Food Microbiology 102 (2022) 103902

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

Biofilm formation by Non-O157 Shiga toxin-producing Escherichia coli in

monocultures and co-cultures with meat processing surface bacteria
Yuan Fang, Jeyachchandran Visvalingam 1, Peipei Zhang, Xianqin Yang *
Agriculture and Agri-Food Canada Lacombe Research and Development Centre, 6000 C & E Trail, Lacombe, Alberta, T4L 1W1, Canada

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

Keywords: This study investigated the impact of meat processing surface bacteria (MPB) on biofilm formation by non-O157
Non-O157 STEC Shiga toxin-producing Escherichia coli (STEC), and potential links between biofilm formation by STEC and
Co-culture biofilms biofilm-related genes in their genomes. Biofilm development by 50 MPB and 6 STEC strains in mono- and co-
Genes related to biofilm formation
cultures was assessed by the crystal violet staining method, and their expression of curli and cellulose was
Meat processing surface bacteria
determined using the Congo red agar method. Genes (n = 141) associated with biofilm formation in the STEC
strains were profiled. Biofilm formation in general correlated with cellulose and curli expression in both mono-
and co-cultures. Most MPB strains had antagonistic effects on the biofilm formation of the STEC strains. Of the
genes investigated, 81% were common among the STEC strains and there seems to be a gene-redundancy in
biofilm formation. The inability of the O26 strain to form biofilms could be due to mutations in the rpoS gene.
Truncation in the mlrA gene in the O145 strain seems not affecting its biofilm formation alone or with MPB. The
O45 strain, despite having the greatest number of biofilm-related genes, did not form measurable biofilms.
Overall, biofilm formation of STEC was affected by curli-cellulose expression and companion strains.

1. Introduction meat can occur by contacting processing equipment surfaces, such as

Shiga toxin-producing Escherichia coli (STEC) are major foodborne
pathogens, causing sporadic and outbreak cases of illness (Crim et al.,
2015). Hemolytic uremic syndrome can be developed in 5–10% cases of
STEC infections (Bielaszewska et al., 2007). E. coli O157:H7/NM and six
additional serotypes of E. coli, known as the “Big Six”, including O26,
O45, O103, O111, O121, and O145, are the most common serotypes
associated with human illnesses in North America (Crim et al., 2015).
Illnesses due to non-O157 STEC strains are increasing worldwide
(Hadler et al., 2011). Estimates from the US Center for Disease Control
and Prevention (CDC) indicated that illness cases caused by non-O157
STEC strains as a group outnumber the cases due to O157 (Hadler
et al., 2011).
STEC, in general, colonize the colon of the gastrointestinal tract of
cattle, but do not cause symptoms in cattle, and as such are found in
feces and on hides of animals presented for slaughter (Barkocy-gallagher
et al., 2003). Transmission of bacteria from hides to skinned carcasses is
inevitable during the dressing process (Bell, 1997). Some bacteria may
also persist in/on meat processing equipment, where recontamination of
conveyor belts (Wang et al., 2018; Yang et al., 2017a, 2017b). Biofilm
formation may play a role in the recurring contamination of meat with
E. coli O157:H7, leading to “high event period”, a time period when beef
processing establishments experience an increased occurrence of prod­
uct contamination by E. coli O157:H7 (Wang et al., 2014). Biofilm is a
structured bacterial community of single or multiple species that adhere
to biotic or abiotic surfaces with self-produced extracellular polymeric
substances (EPS) (Costerton et al., 1999). Floors, walls, and drains in
food processing facilities with routine sanitation have been reported to
be prone to biofilm formation (Sinde and Carballo, 2000). Biofilms, in
general, are more resistant than their planktonic counterparts to bio­
cides commonly used for cleaning and sanitizating, making the elimi­
nation of biofilms in such environment challenging (Wang et al., 2012,
2013).
Bacteria develop biofilms in a complex and coordinated manner that
involves sensing and responding to changes in, including but not limited
to, the cell density, nutrient, accompanying species/strains, and envi­
ronmental stress (Dourou et al., 2011; Uhlich et al., 2014). These tran­
sitions are coordinated by multiple genetic pathways, including flagella

* Corresponding author.
E-mail address: xianqin.yang@canada.ca (X. Yang).
1
Current address: Kane Biotech Inc, 290-100 Innovation Drive, Winnipeg, Manitoba, R3T 2N2, Canada.

https://doi.org/10.1016/j.fm.2021.103902
Received 17 March 2021; Received in revised form 10 August 2021; Accepted 10 September 2021
Available online 14 September 2021
0740-0020/Crown Copyright © 2021 Published by Elsevier Ltd. All rights reserved.
Y. Fang et al. Food Microbiology 102 (2022) 103902

motility, cell-cell signaling, bio-surfactant secretion, cellulose, and curli
biosynthesis (Chapman et al., 2002; Serra et al., 2013a, 2013b). As
important structural elements of biofilms, cellulose provides cohesion
and elasticity that together with cell network encased by curli supports
the formation of high ridges and radial wrinkles (Serra et al., 2013a).
Biofilm formation by STEC and the abundance of genes associated with
biofilms are highly strain-dependent (Biscola et al., 2011; Wang et al.,
2012). Moreover, in real-world scenarios, biofilms may be inhabited by
multiple strains/species where spatial and metabolic interactions
contribute to biofilm properties (Chorianopoulos et al., 2010; Habimana
et al., 2010). When STEC O157:H7 and Salmonella enterica were
co-cultured with microorganisms from meat processing plants, the bio­
film formation was substantially affected by the environmental bacteria,
some of which favored the non-biofilm producer STEC O157:H7 to form
biofilms, and may enhance their persistence in meat processing plants
(Visvalingam et al., 2019a). Information on the effect of bacteria from
meat processing surface on the biofilm development by non-O157 STEC
and the genetic determinants of their biofilm formation is scarce.
Therefore, the objectives of this study were to i) assess the
biofilm-forming ability of the “Big Six” non-O157 STEC in single-species
cultures and co-cultures with bacteria isolated from meat processing
surface, and ii) elucidate the genetic differences associated with the
biofilm formation in these non-O157 STEC strains.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Six non-O157 STEC strains recovered from beef carcasses were
included, and these strains were kindly provided by the Meat Animal
Research Center of the US Department of Agriculture (USDA) (Table 1).
STEC O26:H11, O45:H2, O103:H2, O111:H8, O121:H19 and O145:H28
were referred to as O26, O45, O103, O111, O121, and O145 in this
study, respectively. Meat processing surface bacteria (MPB) included 18
Gram-negative aerobic bacteria (GNA), 8 Gram-positive aerobic bacteria
(GPA), 5 lactic acid bacteria (LAB), 9 Enterobacteriaceae (ENT) (Wang
et al., 2018), and 10 generic E. coli (GEC) (Tables 1 and 2) (Yang et al.,
2015, 2017a, 2017b). The GEC strains were genotyped by multiple-locus
variable-number tandem-repeat analysis and defined as non-persistent
or persistent if they were recovered only on a single sampling or more
than three sampling visits from the same facility, respectively (Table 1)
(Yang et al., 2015).
All bacteria were cultivated in Lennox broth without salt (LB-NS)
(Oxoid Canada Inc., Mississauga, ON, Canada) except for
A. haemolyticus, F. columnare, P. ginsengisoli, and A. urinaeequi, which did
not grow well in LB-NS and thus were grown in half-strength Brain Heart
Infusion (BHI) broth (Oxoid). Stationary-phase cultures were obtained
by incubation at 25 ◦ C with shaking at 80 rpm for 24–120 h (Visva­
lingam et al., 2019b).
2.2. Biofilm development and quantification
To prepare monoculture inoculum, bacterial cultures were diluted
using LB-NS to obtain a cell density of ca. 107 CFU/mL. For bacteria
grown in half-strength BHI broth, 1 mL of culture was centrifuged at
10,000×g for 10 min at 4 ◦ C, and then, the cell pellets were suspended in
1 mL of LB-NS and further diluted using LB-NS. The co-culture inocula
were prepared by mixing two bacterial suspensions in equal volumes.
Biofilms were developed using 96-well round-bottom microtiter
plates (Nunc Immuno TSP lid; Fisher) fitted with pegged lids (Nunc;
Fisher) and quantified using the crystal violet (CV) dye method (Vis­
valingam et al., 2019b). Briefly, 160 μL of bacterial culture was added
into the wells of a 96-well plate in duplicate. LB-NS served as blank.
Inoculated plates were fitted with pegged lids and incubated at 15 ◦ C for
up to 6 days. Biofilms attached to the pegs were quantified by their
ability to take up CV on day 2 and 6. The pegs were washed twice using
200 μL/well of phosphate-buffered saline (PBS, Hardy Diagnostics,
Santa Maria, CA), followed by staining with 180 μL of 0.1% (w/vol) CV
solution for 20 min. Then, the pegs were washed three times with PBS
and immersed into 180 μL/well of 80% ethanol for 20 min to dissolve
the CV absorbed by the biofilms on the pegs. The absorbance of the
resulting solution in each well was measured at 570 nm using a micro­
plate reader (Polarstar Omega, BMG LABTECH GmbH, Ortenberg, Ger­
many). When the absorbance value of a sample at 570 nm (A570) was
greater than 4, the sample was further diluted by 2-fold in 80% ethanol,
and the absorbance of which was determined. The results were calcu­
lated as mean ± standard deviation (SD) of two independent replicates,
with each containing duplicate samples (n = 4).
Biofilm-forming ability of tested bacterial strains was classified to
different levels based on the method described previously, with modi­
fications (Stepanovic et al., 2007). Briefly, the A570 of three times the
standard deviation of the blank was used as the cut-off value (ODc),
which was 0.05 in this study. The A570 ranged from − 0.05 ± 0.03 to
12.17 ± 0.45. Based on the calculation of 2n × ODc, ,the strains were
regarded as non-biofilm former (A570 ≤ 0.05), weak biofilm former
(0.05 < A570 ≤ 0.8), moderate biofilm former (0.8 < A570 ≤ 1.6), strong
biofilm former (1.6 < A570 ≤ 3.2), very strong biofilm former (3.2 <
A570 ≤ 6.4), and extremely strong biofilm former (6.4 < A570 ≤ 12.8).

Table 1 2.3. Expression of cellulose/cellulose-like, and curli/curli-like substances

E. coli strains used in this study.
Group Strain ID Serotype Original strain Origin of strains
Expression of the extracellular polymeric substances cellulose and
used in this ID/genotype thin aggregative fimbriae (curli) by MPB and STEC was evaluated using
study the Congo red indicator (CRI) method (Visvalingam et al., 2017; Wang
Non-O157 O26 O26:H11 Imp 133.1 USDA et al., 2013). Overnight cultures were streaked on CRI plates, followed
STEC O45 O45:H2 F-A 222.5 by incubation at 15 ◦ C for up to 4 days. After incubation, strains were
O103 O103:H2 Mar 125B determined as positive for both cellulose and curli, for curli, for cellu­
O111 O111:H8 C4-462-2_7095
lose, or neither if the colonies on CRI plates were red, brown, pink, or
O121 O121:H19 V2-G2 1-C 16.3
O145 O145:H28 May 109
white, respectively (Uhlich et al., 2014; Zogaj et al., 2001). As the
method has been primarily validated for members of Enterobacteriaceae,
Persistent B0 136 (Yang et al.,
cellulose/cellulose like and curli/curli-like substances were used for
E. coli B1 390 2015, 2017a,
B2 Generic 179 2017b) MPB.
E. coli
B3 541 2.4. Whole genome sequencing and genome assembly of non-O157 STEC
B4 138
strains
Non- B5 518
persistent B6 533
E. coli B7 Generic 545 The six STEC strains were sequenced to explore the potential genes
E. coli contributing to their distinct biofilm forming abilities. Briefly, DNA was
B8 115 extracted from the overnight culture of each STEC strain using a Mas­
B9 235
terPure™ Complete DNA and RNA purification kit (Lucigen, Middleton,

2
Y. Fang et al. Food Microbiology 102 (2022) 103902

Table 2
Bacterial strains isolated from beef processing equipment surfaces and their production of curli/curli-like substances and cellulose/cellulose like substances.
Group Strain ID used in this study Genus/species Original strain ID Curli/like substances Cellulose/like substances Origin of strains

GNA B11 Acidovorax wohlfahrtii 03–11 – + Wang et al. (2018)

B12 Acinetobacter haemolyticus 03–04 – –
B13 Aeromonas sp. 03–09 + –
B14 Brevundimonas staleyi 25–3 ND
B15 Chryseobacterium shigense 25–54 ND
B16 Comamonas koreensis 25–64 – +
B17 Flavobacterium columnare 09–7 – –
B18 Janthinobacterium lividum 09–39 – +
B19 Massilia aurea 03–22 ND
B20 Moraxella osloensis 25–60 – –
B21 Paracoccus aminophilus 09–12 – +
B22 Pedobacter ginsengisoli 03–52 – –
B23 Pseudomonas poae 09–41 + –
B24 Psychrobacter sp. 03–65 – +
B25 Sphingomonas 03–14 ND
B26 Sphingopyxis sp. 03–68 ND
B27 Stenotrophomonas maltophilia 25–39 + –
B28 Acinetobacter lwoffii 09–3 – +

GPA B29 Microbacterium sp. 03–50 – –

B30 Sanguibacter insulinus 25–11 – –
B31 Luteococcus japonicus 03–33 + –
B32 Macrococcus caseolyticus 25–15 + +
B33 Microbacterium phyllosphaerae 09–58 ND
B34 Microbacterium phyllosphaerae 03–25 ND
B35 Plantibacter sp. 09–65 ND
B36 Pseudoclavibacter helvolus 25–1 ND

LAB B37 Aerococcus urinaeequi LAB25-13 – –

B38 Carnobacterium maltaromaticum LAB9-67 – –
B39 Enterococcus sp. LAB3-8 – –
B40 Streptococcus parauberis LAB25-2 ND
B41 Vagococcus fluvialis LAB25-11 ND

ENT B42 Buttiauxella agrestis ENT25-12 – +

B43 Citrobacter gillenii ENT09-29 + +
B44 Enterobacter sp. ENT03-16 – +
B45 Hafnia alvei ENT03-7 – +
B46 Buttiauxella noackiae ENT09-55 – +
B47 Raoultella sp. ENT25-16 + –
B48 Serratia sp. ENT25-20 – +
B49 Yersinia sp. ENT09-26 – +
B50 Salmonella sp. ENT25-1 – +

ND: not determined. No conclusion was made due to the color-reduction of CRI plates by bacteria.

WI, US). The sequencing library was prepared using a NEBNext® Ultra™ for each isolate/genome is listed in Table S1.
II DNA Library Prep Kit, and subsequently sequenced on an Illumina
HiSeq4000 instrument with sequencing depth >300 for 150 × 2 cycles
2.5. Analysis of genes associated with biofilm formation in the STEC
(Genome Quebec, QC, CA). The quality of raw sequencing reads was
genomes
examined using FastQC v0.11.8. The contamination of Phix sequences in
the reads was removed using Bowtie v2.3.4.3 (Langmead and Salzberg,
Genes encoding proteins related to biofilm formation in E. coli were
2012). Trimmomatic 0.39 was used to remove adapter sequences and
collected from GenBank and made into a local database, including genes
sequences with an average quality score <20 or length <100 bases
involved in the biosynthesis of colonic acid, curli, cellulose, β-1,6-N-
(Bolger et al., 2014). Each genome was assembled using SPAdes v3.14.0
acetylglucosamine (PGA), adhesion factors, and quorum sensing
with the size of kmers set at 21, 33, 55, 77, 99, and 127 (Bankevich et al.,
(Table S2). Annotated STEC genomes were searched against the local
2012). The quality of the assembled genomes was assessed using Quast
database using BLASTp (2.6.0+) to identify the genes in each genome,
v5.0.2 (Gurevich et al., 2013). Contigs with length <500 bp or coverage
with >70% identity and >70% coverage of the reference amino acid
<10 were removed (Douglass et al., 2019). The remaining contigs in
sequences (Joshi and Xu, 2007).
each genome were ordered using Mauve (Darling et al., 2004) by
mapping against respective serotypes of STEC, the complete genomes of
which are available in the database of the National Center for Biotech­ 2.6. Data analysis
nology Information (NCBI). The reference strains were E. coli O26:H11
(GenBank assembly accession no: GCA_000091005.1) (Smith et al., The A570 values for the same strain at different incubation times, or a
2017), O45:H2 (GCA_005037845.1), O103:H2 (GCA_000010745.1), MPB strain in mono-cultures and in combination with different STEC
O111:H8 (GCA_003288295.1) (Sekizuka et al., 2019), O121:H19 strains at the same incubation time were analyzed by one-way analysis
(GCA_002844165.1) (Robertson et al., 2018), and O145:H28 of variance (ANOVA). Statistical differences among bacterial combina­
(GCA_000520035.1). The open reading frames (ORFs) in each genome tions and between incubation times were determined by Tukey’s test
were searched using Prodigal (v2.6.3) with default settings (Hyatt et al., using SPSS software, version 21.0 (SPSS Inc., Chicago, IL, USA). To
2010). The raw sequencing reads and draft genomes were submitted to determine the correlation between extracellular property and biofilm
GenBank under the BioProject PRJNA706412, and the accession number forming ability, the A570 values of groups of strains that associated with
the different numbers of curli/curli-like substances and/or cellulose/

3
Y. Fang et al.
cellulose-like substances were pairwise compared using Kruskal-Wallis
test. A significance level of P < 0.05 was used for all statistical analysis.
The interactions between two strains on biofilm formation were
regarded as synergistic, no effect or antagonistic if the A570 value of co-
culture is larger than, equals to, or smaller than the higher A570 value of
the two relevant monocultures (Ren et al., 2015).
3. Results
3.1. Biofilm formation by monocultures of STEC and their co-cultures
with GPA
The biofilm-forming ability of STEC strains in monocultures varied
from non biofilm-forming (O45) to extremely strong biofilm-forming
(O103 and O145), with the latter two strains having A570 values > 8.0
on day 6 (Table 3). The five biofilm forming strains all had significantly
higher (P < 0.05) A570 values on day 6 than on day 2.
Generally, GPA produced no biofilms (A570<0.05) on day 2 and weak
biofilms (A570<0.8) on day 6, except for B32 (M. caseolyticus), which
produced strong biofilms on day 6 (Fig. 1). On day 2, the A570 values for
the co-cultures of B32 and the STEC strains were higher than the A570
values for the co-cultures of other GPA and STEC strains. Of the 48 co-
cultures on day 6, 24 developed extremely strong biofilms (A570 >
6.4), 62.5% of which involved either O103 or O145. B33
(M. phyllosphaerae) and O45, both of which were non-biofilm formers on
their own, formed extremely strong biofilms in their co-cultures on day
6. On the other hand, the A570 values for the day 6 co-culture biofilms of
O111 or O121 with B30 (S. insulinus) or B36 (P. helvolus), a non-biofilm
former on its own, were much lower than the A570 values for their
respective STEC mono-cultures, which were both in the strong biofilm
category. Compared to the O103 and O145, O111 and O121 were more
negatively impacted by the companion GPA strains.
3.2. Biofilm formation by co-cultures of STEC and GNA
Different from GPA, a higher fraction of GNA developed measurable
biofilms on both day 2 and day 6. Unlike the uniformity of co-cultures of
O145 or O103 with GPA in their strong biofilm formation, their
respective co-cultures with GNA show a much higher degree of variation
in this regard, with A570 ranging from much lower to much higher than
that of the respective monocultures (Fig. 2). Even so, as with the GPA-
STEC co-culture biofilms, the co-culture biofilms by O103 or O145
with GNA were mostly stronger than the co-culture biofilms by the other
STEC strains. Among the GNA, B18 (J. lividum) and B11 (A. wohlfahrtii)
developed non- or weak mono-culture and STEC-co-culture biofilms
even with those strong biofilm forming STEC strains.
3.3. Biofilm formation by co-cultures of STEC and LAB or ENT
Despite not being able to form detectable biofilms in monocultures
on both day 2 and day 6, the co-cultures of the LAB strains (B37-41)
Table 3
Biofilm formation by non-O157 Shiga toxin-producing E. coli strains.
Serotype Curli Cellulose Biofilm mass (A570)a
Day 2 Day 6
O26 – + 0.04 ± 0.02 1.5 ± 0.04C*
O45 0 0D
+ –
O103 + + 0.06 ± 0.01 8.22 ± 1.09A*
O111 + + 0.01 ± 0 4.49 ± 0.8B*
O121 + + 0.03 ± 0.01 5.09 ± 0.29B*
O145 + + 0.6 ± 0.06 8.62 ± 0.49A*
a
Means in the same column that do not share a common superscript are
significantly different (P < 0.05). Asterisk indicates significant differences be­
tween means in the same row (P < 0.05).
4
Food Microbiology 102 (2022) 103902
showed STEC strain dependent biofilm formation on day 6 (Fig. 3). The
pattern of biofilm formation of co-cultures of O145, O103, O26 and O45
with LAB was similar to that of the respective STEC monocultures.
However, the co-cultures of O111 or O121 with various LAB strains all
had significantly lower A570 values except for the combination of B41
(V. fluvialis) and O111, compared to their monoculture counterparts. In
contrast to LAB, the biofilm production of STEC-ENT co-cultures was
consistent with the biofilm-forming ability of the ENT strains, in a STEC
strain independent manner (Fig. 3). This trend in that more co-culture
biofilms of O111 and O121 were in the weak category than those of
O145 and O103 was consistent with their respective co-cultures with
GPA and GNA.
3.4. Biofilm formation by co-cultures of STEC and persistent or non-
persistent E. coli
Different from co-cultures of STEC with GPA, LAB or ENT, 30% (two
persistent and one non-persistent strains) of GEC and STEC formed very/
extremely strong co-culture biofilms on day 2, irrespective of the STEC
strains (Fig. 4). Co-culture GEC and STEC O103 and O145 were uniform
on forming very/extremely strong biofilms; however, the biofilm pro­
duction of the co-cultures including other serotypes of STEC exhibited a
high degree of variation depending on the strains of GEC involved,
particularly for non-persistent GEC. Biofilms of persistent GEC (B0-4) in
monocultures and co-cultures with STEC were very/extremely strong in
all cases. However, biofilm formation of non-persistent GEC (B5-9)
varied by non-persistent GEC and STEC. Some non-persistent GEC (B6
and B7) developed very/extremely strong biofilms in monocultures and
together with STEC. Two non-persistent GEC (B5 and B8) did not form
measurable biofilms by themselves or when co-cultures with O111 or
O121 after 6 days, despite that O111 and O121 both formed strong
biofilms at equivalent times.
3.5. Interactions between STEC and MPB on biofilm formation
The interactions between STEC and MPB in biofilm formation were
largely antagonistic, as was observed with 78%, 90%, 81%, and 77% of
the GNA, LAB, ENT, and GEC strains, respectively (Fig. 5). The greatest
antagonistic effects were observed in members of GNA and ENT, in
particular B18 (J. lividum) and B19 (M. aurea), as well as B42 (B. agrestis)
and B44 (Enterbacter sp.), for which no synergistic effects were observed
in any of the combinations with STEC. The greatest synergistic effects
were found in the co-cultures with GPA strains, namely B33
(M. phyllosphaerae) and B34 (M. phyllosphaerae), with differences in the
A570 values being >10.
3.6. Cellulose and curli expression by non-O157 STEC and MPB strains
E. coli O145, O103, O121, and O111 expressed both curli and cel­
lulose, while O26 expressed only cellulose and O45 exhibited only curli
expression. Of the 40 MPB strains, 11 had pigmentation and as such, CRI
could not be used to determine curli or cellulose production. For the
remaining MPB strains, curli (curli-like substance) and/or cellulose
(cellulose-like substance) production was found in all ENT strains, 9/13
of GNA, 2/4 of GPA, and none of LAB, respectively. Of ENT, B43
(C. gillenii) and B47 (Raoultella sp.) were positive for both curli and
cellulose, and curli, respectively. All the other strains expressed only
cellulose. The extent of biofilm formation by mono- and co-cultures, as
determined by A570 values, had a strong correlation (P < 0.05) with
these two types of extracellular substance production (Fig. 6).
3.7. Genome analysis of non-O157 STEC strains
Of the 141 genes that are potentially associated with biofilm for­
mation in E. coli, 114 were found in all six STEC strains including the
clusters bcs, csg, and wca encoding proteins involved in cellulose, curli,
Y. Fang et al. Food Microbiology 102 (2022) 103902

Fig. 1. Biofilm formation by non-O157 STEC strains in co-cultures with Gram-positive aerobic (GPA) bacteria recovered from beef processing equipment surfaces.
The biofilm formation was assessed by CV staining after incubation at 15 ◦ C for 2 and 6 days. Asterisks indicate significant differences in A570 values between the two
incubation days for the same strains. Significant differences among the GPA in monoculture and in combination with different STEC strains are labeled with different
capital letters (P < 0.05). The horizon dash lines are set at A570 at 1.6 (black) and 6.4 (red), representing the cut-off values for strong and extremely strong biofilms,
respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

and colonic acid production, as well as genes encoding for a large
number of known function or putative adhesins and fimbriae (Table S2).
The tibA coding for an adhesin/invasion, commonly found in entero­
toxigenic E. coli (Sherlock et al., 2005), was the only one that was not
found in any of the strains. The 26 genes, which were differentially
abundant in the six STEC genomes, distributed differently, even between
the strains with similar biofilm forming phenotypes (Fig. 7). No unique
genes were found specific to O45, the non-biofilm former, and in fact,
O45 had the least number of biofilm related genes missing in the genome
(5/141).
Some components of the regulatory and functional genes related to
cellulose, c-di-GMP and N-acetyl-D-glucosamine (PGA) biosynthesis
were missing in some strains. The pgaABCD operon for PGA was present
in the genomes of E. coli O45, O103, and O145 but missing in O26,
O111, and O121. The yhjR part of the bcs operon was missing only in
E. coli O145. As part of the synthesis of signalling molecule c-di-GMP,
dgcT encoding for diguanylate cyclase was only present in the genomes
of E. coli O103 and E. coli O45, whereas dosCP encoding for phospho­
diesterase was only absent in E. coli O103. However, additional 9 genes
encoding for diguanylate cyclase were found in all six STEC genomes
(Table S2).
Fimbriae encoding genes were more heterogeneous and diverse than
genes coding for cellulose, curli, PGA, and quorum sensing (Fig. 7;
Table S2). Genes coding for flagella filament (flhDC and fliC), cell
motility (motAB and cheAC), and related genes were all present in the
STEC strains. Genes related to cell-cell communication were present in
all STEC genomes in most cases. lsrG encoding a cytoplasmic kinase
responsible for phosphorylation of AI-2 into an active molecule
following its import into the cell was absent in O26. The gene encoding
the autotransporter Ag43, which mediates cell-cell aggregation and in-
turn leads to cell-aggregates, was not found in O111 or O121.
The two extremely strong biofilm formers, O103 and O145, each had
genes for the biosynthesis of surfactin and colicin (Fig. 7). The O145
strain had a truncated MlrA (Fig. S1) and the O26 strain differed by 40
amino acids in its RpoS from the wild type strain ATCC 43895 and the
other STEC strains (Fig. S2).
4. Discussion
Biofilm formation is an essential strategy for bacteria to survive
under adverse conditions, including food-processing environments
(Burmølle et al., 2006; Habimana et al., 2010; Wang et al., 2013). Bio­
films developed by mixed-species rather than single species represent
the most frequent form of microbiological contamination and persis­
tence in food industries (Yuan et al., 2020). Observations made on
monoculture biofilms might not reflect the mixed-species biofilm for­
mation as interspecies interactions may alter the behavior of the indi­
vidual bacterial strains in biofilm formation (Burmølle et al., 2014). This
observation was confirmed in this study, which shows that biofilm for­
mation of non-O157 STEC was differentially affected by bacterial strains
isolated from beef processing equipment surfaces. Importantly, this
study provides pertinent information on the biofilm formation in meat
processing environment by evaluating biofilm formation of 50 bacterial
strains isolated from beef processing plants, which was considered as a
representative composition of microbiome of meat processing environ­
ment (Wang et al., 2018). The findings that biofilm formation of STEC
strains was negatively or positively influenced by companion strains
highlight the importance, from a food safety standpoint, of addressing
biofilms under conditions simulating their natural ecological niches.
Production of cellulose and curli determines the morphology and
biofilm strength in Enterobacteriaceae (Zogaj et al., 2001). Genome
analysis has demonstrated that functional amyloid systems are

5
Y. Fang et al. Food Microbiology 102 (2022) 103902

Fig. 2. Biofilm formation by non-O157 STEC strains in co-cultures with Gram-negative aerobic bacteria (GNA) recovered from beef processing equipment surface.
The biofilm formation was assessed by CV staining after incubation for 2 and 6 days. Asterisks indicate significant differences in A570 values between the two in­
cubation days for the same strains. Significant differences among the GNA in a single culture and combination with different STEC are labeled with different su­
perscript (P < 0.05). The horizon dash lines are set at A570 at 1.6 (black) and 6.4 (red), representing the cut-off values for strong and extremely strong biofilms,
respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

wide-spread in bacteria (Dueholm et al., 2012), and that amyloids are
abundant in natural biofilms (Larsen et al., 2007). Thus, this study used
the Congo red assay to determine the extracellular substance production
in non-Enterobacteriaceae species as well as in Enterobacteriaceae. The
ability to uptake Congo red by MPB and STEC had a positive correlation
with the degree of their biofilm formation in mono- and co-cultures, as
determined by A570. This suggests that CRI can be a useful method for
screening bacterial strains for biofilm forming potentials.
Despite the wide-spread of genes encoding functional amyloids in
bacterial genomes (Biscola et al., 2011; Dueholm et al., 2012), the
production of curli and cellulose varies with species and even strains, in
the case of E. coli (Visvalingam et al., 2017; Wang et al., 2012). The
strain-dependent curli and cellulose production was also noted in this
study (O26, curli-negative; O45, cellulose-negative; the other four
strains positive for both curli and cellulose), even though both O45 and
O26 harbored essential genes for the biosynthesis of curli and cellulose.
CsgD is a transcriptional regulator, which positively regulates the
expression of the structural genes (csgBC) and the export of proteins for
assembling curli (Brombacher et al., 2003). The diguanylate cyclase
ArdA indirectly activates cellulose production (Zogaj et al., 2001). The
transcription of CsgD is controlled by the alternative Sigma factor RpoS
and MlrA, the latter of which binds to the former to enhance the
expression of CsgD; and mutations in RpoS and insertions in MlrA by the
Stx1-encoding prophage have been attributed to the limited curli
expression and subsequent biofilm formation in some E. coli O157
strains (Uhlich et al., 2013). The inability of the O26 strain to produce
curli in this study may be attributable to the multiple mutations in the
RpoS in O26. Interestingly, a truncated MlrA did not seem to affect the
curli or cellulose production, or biofilm formation by O145. AdrA is
responsible for the synthesis of the secondary signaling molecule
c-di-GMP, which activates cellulose biosynthesis (bcsABEC and bcsEFG)
(Povolotsky and Hengge, 2015; Simm et al., 2004). The homeostasis of
c-di-GMP is controlled by the antagonistic activities between diguany­
late cyclase with GGDEF domains and the phosphodiesterase with EAL
domains (Simm et al., 2004). However, E. coli encodes multiple proteins
containing GGDEF or EAL domains, making the synthesis and signaling
network of c-di-GMP more complicated (García et al., 2004; Povolotsky
and Hengge, 2015; Weber et al., 2006). The redundancy of genes
encoding diguanylate cyclases and phosphodiesterase was also observed
in the six STEC genomes in this study and missing some of the genes
(dgcT/ycdT, dosP, dosC) in O111, O121, O103 and O145 did not impact
their curli or cellulose production or biofilm formation. In addition,
production of curli and cellulose by Enterobacteriaceae was affected by
growth conditions through the differential expression of csgD and/or
adrA (García et al., 2004), and the level of c-di-GMP (Reinders et al.,
2015). These factors may have contributed to the findings that the O45
strain did not produce cellulose on CRI despite having all the essential
genes.
In mixed-species biofilms, microorganisms interact through jamming
of quorum sensing molecules (Jahid et al., 2018) and the production of
secondary metabolites or toxins that inhibit/promote the growth of
coexisting microorganisms (Chorianopoulos et al., 2010; Habimana
et al., 2010). Biofilm formation of a non-biofilm producer E. coli O157:
H7 was enhanced by A. calcoaceticus in dual-species biofilms (Habimana
et al., 2010). In contrast, H. alvei and/or Serratia sp. inhibited the bio­
films formed by S. enterica and Listeria monocytogenes (Alavi et al., 2013;
Visvalingam et al., 2019b) and non-O157 STEC strains (this study). The
presence of unknown compounds in the culture supernatant of H. alvei
contributed to the inhibition of metabolic activity and biofilm produc­
tion by S. Typhimurium (Chorianopoulos et al., 2010). Production of
antimicrobial compounds by Serratia plymuthica is one of the factors
contributing to its inhibitory effects on the biofilm formation by E. coli
MG1655, which is regulated by the quorum-sensing molecule
N-acyl-L-homoserine lactone (AHL) (Moons et al., 2006). Such

6
Y. Fang et al. Food Microbiology 102 (2022) 103902

Fig. 3. Biofilm formation by non-O157 STEC strains in co-cultures with lactic acid bacteria (LAB; B37-41) or Enterobacteriaceae (ENT; B42-50) recovered from beef
processing equipment surface. The biofilm formation was assessed by CV staining after incubation for 2 and 6 days. Asterisks indicate significant differences in A570
values between the two incubation days for the same strains. Significant differences among the LAB and ENT in a single and combination with different STEC are
labeled with different superscript (P < 0.05).). The horizon dash lines are set at A570 at 1.6 (black) and 6.4 (red), representing the cut-off values for strong and
extremely strong biofilms, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

antagonistic effects on the biofilm formation were also found in the
dual-species biofilms of Aeromonas sp. (B13) and E. coli O103, O111,
O121 or O145, as well as of A. urinaeequi (B37) or C. maltaromaticum
(B38) and E. coli O111 or O121 in this study. E. coli do not produce AHLs,
but produce autoinducer-2 (AI-2) as quorum sensing molecules (Walters
and Sperandio, 2006). However, they can detect AHLs from other spe­
cies by a receptor-like protein (SdiA) (Lindsay and Ahmer, 2005), which
can modulate their gene expression (Van Houdt et al., 2006). Different
from AHLs, AI-2 was employed to increase the biofilm formation in
E. coli by stimulation of flagella motility via the motility quorum-sensing
regulator MqsR (González Barrios et al., 2006). Internalization of
extracellular AI-2 requires the phosphorylation by LsrK kinase, and then
the phosphorylated AI-2 is further processed by LsrG and LsrF (Xavier
and Bassler, 2005). The six STEC genomes harbored all genes for the
AI-2 system and SdiA except for E. coli O26, which did not have LsrG.
Despite this, different patterns in the biofilm formation by different
STEC strains and the same MPB were noted. In general, compared to the
A570 values for the respective strong biofilm forming STEC strains
(O145, O103, O121, and O111) in monocultures, the A570 values for the
O145 and O103 in co-cultures with MPB were less affected than those
for the MPB co-cultures with O121 and O111, where in many cases the
co-cultures behaved similarly with the non-biofilm forming MPB (B5,
B6, B11, B16, B20, B24, B30, B36–40, B44). The exact reason for this
behavior is unclear. One noted difference in the genomes was that O145
and O103 each harbored a gene encoding for surfactin and colicin, the
former of which is a biosurfactant and the latter a bacteriocin and both
of which can inhibit biofilm formation of other bacterial species (Banat
et al., 2014; Rendueles et al., 2014). In addition, O121 and O111 did not
harbor flu, a gene encoding for the autotransporter Ag43. The exact role
of Ag43 in the biofilm formation of E. coli is a matter for debate (Danese
et al., 2000; Taghadosi et al., 2017). However, Kjaergaard et al.
demonstrated that Ag43 induced intra- and inter-species cell
aggregation and enhanced the biofilm formation by Pseudomonas fluo­
rescens (Kjaergaard et al., 2000). Whether or not these factors play a role
in the different patterns of biofilm formation in the STEC-MPB co-­
cultures would warrant further investigations.
Cell to cell adhesion also plays a role in biofilm formation (Van
Houdt and Chris, 2005; Wang et al., 2004). The PGA polymers, fimbriae,
membrane auto-transporters, and adhesins mediate cell-cell or cell to
surface adhesion (Hammer and Bassler, 2003; Schembri et al., 2003;
Wang et al., 2004). Type 1 fimbriae contributed to surface colonization
and biofilm formation of E. coli K12 (Beloin et al., 2004); however, the
disruption of type 1 fimbriae did not affect the biofilm formation
significantly (Reisner et al., 2003). Homologous sequences of fim were
found in many E. coli, including yra, yfc, yad, and yeh, which promoted
biofilm formation in the absence of type 1 fimbriae (Korea et al., 2010).
On the other hand, EhaA was physically shielded by the expression of
type 1 fimbriae (Wells et al., 2008); flagella encoded by fliC were not
essential for biofilm formation when the curli was expressed (Pri­
gent-Combaret et al., 2000). The depolarization of PGA resulted in the
dispersal of biofilms of E. coli (Itoh et al., 2005). The pga operon was
present in the extremely strong biofilm formers E. coli O103 and O145
and non-biofilm former E. coli O45, but not in the other two strong
biofilm formers E. coli O111 and O121. It suggested that other adhesion
mechanisms are compensated to the PGA-dependent biofilm formation
in E. coli O111 and O121.
In conclusion, this study found that production of cellulose and curli
was positively correlated with the degree of biofilm formation in both
mono- and co-cultures of STEC and MPB. Although a plethora of genes
encoding for proteins involved in essential steps in biofilm formation
were present in all STEC genomes investigated; finesse of biofilm pro­
duction is likely on the regulatory level, rather than the presence or
absence of structural genes. Phenotypic characterization of biofilm
formation under relevant conditions is then indispensable. Some MPB,

7
Y. Fang et al. Food Microbiology 102 (2022) 103902

Fig. 4. Biofilm formation by non-O157 STEC strains in co-cultures with persistent (B0-4) and non-persistent (B5-9) generic E. coli recovered a beef plant. The biofilm
formation was assessed by CV staining after incubation for 2 and 6 days. Asterisks indicate significant differences in A570 values between the two incubation days for
the same strains. Significant differences among the generic E. coli in a single culture and combination with different STEC are labeled with different superscript (P <
0.05). The horizon dash lines are set at A570 at 1.6 (black) and 6.4 (red), representing the cut-off values for strong and extremely strong biofilms, respectively. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. Interactions between non-O157 STEC strains and MPB on the biofilm formation. The interactions were determined as synergistic, antagonistic, and neutral if
the A570(co-culture – mono-culture) is greater than, smaller than and equals to zero, respectively. Strains of B0-4, B5-9, B11–B28, B29–B36, B37–41, B42-50, are groups of
persistent and non-persistent E. coli, GNA, GPA, LAB, and ENT, respectively.

particularly some members of Gram-negative aerobic bacteria, lactic and health effects.
acid bacteria, and Enterobacteriaceae have strong antagonistic effects on
the biofilm formation by various STEC strains, which could be further
explored as biocontrol agents for biofilms, depending on their and safety

8
Y. Fang et al. Food Microbiology 102 (2022) 103902

Fig. 6. Correlation between the biofilm formation and expression of curli and cellulose in mono-cultures (Panel A) and dual-cultures (B). Phenotype for mono-
cultures is defined as 0 (no curli or cellulose), 1 (curli or cellulose), and 2 (curli and cellulose). For dual-cultures in panel B, number 1–4 was defined as the
number of positive determinants expressed by the two bacterial strains in the dual-culture.

Fig. 7. The presence (Y; light blue) or absence (N; dark blue) of genes related to biofilm formation which showed difference in distribution in the six STEC genomes.
(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Declaration of competing interest Appendix A. Supplementary data

None. Supplementary data to this article can be found online at https://doi.

org/10.1016/j.fm.2021.103902.
Acknowledgments
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