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Frontiers in Microbiology | www.frontiersin.org 1 June 2022 | Volume 13 | Article 880043
ORIGINAL RESEARCH
published: 23 June 2022
doi: 10.3389/fmicb.2022.880043
Edited by:
Laurent Dufossé,
Université de la Réunion, France
Reviewed by:
Yujie Zhang,
UnitedStates Department of
Agriculture, UnitedStates
Christian Menge,
Friedrich-Loefer-Institute, Germany
*Correspondence:
Catherine D. Carrillo
catherine.carrillo@inspection.gc.ca
†Deceased
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 20 February 2022
Accepted: 18 May 2022
Published: 23 June 2022
Citation:
McMahon TC, Kingombe CB,
Mathews A, Seyer K, Wong A,
Blais BW and Carrillo CD (2022)
Microbial Antagonism in Food-
Enrichment Culture: Inhibition of
Shiga Toxin-Producing Escherichia
coli and Shigella Species.
Front. Microbiol. 13:880043.
doi: 10.3389/fmicb.2022.880043
Microbial Antagonism in
Food-Enrichment Culture: Inhibition
of Shiga Toxin-Producing Escherichia
coli and Shigella Species
TanisC.McMahon
1,2, CesarBinKingombe
3†, AmitMathews
4, KarineSeyer
5, AlexWong
2,
BurtonW.Blais
1 and CatherineD.Carrillo
1
*
1 Research and Development, Ottawa Laboratory (Carling), Ontario Laboratory Network, Canadian Food Inspection Agency,
Ottawa, ON, Canada, 2 Department of Biology, Carleton University, Ottawa, ON, Canada, 3 Independent Researcher, Ontario,
ON, Canada, 4 Microbiology, Greater Toronto Area Laboratory, Ontario Laboratory Network, Canadian Food Inspection
Agency, Toronto, ON, Canada, 5 Microbiology (Food), St-Hyacinthe Laboratory, Eastern Laboratories Network, Canadian
Food Inspection Agency, St-Hyacinthe, QC, Canada
Bacterial pathogens, such as Shiga toxin-producing Escherichia coli (STEC) and Shigella
spp., are important causes of foodborne illness internationally. Recovery of these organisms
from foods is critical for food safety investigations to support attribution of illnesses to
specic food commodities; however, isolation of bacterial cultures can bechallenging.
Methods for the isolation of STEC and Shigella spp. from foods typically require enrichment
to amplify target organisms to detectable levels. Yet, during enrichment, target organisms
can beoutcompeted by other bacteria in food matrices due to faster growth rates, or
through production of antimicrobial agents such as bacteriocins or bacteriophages. The
purpose of this study was to evaluate the occurrence of Shigella and STEC inhibitors
produced by food microbiota. The production of antimicrobial compounds in cell-free
extracts from 200 bacterial strains and 332 food-enrichment broths was assessed. Cell-
free extracts produced by 23 (11.5%) of the strains tested inhibited growth of at least one
of the ve Shigella and seven STEC indicator strains used in this study. Of the 332
enrichment broths tested, cell-free extracts from 25 (7.5%) samples inhibited growth of
at least one of the indicator strains tested. Inhibition was most commonly associated with
E. coli recovered from meat products. Most of the inhibiting compounds were determined
to beproteinaceous (34 of the 48 positive samples, 71%; including 17 strains, 17 foods)
based on inactivation by proteolytic enzymes, indicating presence of bacteriocins. The
cell-free extracts from 13 samples (27%, eight strains, ve foods) were determined to
contain bacteriophages based on the observation of plaques in diluted extracts and/or
resistance to proteolytic enzymes. These results indicate that the production of inhibitors
by food microbiota may bean important challenge for the recovery of foodborne pathogens,
particularly for Shigella sonnei. The performance of enrichment media for recovery of
Shigella and STEC could beimproved by mitigating the impact of inhibitors produced by
food microbiota during the enrichment process.
Keywords: bacteriocin, bacteriophage, Shiga toxin-producing Escherichia coli, Shigella, foodborne pathogen
Frontiers in Microbiology | www.frontiersin.org 2 June 2022 | Volume 13 | Article 880043
McMahon et al. Microbial Antagonism in Food Enrichment
INTRODUCTION
Foodborne illnesses due to Shiga toxin-producing Escherichia
coli (STEC) and Shigella spp. are an important public health
concern around the world (EFSA, 2013; Gould et al., 2013;
omas et al., 2015; Adam and Pickings, 2016; e et al.,
2016). In Canada, it is estimated that there are approximately
1,200 domestically-acquired cases of foodborne shigellosis and
approximately 33,000 illnesses attributed to STEC per year
(omas et al., 2013). Infections by both STEC and Shigella
can result in serious illnesses. Complications such as hemolytic-
uremic syndrome (HUS), hemorrhagic colitis and Reiter’s
syndrome can have long-term eects and are sometimes fatal
(Donnenberg and Whittam, 2001; Perelle et al., 2004; Warren
et al., 2006; Smith et al., 2014; e et al., 2016). Food
contamination with Shigella is exclusively from human sources
(Warren etal., 2006), whereas animals are important reservoirs
associated with foodborne STEC (Kim et al., 2020).
Infection with STEC or Shigella spp. can occur due to
ingestion of as little as 10–100 cells (Kothary and Babu, 2001;
orpe, 2004). erefore, methods used to test foods for these
pathogens must bevery sensitive, and must beable to identify
a small number of target cells that are likely present as a
miniscule portion of the food microbiota. Current methods
used to detect foodborne STEC and Shigella in Canada,
UnitedStates, and Europe involve an enrichment step followed
by screening of enrichment cultures for characteristic virulence
genes (Bin Kingombe et al., 2006; Warren et al., 2006; ISO,
2012; Blais et al., 2014; USDA-FSIS, 2019). Typically, an
enrichment procedure is conducted with the intention of favoring
the growth of the target bacteria while limiting the growth
of non-target or background bacteria present in the sample.
Nonetheless, the non-target bacteria can oen outcompete the
target pathogen under these conditions due to faster growth
rates, or production of compounds that actively interfere with
their growth (Uyttendaele etal., 2001; Blais et al., 2019). is
can lead to false-negative results in cases where the pathogen
dies, or is present at a proportionally lower level aer enrichment,
and cannot be detected or isolated in downstream analyses
(Kozak et al., 2013). ere are many examples of foodborne
outbreaks associated with STEC or Shigella spp. in which
pathogens were not successfully recovered from implicated
foods despite strong epidemiological evidence supporting food
attribution (Heier et al., 2009; Lewis et al., 2009; Marshall
et al., 2020; Mikhail et al., 2021). Failure to recover target
pathogens may result in delays in attribution of an outbreak
to a food commodity.
A number of studies have investigated growth dynamics in
food enrichment cultures to gain a better understanding of
the growth of interfering organisms in these environments
(Jarvis et al., 2015; Margot et al., 2016; Ottesen et al., 2016;
Kang etal., 2021). e aim of many of these studies has largely
been to catalog species in the food microbiome that can interfere
with the successful recovery of target pathogens in enrichment
cultures, and to evaluate the strengths and weaknesses of dierent
methodological approaches in reducing the growth of non-target
organisms. Few studies have looked specically at the role of
antimicrobial compounds produced by food microbiota in food
enrichment culture. Although, one study of tomato enrichment
culture microbiomes identied Paenibacillus as a potential
Salmonella-inhibiting organism in this system, based on known
activity of this organism (Ottesen et al., 2013).
Bacteria can produce a variety of antimicrobial compounds.
Almost all bacteria encode bacteriocins, which are antimicrobial
peptides (Kim et al., 2014; Yang et al., 2014; Simons et al.,
2020). Bacteriocins produced by Gram-negative bacteria that
target related species are classied as colicins (25–80 kDa) or
microcins (10 kDa; Duquesne et al., 2007; Yang et al., 2014;
Mader etal., 2015). ese bacteriocins damage host cells through
pore formation, DNA/RNA degradation, protein synthesis
inhibition or DNA replication inhibition (Alonso et al., 2000;
Duquesne et al., 2007; Yang et al., 2014). Another mechanism
of microbial antagonism is through the production of
bacteriophages that can infect and kill bacteria (Penadés et al.,
2015; Shahin et al., 2019). Bacteriophages have two cycles of
viral reproduction: the lysogenic and lytic cycles. In the lysogenic
cycle, the bacteriophage genome is integrated into the bacterial
host’s genome as a prophage and is propagated through replication
of the host’s chromosome without damaging the host cell (Parasion
et al., 2014; Penadés et al., 2015). Induction involves conversion
of the lysogenic infection into a lytic infection, where the host’s
machinery is used to produce mature phages, which are released
through lysis of the infected cells. Bacteriophages in the lysogenic
cycle can be induced into the lytic cycle when the bacteria
undergo stress or through induction of the SOS response. Most
phages can only aect a subset of bacteria within a species, and
specicity of the phage depends on the receptors to which it
binds (Koskella and Meaden, 2013; Parasion etal., 2014). Finally,
other antibiotic compounds (e.g., lipopeptides, aminoglycosides,
tetracyclines, and aminocoumarins) may beproduced by bacterial
groups such as Actinomycetales, Bacillales, and Enterobacterales
(Mandal et al., 2013; Challinor and Bode, 2015; Mohr, 2016).
e purpose of this study was to evaluate the occurrence
of antimicrobial compounds produced by food microbiota in
food enrichment cultures. Shigella spp. and STEC were selected
for this study as they are related genera that have low infectious
doses, and are among the most dicult pathogens to recover
from foods. Cell-free extracts derived from food-associated
bacterial strains and from food enrichments [modied Tryptone
Soya Broth (mTSB) or Shigella broth (SB)] were tested for
inhibitory activity against STEC and Shigella. Samples containing
inhibitors were further characterized to determine the likely
mechanisms of inhibition. Results of this study will beof great
value in the development of improved methods for a more
reliable recovery of STEC and Shigella spp. from foods.
MATERIALS AND METHODS
Growth and Maintenance of Bacterial
Strains
A selection of 200 predominantly Enterobacteriaceae strains,
most of which were previously isolated from food enrichment
broths, were selected for this study (Supplementary Table S1).
McMahon et al. Microbial Antagonism in Food Enrichment
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Seven E. coli strains representing clinically important STEC
serotypes and ve Shigella strains were used as the indicator
strains for testing sensitivity to inhibition from cell-free extracts
(Table 1). All strains were stored at −80°C in 15% glycerol
and were plated on Brain-Heart Infusion agar (BHI; OXOID,
Nepean, ON, Canada) overnight (14–16 h) at 37°C prior to use.
Preparation of Cell-Free Extracts
Cell-free extracts were prepared from (1) overnight cultures
of bacterial strains, and (2) food enrichment cultures from
negative samples collected by the CFIA’s food testing laboratories
in Ottawa (ON), Toronto (ON), and Saint-Hyacinthe (QC).
Bacterial strains (n = 200; Supplementary Table S1) were
grown overnight at 37°C in 10 ml of Nutrient broth (OXOID)
and broths were ltered using a 0.22 μm Vacuum lter (EMD
Millipore Steriip™ Sterile Disposable Vacuum Filter Units;
ermo Fisher Scientic, Ottawa). e cell-free extracts were
stored at 4°C for up to 3 months and at −20°C for longer
storage (Figure 1).
Food products were sampled between the fall 2016 and winter
2017 and were representative of the types of foods tested in
regulatory food testing programs (Supplementary Table S2).
Extracts were prepared from 332 enrichment cultures derived
from 235 food samples. Categories of food products tested
included fruits [46 (14%)], salads and coleslaws [78 (23%)],
meats [85 (26%)] and vegetables, cheese and other [123 (37%)].
Enrichment cultures were generated using methods described
in the Canadian Compendium of Analytical Methods: either
the method for detection of Shigella spp. in foods (MFLP-26;
n = 23; Bin Kingombe et al., 2006) or the method for detection
of STEC in foods (MFLP-30; n = 115; Microbiological Methods
Committee, 2012) or both (n = 97). e meats and cheeses were
only enriched in mTSB and the rest were enriched in SB or
both broths. For the MFLP-26 method, samples are enriched
in a 1:10 dilution of sample to Shigella broth (SB; SB base,
OXOID) with Tween-80 (Sigma, Markham, ON, Canada)
containing 0.5 μg/ml of novobiocin (Sigma-Aldrich, Oakville,
ON). e SB is enriched for 20 h at 42°C in a CO2 incubator
or CO2 jar system. e MFLP-30 method involves a 1:10 dilution
of sample to modied Tryptone Soya Broth (mTSB, OXOID)
containing 20 μg/ml of novobiocin, followed by aerobic incubation
of the enrichment at 42°C for 18–24 h. Typically, 25 g of food
are enriched in 225 ml of enrichment broth using both methods.
Note that enrichments were negative for targeted pathogens and
that bacterial growth was observed for all samples based on
turbidity of broths.
Enrichment broths were stored at 4°C prior to use. Cell-
free extracts were prepared from 25 to 50 ml of enrichment
broth by centrifugation at 500 × g for 5 min, transferring the
supernatant to a new tube, followed by centrifugation at 14000
× g for 10 min (Figure 1). e supernatant was then ltered
as described above and stored at 4°C or −20°C. e pellets
from the high-speed spin were resuspended in 30% glycerol
and stored at −80°C.
Detection of Inhibitors in Cell-Free
Extracts
The methods for evaluating the inhibitory activity of cell-
free extracts were modified from methods developed by
(Arici et al., 2004) and (Vijayakumar and Muriana, 2015;
Figure1). Shigella and STEC were grown overnight in 10 ml
of Nutrient Broth (NB, OXOID) at 37°C to obtain a
concentration of approximately 108 cells/ml. Overnight cultures
of each of the indicator strains were enumerated to assess
reproducibility of this approximation for initial experiments,
but enumeration was not done routinely (data not shown).
The overnight Shigella or STEC culture was added at a
concentration of approximately 1 × 106 cells/ml into soft agar
cooled to 40°C [NB (OXOID) containing 0.5% (w/v)
bacteriological agar (Sigma-Aldrich)] and poured into a Petri
dish. Once the plates were solidified, 3 μl spots of the cell-
free extracts were added and left to absorb into the agar
before incubating plates overnight at 37°C. To enable high-
throughput analyses, multichannel pipets were used to generate
36 spots on square Petri plates. All extracts were tested
in triplicate.
Isolation of Bacteria Producing Inhibitors
From Food Enrichments
e method from Henning et al. (2015) was used to isolate
the inhibitor-producing bacteria from a subset of 13 of the
food enrichment broths that were active against at least one
indicator organism. Dilutions of the enrichment broths were
spread plated on nutrient agar (OXOID) then immediately
overlaid with a thin nutrient agar sandwich layer. e plates
were incubated at 37°C overnight before a so agar containing
~1 × 106 cells/ml of the indicator species (Shigella sonnei OLC2340)
was layered on top (see above). e triple layer agar was
incubated overnight at 37°C. Note that only S. sonnei OLC2340
TABLE1 | STEC and Shigella strains used as indicator organisms.
Isolate Genus Species Serotype Description/Isolation
source
OLC0024 Shigella sonnei ATCC29930/feces
OLC2340 Shigella sonnei Pasta salad outbreak/
human feces
OLC0603 Shigella exneri 1a ATCC25929/human
feces
OLC1597 Shigella exneri 1b ATCC12022/missing
OLC0608 Shigella dysenteriae Human feces
OLC0455 Escherichia coli O111:H11 STEC stx1a, eae/
missing
OLC0464 Escherichia coli O26:H11 STEC stx1a, eae/
missing
OLC0675 Escherichia coli O145:NM STEC stx1a, eae/
human feces
OLC0679 Escherichia coli O103:H2 STEC stx1a, eae/
human feces
OLC0710 Escherichia coli O121:H19 STEC stx2a, eae/
human feces
OLC0716 Escherichia coli O45:H2 STEC stx1a, eae/
human feces
OLC0797 Escherichia coli O157:H7 STEC stx1a, sxt2a,
eae/human feces
McMahon et al. Microbial Antagonism in Food Enrichment
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was used in this experiment, due to the complexity of the
method, and the susceptibility of this strain to most of the
cell-free extracts.
Following incubation, the triple agar plates were inverted
and placed onto their Petri dish covers, and colonies surrounded
by zones of clearing were streaked onto a new nutrient agar
plate. e streaked plates were incubated overnight at 37°C
and isolated colonies were patched onto two nutrient agar
plates. e two plates were incubated at 37°C for 4 h and
then one of the duplicate plates was overlaid with so agar
containing ~1 × 106 cells/ml of S. sonnei OLC2340. e plates
were again incubated at 37°C overnight. Isolates recovered
from these plates were streaked for purity, then re-tested to
conrm inhibitory activity. Isolates conrmed to cause inhibition
were stored and maintained as described above.
Testing Cell-Free Extracts for
Proteinaceous Properties, Bacteriophage,
and pH
Proteolytic enzymes were used to assess the proteinaceous
nature of the inhibitory cell-free extracts (Elayaraja etal., 2014).
Proteinase K (ermo Fisher Scientic) and trypsin (Sigma-
Aldrich) were added to the extracts at a nal concentration
of 1 mg/ml or 1X, respectively. e extracts were incubated
for 2 h at 30°C before spotting on so agar containing target
bacteria as described above, alongside an untreated control.
e presence of bacteriophage was assessed using the dilution
method from Hockett and Baltrus (2017). Cell-free extracts
were serially diluted two times in nutrient broth (1:10 and
1:100). Dilutions were spotted on so agar as described above.
Bacteriophage presence was conrmed if individual plaques
were visible in the diluted samples and/or the clearing was
unaected by proteolytic enzymes. e pH of the cell-free
extracts were measured with pH indicator strips (ermo
Fisher Scientic).
RESULTS
Inhibition of Growth of Shigella and STEC
by Cell-Free Extracts Derived From
Bacterial Strains
Cell-free extracts from 200 bacterial strains, primarily
recovered from food (Supplementary Table S1), were tested
to detect inhibition of growth of five strains of Shigella and
seven strains of STEC (Tab l e 1). Twenty-three of the 200
strains tested (11.5%) inhibited the growth of at least one
of the 12 indicator organisms (Shigella and EHEC) used in
this study. Most of the strains tested were Enterobacteriaceae
(194/200), except for six strains (Pseudomonadaceae and
Aeromonadaceae; Figure 2; Supplementary Table S1). The
three main genera evaluated were Escherichia, Enterobacter
and Hafnia [118 (48.5%), 31(14.5%), and 20(10%),
respectively]. Cell-free extracts produced by 21 E. coli (10.5%
of all strains, 17.8% of E. coli) inhibited growth of at least
one strain of Shigella, and cell-free extracts from two
Enterobacter spp. strains (1% of all strains, 6.5% of Enterobacter
spp.) inhibited growth of at least one strain of STEC
(Figure 2). Most of the inhibitor-producing E. coli affected
S. sonnei (18 of 21, 86%), with a smaller proportion (n = 7,
33%) affecting Shigella flexneri. E. coli classified as STEC
were more likely to produce bacteriocins (10 of 19 strains,
FIGURE1 | Detection of inhibitors in cell-free extracts from food
enrichments or bacterial strains. Bacterial strains were grown overnight in
10 ml of Nutrient broth at 37°C (right side) and food enrichments were
incubated according to MLFP-26 or MLFP-30 methods. For food
enrichments (left side), an aliquot of 25–50 ml was taken and centrifuged rst
at 500 × g to remove large food particles and then at 14,000 × g to remove
bacterial cells and debris. Supernatants from cell enrichments or overnight
bacterial cultures were then ltered through a 0.22 μm vacuum lter to remove
remaining food particles and bacterial cells. Filtrates were spotted on soft
agar containing indicator strains. Filtrate spots were absorbed into the agar,
and plates were incubated overnight at 37°C. The following day, pictures
were taken to observe the clearings and the diameter of the clearings were
measured in millimeters. A representative plate with 35 different cell-free
extracts and a negative control spotted in a 6 × 6 grid is shown.
McMahon et al. Microbial Antagonism in Food Enrichment
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52.6%), compared to other E. coli (11 of 99 strains, 11.1%).
In most samples, both strains of S. sonnei (OLC0024 and
OLC2340) were inhibited (16 out of 18) whereas only three
out of the seven extracts affecting S. flexneri inhibited both
of the strains used in this study (Figure 3). The inhibitor-
producing Enterobacter spp. affected E. coli O45 (two extracts)
and E. coli O103 (one extract; Figure3). None of the strains
evaluated in this study inhibited growth of Shigella dysenteriae.
e relative strength of inhibition varied among cell-free
extracts and was assessed based on the diameter of the zone
of inhibition and the opacity of the clearing (Figure 3A).
Samples were designated as very weak (3 mm diameter) to
very strong (11 mm diameter; Figure3; Supplementary Table S3).
e inhibitory activity of cell-free extracts from E. coli on
S. sonnei was generally categorized as strong and very strong,
whereas inhibitory activity on S. exneri was largely determined
to be medium or weak. Similarly, the cell-free extracts from
Enterobacter spp. that inhibited STEC produced medium or
weak inhibition of growth.
Inhibition of Shigella and STEC From
Cell-Free Extracts From Food Enrichments
Cell-free extracts from 332 food-enrichment broths derived
from 235 food products (Figure 4; Supplementary Table S2)
were evaluated to determine prevalence of inhibitors to Shigella
spp. or STEC in food enrichments. Of the 332 enrichment
broths tested, cell-free extracts from 25 (7.55%) samples inhibited
growth of at least one of the 12 Shigella or STEC strains used
in this study (Figure 5). Twenty-one samples (6%) inhibited
growth of Shigella spp. and seven (2%) inhibited growth of
STEC (Figure 5). Among the 25 cell-free extracts containing
inhibitors, 21 (84%) aected S. sonnei, six (24%) aected
S. exneri and seven (28%) aected STEC growth (Figure 5).
One of the extracts (GTA-1452) inhibited growth of all STEC
strains tested in this study and three extracts inhibited both
Shigella and STEC strains. None of the extracts aected growth
of S. dysenteriae.
As with the extracts from bacterial strains, the relative
strength of inhibition was assessed based on the diameter of
FIGURE2 | Species and inhibitory activity of foodborne bacterial strains tested against Shigella and STEC. In total cell-free extracts from 200 bacterial strains were
tested on ve Shigella spp. strains (two Shigella sonnei, two Shigella exneri, and one Shigella dysenteriae) and seven STEC (Serotypes O26, O45, O103, O111,
O121, O145, and O157) samples. Relative proportion of strains producing inhibitors are indicated according to genus impacted (Shigella spp. vs. STEC), and
predicted inhibitor (bacteriocin vs. bacteriophage; see legend).
McMahon et al. Microbial Antagonism in Food Enrichment
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the zone of inhibition and the opacity of the clearing (Figure3A).
Strength of inhibition varied among the extracts and among
indicator organisms tested (Figure5; Supplementary Table S3).
Inhibitory activity against S. sonnei tended to bestrong, whereas
inhibitory activity against S. exneri and STEC was weaker
(Figure 5). Shigella inhibitors were found in two (1.7%) of
A
B
FIGURE3 | Strengths of inhibitory activity of bacterial strains. (A) Representative image of the different strengths of the inhibitory activity observed in the strains
used in this study. Level of activity (e.g., very weak to very strong) was classied based on diameter of the zone of clearing and opacity of the spot. (B) Relative
strength of inhibitory activity of cell-free extracts from bacterial strains on Shigella spp. and STEC is indicated. The strains are grouped based on predicted inhibitor
present in the sample (bacteriocins, bacteriophage, or both).
McMahon et al. Microbial Antagonism in Food Enrichment
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the 120 SB extracts (curry leaves and broccoli slaw) and STEC
inhibitors were present in seven (3.2%) of 213 mTSB extracts
tested. In contrast, mTSB extracts examined in this study were
more likely to contain Shigella spp. inhibitors (8.9%), whereas
none of the SB extracts contained STEC inhibitors. All the
food enrichments had relatively neutral pH values between
6.0 and 7.5.
Cell-free extracts derived from the 85 meat mTSB extracts
were most likely to contain inhibitors to either Shigella and/
or STEC (19 extracts, 22% of meat enrichments; Figure 6;
Supplementary Table S2). ese extracts inhibited growth of
Shigella spp. (14 extracts), STEC (two extracts) or both (three
extracts). Two of the 23 cell-free extracts derived from cheese
mTSB enrichments inhibited growth of either Shigella spp. or
STEC. In contrast, only four of the 224 extracts derived from
plant products (ax seeds, fruit, and vegetables) enriched in
mTSB (n = 104) or SB (n = 120) contained inhibitors. In the
97 samples where plant products were enriched in both mTSB
and SB, inhibition was only observed in one of the broths.
For two of these samples (curry leaves and broccoli slaw),
inhibitory compounds were detected in the SB extracts, but
not the mTSB extracts. For two samples (both leafy greens)
inhibitory compounds were detected the mTSB extracts, but
not in the SB extracts.
Characterization of Inhibitory Properties of
Cell-Free Extracts
All the cell-free extracts causing inhibition in at least one of
the indicator Shigella or STEC strains were treated with proteolytic
enzymes (proteinase-K and trypsin) to determine if inhibition
was eliminated by the removal of the protein components of
the extracts indicating that inhibitor was likely to bea bacteriocin
(Figure 7A). e inhibitory activity of the cell-free extracts
from most of the food strains [17 (74%)] and the food
enrichments [15 (60%)] were aected by at least one proteolytic
enzyme (suspected bacteriocins in Figures 3, 5). For the food
enrichment broths, there were two samples that were
indeterminate for all inhibited strains and one sample
indeterminate for S. exneri due to lack of inhibition in the
untreated control. Inconclusive results were likely associated
with prolonged storage of these extracts as inhibitory activity
FIGURE4 | Food product enrichment cultures tested for production of inhibitors. Gray pie chart: Categories. Blue pie chart: Salads and Coleslaws. Red pie chart:
Meats. Green pie chart: Vegetables, Cheese, and Other. Purple pie chart: Fruits. Total food enrichment samples tested was 332 samples.
McMahon et al. Microbial Antagonism in Food Enrichment
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was found to generally decrease over time during storage at
4°C (data not shown). For the cell-free extract from OLC1219,
the proteinase treatment reduced inhibition for S. sonnei strains
but not for S. exneri (Figure 3) indicating the presence of
dierent inhibition mechanisms for these two species. Most
of the cell-free extracts that inhibited Shigella (particularly
S. sonnei) were aected by proteolytic enzymes (suspected
bacteriocins in Figures 3, 5). Cell-free extracts that inhibited
STEC were generally not aected by proteolytic enzymes
(Figures 3, 5).
To identify bacteriophages, the inhibitory cell-free extracts
from the food strains and food enrichments that were resistant
to proteolytic digestion were also diluted to assess the presence
of plaques (Figure 7A). Plaques were observed in dilutions
derived from eight of the cell-free extracts from bacterial strains
(suspected bacteriophage in Figures3, 7B). For strain OLC1219
FIGURE5 | Strengths of inhibitory activity of food enrichments on Shigella and STEC. Relative strength of inhibitory activity of extracts from mTSB (“m” in sample
name) and SB enrichment (“s” in sample name) on Shigella spp. and STEC is indicated (see Figure3A for representative images of different strengths of inhibitory
activity). The enrichments are grouped based on predicted inhibitor present in the sample (bacteriocins, bacteriophage, or both). Samples were deemed to
beindeterminate if observed inhibition of untreated samples was weak and impacts of proteolytic enzymes and dilutions could not beobserved. Stars indicate
samples for which attempts were made to recover isolates of the bacteria responsible for inhibition. Yellow stars indicate that an isolate producing an inhibitor was
recovered, blue stars indicate attempts to recover isolates from samples were unsuccessful.
McMahon et al. Microbial Antagonism in Food Enrichment
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plaques were observed with S. exneri but not S. sonnei indicating
that inhibition of S. exneri growth was likely due to
bacteriophage, and inhibition against S. sonnei growth was
likely due to production of bacteriocins (see above). Extracts
from the OLC1028 strain were not completely aected by
proteolytic enzymes and plaques were observed following
treatment with proteolytic enzymes indicating production of
both bacteriocin and bacteriophage inhibitors of S. sonnei.
Plaques were observed in extracts from ve of the food
enrichment broths (bacteriophage in Figure 5, 7C).
Recovery of Bacteria Producing Inhibitory
Compounds From Food Enrichment
Broths
Attempts were made to recover isolates of bacteria producing
inhibitors from 13 of the mTSB enrichment broths producing
inhibitory compounds (Figure5, stars) using the triple overlay
method (Henning et al., 2015). Inhibiting organisms were
successfully recovered from seven of these samples (Figure 5,
yellow stars and Table2). All recovered isolates were determined
to be E. coli based on whole-genome sequence analysis (data
not shown). Cell-free extracts from isolates generally exhibited
similar inhibitory properties to the cell-free extracts from
enrichment cultures (Tab l e 2 ; Figure 5, yellow stars). For
example, all seven isolates inhibited growth of both strains of
S. sonnei. Activity of the extracts from six of the E. coli isolates
was aected by proteolytic enzymes indicating that inhibition
was likely due to the production of bacteriocins (Table 2 ).
e isolate recovered from STH-2768 m produced an inhibitor
that aected both S. sonnei and S. exneri, and was determined
to produce bacteriophage as the inhibition was not impacted
by proteolytic enzymes.
DISCUSSION
Recovery of the Shigella spp. and STEC from foods is important
to support food safety investigations and to ensure the timely
recall of implicated foods. Yet, this can beextremely challenging,
in part due to problems culturing pathogens to detectable
levels relative to non-target organisms. Studies conducted using
current microbiological methods may underestimate the
prevalence of Shigella spp. as this organism is easily outcompeted
by other Enterobacteriaceae (Fishbein etal., 1971; Pollock and
Dahlgren, 1974; Uyttendaele et al., 2001). Similarly, detection
of STEC using existing methodology can be dicult due to
lack of selective enrichment media, and competition with
non-target organisms during food enrichment culture (Duy
et al., 1999; Catarame et al., 2003; Vimont et al., 2006;
FIGURE6 | Inhibitory activity of food enrichments on Shigella and STEC. The number of enrichment broths from each food category that did not produce
inhibitors, or are inhibitory to either Shigella, STEC or both are indicated.
McMahon et al. Microbial Antagonism in Food Enrichment
Frontiers in Microbiology | www.frontiersin.org 10 June 2022 | Volume 13 | Article 880043
Knowles etal., 2016; Verhaegen etal., 2016; Blais etal., 2019).
Very few studies have examined the impact of antimicrobial
compounds produced by food microbiota on pathogen detection.
is study was performed to determine whether antimicrobial
compounds impacting STEC, or Shigella spp. were commonly
produced by the microbiota associated with various foods.
Inhibition of Target Foodborne STEC and
Shigella spp. by Food Microbiota
A collection of 200 food-associated bacterial strains
(Supplementary Table S1) was tested for the production of
inhibitory activity against a panel of seven STEC and ve
Shigella strains. e panel of indicator strains was selected to
represent clinically important species and strains of pathogens
that are targeted in food testing programs. e Gram-negative
foodborne bacteria included in this study would typically
be highly represented in food-enrichment cultures aimed at
recovery of Shigella/STEC. Most methods integrate antibiotics
such as novobiocin to reduce growth of Gram-positive bacteria,
but generally do not include selective agents that reduce growth
of Gram-negative bacteria (Vimont et al., 2007). Only two of
A
BC
FIGURE7 | Characterization of inhibitors in isolate and food-enrichment cell-free extracts. Cell-free extracts from food strains and enrichments which inhibited
growth of at least one of the indicator strains tested were further evaluated to characterize inhibitors present. (A) The cell-free extracts were treated with two
proteolytic enzymes (proteinase-K or trypsin) or were diluted (1:10 and 1:100) then spotted on soft agar containing indicator strains. Loss of activity following protein
digestion indicated that growth inhibition was likely due to presence of bacteriocins (suspected bacteriocins bottom row), and resilience to protein digestion along
with presence of plaques in diluted extracts indicated bacteriophage (suspected bacteriophage, top row). (B,C) Inhibitory activity of bacterial isolate extracts (B) or
food enrichment extracts (C) against indicator organisms. Predicted bacteriocins are indicated by blue bars, bacteriophages (orange bars) or both (gray bars).
Indeterminate samples could not beassessed due to weak zones of clearing (yellow bars).
TABLE2 | Inhibitor-producing organisms recovered from food enrichment
cultures.
Strain Enrichment
culture Serotype Activity Species
inhibited
OLC3028 STH-2577 O174:H8 Bacteriocin Shigella sonnei
OLC3029 STH-2682 O23:H9 Bacteriocin Shigella sonnei
OLC3030 STH-2725 O123:H16 Bacteriocin Shigella sonnei
OLC3032 STH-2768 O105:H7 Bacteriophage Shigella sonnei
Shigella exneri
OLC3035 STH-2777 O153/
O178:H11
Bacteriocin Shigella sonnei
OLC3094 OTT-1094 O81:H7 Bacteriocin Shigella sonnei
OLC3095 OTT-1019 O51:H10 Bacteriocin Shigella sonnei
McMahon et al. Microbial Antagonism in Food Enrichment
Frontiers in Microbiology | www.frontiersin.org 11 June 2022 | Volume 13 | Article 880043
the 36 Enterobacter strains tested inhibited growth of STEC
(Figure3B; OLC1633 and OLC1667). In previous investigations
organisms such as Clostridium spp., E. coli, Hafnia alvei,
Brochothrix thermosphacta, and Pediococcus acidilactici present
in meats were shown to outcompete STEC; however, the
mechanism for this was not identied (Duy et al., 1999;
Kang et al., 2021). Similarly, Paquette et al. (2018) identied
E. coli strains capable of inhibiting STEC due to production
of diusible antimicrobial compounds (Paquette et al., 2018).
While E. coli with this activity was not found in the present
study, analysis of a larger number of strains or food samples
may have led to the identication of strains with similar
properties. In contrast, extracts from 20 of the 118 E. coli
strains tested were found to inhibit growth of Shigella spp.,
particularly S. sonnei, indicating that this species is highly
susceptible to antimicrobial compounds produced by food
microbiota. e inhibition of Shigella spp. by antibiotics produced
by E. coli recovered from human sources was rst described
over 70 years ago (Halbert, 1948; Halbert and Gravatt, 1949),
and S. sonnei strains have been used as indicator organisms
for bacteriocins detection due to known susceptibility to a
variety of colicins (Šmarda and Obdržálek, 2001; Micenková
et al., 2014). It is interesting to note that STEC strains were
more likely to produce inhibitors impacting growth of Shigella
spp. compared to non-pathogenic strains. e association of
bacteriocins and virulence factors in E. coli has been previously
reported (Bradley et al., 1991; Micenková et al., 2014). Both
of these traits may provide a competitive advantage in
certain environments.
Analysis of cell-free extracts derived from a variety of
food enrichments was done to evaluate the production of
inhibitors by more complex communities of representative
food microbiota. While Shigella broth would not be used for
detection of STEC, and mTSB would not beused for detection
of Shigella, similar analyses conducted with both types of
enrichment broths enabled comparison of inhibitor production
by organisms growing in the two media. Inhibition of at
least one of the 12 indicator organisms tested was observed
for 7.5% of the enrichment cultures tested, making it a
relatively common occurrence overall. Inhibitors were more
commonly associated with raw meat products enriched in
mTSB (22% of raw meat enrichments) relative to plant products
(1.8%). Similarly, a recent study found pathogen-killing
bacteriophage to be more prevalent in raw beef and chicken
than in vegetables and seafood (Premarathne et al., 2017).
e relatively high prevalence of Shigella inhibition in the
mTSB enrichments may be due to the species of bacteria
present in the food matrix rather than dierences in the
enrichment broths. For example, E. coli is known to
be associated with animal products (Barco et al., 2015).
Shigella-inhibiting E. coli has long been known to beassociated
with human clinical samples (Halbert, 1948; Halbert and
Gravatt, 1949; Levine and Tanimoto, 1954), but to conrm
animal association, samples of animal fecal matter should
be further examined. e association of inhibition to mTSB
is partly due to the fact that meat and cheese products were
not enriched in Shigella broth in this study. Paired enrichments
of raw meat products in both broths could be evaluated to
conrm the association of inhibitor-production with raw meats
rather than enrichment broths. In the 97 plant products
enriched with both broths, no association of the inhibiting
organisms to the enrichment broths was observed; however,
only four of the 224 broths examined produced inhibitors.
Similar to what was observed with the bacterial strains,
S. sonnei was the indicator species that was the most sensitive
to inhibition (21 out of 25 extracts), followed by S. exneri
and STEC (Figure5). e observation of similar trends between
the bacterial strains and enrichments indicates that the bacterial
strains used in this study are representative of the bacteria in
food enrichments. Most enrichments aected only one species
(e.g., S. sonnei or STEC for 19 of 25 extracts) however there
were some samples that showed a broader activity. For instance,
cell-free extracts from one enrichment culture inhibited all
seven STEC strains (GTA-1452, Figure 5). ere were also
three enrichment broths that inhibited both STEC and Shigella
strains (GTA-1549, STH-2577 and STH-2777). is is likely
indicative of an inhibitor that has a larger spectrum of activity,
or potentially more than one strain with this activity in the
samples (see below). Bacteriocins and bacteriophages can have
broad or narrow spectrums of activity (Penadés et al., 2015;
Simons et al., 2020). Note that the approach used to evaluate
cell-free extracts was only semi-quantitative, as interpretation
could besomewhat subjective, particularly for “weak” and “very
weak” spots. Size of spots was also inuenced by length of
storage, with reduced activity over time (data not shown).
Nonetheless, general trends could be determined with the
screening approach used in this study.
Recovery of inhibiting organisms from enrichment cultures
was challenging. Out of 13 enrichment cultures, seven E. coli
isolates were recovered from cultures derived from raw meat
products using a triple agar overlay method (Henning et al.,
2015; Tab l e 2 ). Recovery of inhibitor-producing E. coli is
consistent with observations for the food-associated bacterial
strains, and further supports the nding that E. coli may bean
important source of inhibition, particularly for growth of S. sonnei.
Results may be biased by the use of only one of the indicator
strains (S. sonnei, OLC2340) for the recovery of inhibitory
isolates, which were all derived from meat enrichments. E. coli
is an indicator of fecal matter contamination which could occur
during the slaughter process, potentially explaining the association
of these organisms with raw meat products (Tallon etal., 2005;
Odonkor and Ampofo, 2013; Barco et al., 2015). Failure to
recover isolates in six samples may be because the method
required recovery of a bacterial strain producing the inhibition.
is approach would not work for the recovery of bacteriophage
that may have been present in the foods, but not associated
with a bacterial host. Cell-free extracts from the isolated E. coli
strains had similar inhibitory properties to the extracts from
the food samples from which they were isolated (Tabl e 2 ;
Supplementary Table S3), particularly to S. sonnei, which was
the organism used for the recovery of these isolates. However,
dierences in activity against other indicator strains were observed.
For example, OLC3032 was active against S. exneri, unlike
in the original enrichment culture where only S. sonnei was
McMahon et al. Microbial Antagonism in Food Enrichment
Frontiers in Microbiology | www.frontiersin.org 12 June 2022 | Volume 13 | Article 880043
aected and conversely OLC3028 did not inhibit growth of
S. exneri, unlike the original food enrichment culture. is
indicates that multiple strains may have been responsible for
inhibitory activity in the more complex enrichment cultures.
Use of multiple indicator strains, and characterization of additional
bacterial isolates may have led to recovery of additional strains.
S. sonnei and S. exneri were included as indicator organisms
in this study as they are the Shigella species most commonly
associated with outbreaks in North America (ompson etal.,
2015; Centers for Disease Control and Prevention (CDC), 2016;
e et al., 2016). Shigella sonnei was most commonly aected
by antimicrobial compounds, followed by S. exneri (Figures3, 5).
Previous studies have that shown that S. exneri was more
highly aected by organic acids (e.g., by-products from food
and food microbiota) than S. sonnei (Hentges, 1969; Uyttendaele
etal., 2001; Zhang etal., 2011). S. dysenteriae was not aected
by any of the cell-free extracts, so it is possible this species
has a tolerance or resistance to inhibitors such as bacteriocins
due to dierences in targeted receptors in this species (Alonso
et al., 2000). e acidity of the cultures was not likely to
be an important factor in the observed growth inhibition. e
pH range of Shigella growth is between 4.8–9.3 and the range
of STEC growth is 4.0–10.0 (Food and Drug Administration,
2011). e pH of the bacterial isolate cell-free extracts (6.5–7.5)
and food enrichment extracts (6.0–7.5) was well within
these ranges.
Foodborne Shigella outbreaks are oen associated with plant
products (Kozak et al., 2013; Nygren et al., 2013). While
enrichment broths derived from plant products were less likely
to contain bacteriocins than those derived from meats in this
study, this does not mean that recovery of Shigella spp. from
plant products would not beproblematic. Shigella contamination
of foods is exclusively from human sources, and such
contamination would likely also include E. coli. Given that
human isolates of E. coli commonly inhibit Shigella spp. (Halbert,
1948; Levine and Tanimoto, 1954), the combination of organisms
could impact recovering Shigella from the implicated foods.
In fact, in a foodborne outbreak associated with baby corn
in 2007, Shigella was not recovered from implicated samples
despite strong epidemiological evidence, however, samples had
high levels of E. coli (100–350 cfu/g) indicating potential issues
with food hygiene (Lewis et al., 2009).
Identication of Predicted Bacteriocins
and Bacteriophage in Cell-Free Extracts
To gain a better understanding of the mechanism of the
inhibition, cell-free extracts were digested with proteolytic
enzymes to determine if the inhibitor was impacted, as would
beexpected for bacteriocins which are proteinaceous compounds
(Elayaraja et al., 2014), and extracts were diluted to identify
the plaques that would beattributed to bacteriophages (Hockett
and Baltrus, 2017; Figure7). Most of the inhibitors, particularly
those aecting S. sonnei, were aected by proteolytic enzymes
and are likely to be bacteriocins (Figures 3, 5, 7). Notably,
there were two E. coli strains (OLC1028 and OLC1219) that
were predicted to produce both bacteriocins and bacteriophages
with antimicrobial activity against Shigella spp. (Figure 3). In
contrast, STEC inhibition was more commonly associated with
bacteriophages (Figures 3, 5). e Enterobacter strains that
inhibited growth of the O45 and O103 were both predicted
to produce bacteriophages (Figure 3). Similarly, the broad
spectrum inhibition of STEC observed for sample GTA-1452
was also determined to beassociated with bacteriophages. is
inhibition could be due to the presence of a bacteriophage
that has a wide spectrum of activity, or multiple bacteriophages
targeting a few strains (Penadés et al., 2015; Simons et al.,
2020). For example, STEC-killing phage can have broad or
narrow host ranges (Litt et al., 2018; Mangieri et al., 2020).
Interestingly, the inhibition produced by the isolate recovered
from the STH-2768 m (OLC3032) was determined to be due
to bacteriophages (Table2) rather than the bacteriocins observed
in the original culture (Supplementary Table S3), indicating
that there was likely at least one bacteriocin-producing organism
in the original culture that was not recovered. e identication
of multiple inhibiting organisms within a single enrichment
culture is further evidence that these strains may bean important
factor in the success of competitive enrichment for food
microbiology methods.
Bacteriophages active against STEC have been isolated from
numerous zoonotic sources and may be relatively common
indicating potential importance for enrichment cultures (Litt
et al., 2018; Mangieri et al., 2020; Pinto etal., 2021). Presence
of bacteriophages active against target species in enrichment
cultures has been shown to reduce relative proportions of target
Salmonella and S. sonnei by over a log indicating their critical
impact on food microbiological methods (Muniesa etal., 2005).
Bacteriocins would be expected to have similar inhibitory
activity in food enrichment cultures. e ability to produce
bacteriocins impacting closely related species is a common
feature of E. coli enabling their survival in competitive
environments (Micenková et al., 2016; Cameron etal., 2019).
E. coli, particularly human isolates, frequently produce colicins,
some of which have been shown to specically target Shigella
spp. (Halbert and Gravatt, 1949; Levine and Tanimoto, 1954).
Only two colicins (colicin Z and Js) that specically target
Enteroinvasive E. coli (EIEC) and Shigella species have been
characterized (Micenková etal., 2019). Further analysis of the
E. coli recovered in this study will be conducted to determine
if the strains recovered encode related colicins.
In this study, S. sonnei were more susceptible to bacteriocins
than S. exneri and STEC. Due to practical considerations,
the number of indicator strains used in this study was necessarily
limited. It is possible that some of the indicator organisms
used had higher resilience to phage and bacteriocins due to
biological factors such as presence of immunity proteins, or
other defense mechanisms (Rendueles et al., 2022). Use of a
larger panel of Shigella strains, particularly strains associated
with food outbreaks, will be necessary to establish whether
S. sonnei strains are more susceptible than others. An
understanding of the impact of bacteriocins on enrichment
dynamics could lead to approaches to mitigating their impact.
For example, fermentable sugars have been shown to reduce
colicin production by S. sonnei (Lavoie et al., 1973). Future
McMahon et al. Microbial Antagonism in Food Enrichment
Frontiers in Microbiology | www.frontiersin.org 13 June 2022 | Volume 13 | Article 880043
studies will target enrichment dynamics associated with
bacteriocins in food matrices.
While outside of the scope of the current study, there is
currently signicant interest in alternatives to antibiotics for
the prevention of food contamination with these Shigella spp.
and STEC, and for the development of therapeutics (Yan g
et al., 2014; Mangieri et al., 2020; Telhig et al., 2020; Jiang
et al., 2022). e bacteria recovered in this study are highly
active against pathogenic strains of Shigella spp. and STEC,
with potential for future use in the production of alternatives
to antibiotics.
CONCLUSION
Isolation of a foodborne bacterial pathogen is an important
component of a food safety investigation that is sometimes
dicult to achieve. is study provides evidence that production
of antimicrobial compounds such as bacteriocins and
bacteriophages by food microbiota is a relatively common
occurrence which could impede growth of Shigella and STEC
in enrichment cultures, reducing eectiveness of microbiological
methods aimed at recovering these organisms. Presence of
bacteriocin-producing E. coli may be an important challenge
for the recovery of Shigella spp. and presence of bacteriophage
may be of greater concern for STEC. Development of new
methods should mitigate the potential interference due to
inhibitor production by food microbiota. Organisms recovered
in this study could be included as interfering organisms in
future method validations, leading to a more rigorous assessment
of methods aimed at recovery of Shigella and STEC from foods.
DATA AVAILABILITY STATEMENT
e original contributions presented in the study are included
in the article/Supplementary Material, and further inquiries
can be directed to the corresponding author.
AUTHOR CONTRIBUTIONS
CK originally identied bacteriocins as an important inhibitor
for Shigella spp. TM, CC, AW, and BB conceived and designed
the experiments and contributed to writing of the manuscript.
TM performed laboratory experiments. TM and CC analyzed
the data and wrote the rst dra of the manuscript. TM, CC,
AM, KS, and BB contributed reagents, materials, and analysis
tools. All authors contributed to the article and approved the
submitted version.
FUNDING
is study has received funding from the Canadian Food
Inspection Agency and the Government of Canada
interdepartmental Genomic Research Development Initiative
(GRDI).
ACKNOWLEDGMENTS
e authors gratefully acknowledge sta at the CFIA food
microbiology laboratories in Ottawa (Austin Markell),
Scarborough (Katherine Han), and Saint-Hyacinthe (José Riva,
Julie Plamondon, Lyne Laamme) for providing food enrichment
cultures. e authors also appreciate technical assistance from
Bridgette Kelly, Susan Van Zanten, Hilda Hoo, Paul Manninger,
Martine Dixon, and Mylène Deschênes. e authors would
also like to thank Sam Mohajer, Chloe Anastasiadis and Lang
Yao for critical review of the manuscript.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.880043/
full#supplementary-material
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