga toxin–producing Escherichia coli infections in the United States during
2000–2010: epidemiologic features and comparison with E. coli O157 infections.
Foodb. Pathog. Dis. 10, 453–460.
Guillon, A., Fouquenet, D., Morello, E., Henry, C., Georgeault, S., Si-Tahar, M., Hervé, V.,
2018. Treatment of Pseudomonas aeruginosa biofilm present in endotracheal tubes by
poly-L-lysine. Antimicrob. Agents Chemother. 62 e00564-00518.
Hungaro, H.M., Mendonça, R.C.S., Gouvêa, D.M., Vanetti, M.C.D., de Oliveira Pinto, C.L.,
2013. Use of bacteriophages to reduce Salmonella in chicken skin in comparison with
chemical agents. Food Res. Int. 52, 75–81.
Joseph, A.P., Latha, C., Vinodkumar, K., Vinod, V.K., Sathu, T., 2018. An important ap-
proach for control of Enterohaemorrhagic Escherichia coli by identification of con-
taminating sources in beef production line. Int J Curr Microbiol App Sci 7,
1921–1929.
Kocharunchitt, C., Ross, T., Mcneil, D.L., 2009. Use of bacteriophages as biocontrol agents
to control Salmonella associated with seed sprouts. Int. J. Food Microbiol. 128 (3),
453–459.
Korber, D., Choi, A., Wolfaardt, G., Ingham, S., Caldwell, D., 1997. Substratum topo-
graphy influences susceptibility of Salmonella enteritidis biofilms to trisodium phos-
phate. Appl. Environ. Microbiol. 63, 3352–3358.
Kühne, A., Bouwknegt, M., Havelaar, A., Gilsdorf, A., Hoyer, P., Stark, K., Werber, D.,
Infection, 2016. Estimating true incidence of O157 and non-O157 Shiga toxin-pro-
ducing Escherichia coli illness in Germany based on notification data of haemolytic
uraemic syndrome. Epidemiology 144, 3305–3315.
Kutter, E., 2009. Phage host range and efficiency of plating. In: Clokie, Martha R.J.,
Kropinski, Andrew M. (Eds.), Bacteriophages: Methods and Protocols, Volume 1:
Isolation, Characterization, and Interactions. Humana Press, New York, pp. 131–150
(chapter 14).
Li, W., Liu, H., Xu, Q., 2012. Extracellular dextran and DNA affect the formation of
Enterococcus faecalis biofilms and their susceptibility to 2% chlorhexidine. J. Endod.
38, 894–898.
Li, Y.-Q., Han, Q., Feng, J.-L., Tian, W.-L., Mo, H.-Z., 2014. Antibacterial characteristics
and mechanisms of ϵ-poly-lysine against Escherichia coli and Staphylococcus aureus.
Food Contr. 43, 22–27.
Lin, L., Gu, Y., Li, C., Vittayapadung, S., Cui, H., 2018. Antibacterial mechanism of ε
-Poly-lysine against Listeria monocytogenes and its application on cheese. Food Contr.
91, 76–84.
Ma, Z., Bumunang, E.W., Stanford, K., Bie, X., Niu, Y.D., McAllister, T.A., 2019. Biofilm
formation by Shiga Toxin-Producing Escherichia coli on stainless steel coupons as
affected by temperature and incubation time. Microorganisms 7 (95), 1–11.
Ma, Z., Stanford, K., Bie, X.M., Niu, Y.D., McAllister, T.A., 2020. Effects of beef juice on
biofilm formation by Shiga toxin–producing Escherichia coli on stainlesss steel.
Foodborne Path.and Dis 17, 235–242.
Morgan, A.D., Bonsall, M.B., Buckling, A., 2010. Impact of bacterial mutation rate on
coevolutionary dynamics between bacteria and phages. Evolution: International
Journal of Organic Evolution 64, 2980–2987.
Food Microbiology 92 (2020) 103572
Pang, X., Yuk, H.G., 2019. Effects of the colonization sequence of Listeria monocytogenes
and Pseudomonas fluorescens on survival of biofilm cells under food-related stresses
and transfer to salmon. Food Microbiol. 82, 142–150.
Sadekuzzaman, M., Yang, S., Mizan, M.F.R., Ha, S.D., 2017a. Reduction of Escherichia coli
O157:H7 in biofilms using bacteriophage BPECO 19. J. Food Sci. 82, 1433–1442.
Sadekuzzaman, M., Yang, S., Mizan, M.F.R., Kim, H.S., Ha, S.D., 2017b. Effectiveness of a
phage cocktail as a biocontrol agent against L. monocytogenes biofilms. Food Contr.
78, 256–263.
Sharma, M, Ryu, J.-H, Beuchat, L.R., 2005. Inactivation of Escherichia coli O157:H7 in
biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage.
J. Applied Microbiol. 99, 449–459. https://doi.org/10.1111/j.1365-2672.2005.
02659.x.
Silagyi, K., Kim, S.H., Lo, Y.M., Wei, C.I., 2009. Production of biofilm and quorum sensing
by Escherichia coli O157:H7 and its transfer from contact surfaces to meat, poultry,
ready-to-eat deli, and produce products. Food Microbiol. 26, 514–519.
Singh, P., Liu, Y., Bosilevac, J.M., Mustapha, A., 2019. Detection of Shiga toxin-producing
Escherichia coli, stx1, stx2 and Salmonella by two high resolution melt curve multiplex
real-time PCR. Food Contr. 96, 251–259.
Stopforth, J., Samelis, J., Sofos, J., Kendall, P., Smith, G., 2002. Biofilm formation by acid-
adapted and nonadapted Listeria monocytogenes in fresh beef decontamination
washings and its subsequent inactivation with sanitizers. J. Food Protect. 65,
1717–1727.
Tait, K., Skillman, L.C., Sutherland, I.W., 2002. The efficacy of bacteriophage as a method
of biofilm eradication. Biofouling 18, 305–311.
Tarr, P.I., Gordon, C.A., Chandler, W.L., 2005. Shiga-toxin-producing Escherichia coli and
haemolytic uraemic syndrome. Lancet 365, 1073–1086.
Vidakovic, L., Singh, P.K., Hartmann, R., Nadell, C.D., Drescher, K., 2018. Dynamic
biofilm architecture confers individual and collective mechanisms of viral protection.
Nat Microbiol 3, 26–31.
Wang, R., Bono, J.L., Kalchayanand, N., Shackelford, S., Harhay, D.M., 2012. Biofilm
formation by Shiga toxin–producing Escherichia coli O157: H7 and Non-O157 strains
and their tolerance to sanitizers commonly used in the food processing environment.
J. Food Protect. 75, 1418–1428.
Wang, R., Luedtke, B.E., Bosilevac, J.M., Schmidt, J.W., Kalchayanand, N., Arthur, T.M.,
2016b. Escherichia coli O157:H7 strains isolated from high-event period beef con-
tamination have strong biofilm-forming ability and low sanitizer susceptibility, which
are associated with high pO157 plasmid copy number. J. Food Protect. 79,
1875–1883.
Wang, C., Nie, T., Lin, F., Connerton, I.F., Lu, Z., Zhou, S., Hang, H., 2019. Resistance
mechanisms adopted by a Salmonella Typhimurium mutant against bacteriophage.
Virus Res. 273, 197759.
Wang, J., Stanford, K., McAllister, T.A., Johnson, R.P., Chen, J., Hou, H., Zhang, G., Niu,
Y.D., 2016a. Biofilm formation, virulence gene profiles, and antimicrobial resistance
of nine serogroups of Non-O157 Shiga Toxin-Producing Escherichia coli. Foodb.
Pathog. Dis. 13, 316–324.
Wang, H., Zhang, X., Zhang, Q., Ye, K., Xu, X., Zhou, G., 2015. Comparison of microbial
transfer rates from Salmonella spp. biofilm growth on stainless steel to selected pro-
cessed and raw meat. Food Contr. 50, 574–580.
Watson, B.N., Vercoe, R.B., Salmond, G.P., Westra, E.R., Staals, R.H., Fineran, P.C., 2019.
Type IF CRISPR-Cas resistance against virulent phages results in abortive infection
and provides population-level immunity. Nat. Commun. 10, 1–8.
Wommack, K.E., Williamson, K.E., Helton, R.R., Bench, S.R., Winget, D.M., 2009.
Methods for the isolation of viruses from environmental samples. In: Clokie, M.R.J.,
Kropinski, A.M. (Eds.), Bacteriophages: Methods and Protocols, Volume 1: Isolation,
Characterization, and Interactions. 1. Humana Press, New Jersey, pp. 3–14. https://
doi.org/10.1007/978-1-60327-164-6.
Yang, X., He, A., Badoni, M., Tran, F., Wang, H., 2017. Mapping sources of contamination
of Escherichia coli on beef in the fabrication facility of a commercial beef packing
plant. Food Contr. 75, 153–159.
Yin, B., Zhu, L., Zhang, Y., Dong, P., Mao, Y., Liang, R., Niu, L., Luo, X., 2018. The
characterization of biofilm formation and detection of biofilm-related genes in
Salmonella isolated from beef processing plants. Foodb. Pathog. Dis. 15, 660–667.
Zhang, Q.Q., Zhang, Y.H., Cai, F.Y., Liu, X.L., Chen, X.H., Jiang, M., 2019. Comparative
antibacterial and antibiofilm activities of garlic extracts, nisin, ε‐polylysine, and citric
acid on Bacillus subtilis. J. Food Process. Preserv. 43, e14179.