Water Air Soil Pollut (2021) 232: 296

https://doi.org/10.1007/s11270-021-05241-w

A Novel Comparison of Virulence Genes, Biofilm‑Forming

Capacity, Antibiotic Resistance, and Level of Reactive
Oxygen Species of Sediment, Sewage, and O157 E. coli
Jocelyn Susan Bel · Neelam Khaper · Sreekumari Kurissery ·
Kam Tin Leung

Received: 2 March 2021 / Accepted: 13 June 2021 / Published online: 6 July 2021
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021

Abstract Even though sediment Escherichia coli the planktonic cellular ROS levels of the three sam-
is a common source of contamination in freshwater ple groups tested were not different from each other,
environments, their characteristics such as virulence the level of ROS in biofilm cells was eightfold lower
genes, biofilm forming capacity, antibiotic resist- than their planktonic counterparts (p < 0.001). Cells
ance, and level of reactive oxygen species (ROS) with higher ROS tolerance have been shown to have
are not well understood. This study examined E. coli more tolerance to bactericidal antibiotics; how-
from freshwater sediments, sewage, and a collection ever, the antibiotic-resistance levels of the bacteria
of O157 isolates. Multiplex PCR analysis revealed in this study did not correlate to their cellular ROS
that none of the sediment and 3% of the sewage iso- concentrations.
lates contained diarrheagenic E. coli genes. However,
12.5% of sediment and 42.4% of sewage isolates Keywords Sediment and sewage E. coli · Virulence
contained one or more uropathogenic genes. Biofilm genes · Biofilms · Antibiotic resistance · ROS
assays determined that sediment E. coli were sig-
nificantly better biofilm formers (p < 0.001), form-
ing 2- and 3.5-times as much biofilm as sewage and 1 Introduction
O157 E. coli, respectively. The antibiotic-resistance
patterns illustrated that the E. coli belonged to three With E. coli contamination in fresh water being an
distinct groups with sediment being most susceptible important concern for health reasons, it is essential
and sewage being most resistant (p < 0.05). Although to know the sources and characteristics of this group
of bacteria. Sewage is one of the major sources of E.
coli contamination in freshwater environments. Sew-
J. S. Bel · N. Khaper · K. T. Leung (*)
age effluents may also carry other human pathogens
Department of Biology, Lakehead University, 955 Oliver
Road, Thunder Bay, ON P7B 5E1, Canada that increase the health risk of this kind of contamina-
e-mail: [email protected] tion (Merkx-Jacques et al., 2013). However, another
important source of contamination can be from natu-
N. Khaper
ralized E. coli residing in sediments and other envi-
Medical Sciences Division, Northern Ontario School
of Medicine, Lakehead University, Thunder Bay, ON, ronmental sources such as periphyton (Ksoll et al.,
Canada 2007; Moreira et al., 2012; Quero et al., 2015; Sherer
et al., 1992). In the environment, bacteria are able to
S. Kurissery
form biofilms on the interface of sediment and water
Department of Sustainability Sciences, Lakehead
University, Orillia Campus, Orillia, ON, Canada allowing for increased survival (Sherer et al., 1992).

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E. coli can have increased survival in sediments due environment is a growing concern (Canadian Insti-
to the existence of fine soil particles, nutrients, and tute for Health Informaiton, 2017). Most antibiotic
high organic matter contents, and these sediment bac- compounds are not fully metabolized by our bodies
teria can be found in higher concentrations compared or broken down at wastewater treatment plants, and
to the water column (Badgley et al., 2010; Sherer therefore become a source of antibiotic contamina-
et al., 1992). Many studies have demonstrated that tion in the freshwater environments (Watkinson et al.,
E. coli in sediments were able to live considerably 2007; Kümmerer, 2009). This increase in the concen-
longer compared to living in the water column (Bur- tration of antibiotics entering the natural water sys-
ton et al., 1987; Hood & Ness, 1982; Sherer et al., tem presents a health concern where there is a risk of
1992). However, other characteristics (such as the bacteria within the environment becoming resistant to
possession of virulence gene and population diver- these antibiotics (Watkinson et al., 2007). Therefore,
sity) and environmental factors that influence survival it is important to determine the antibiotic-resistance
of this group of bacteria have only been examined by patterns of the freshwater sediment E. coli.
a few studies (Luna et al., 2010; Quero et al., 2015; Other than the conventional cellular target-
Sherer et al., 1992; Vignaroli et al., 2015). Therefore, related antibiotic killing mechanisms generally
to determine the potential health risk caused by the known, a new model has been proposed in which
naturalized sediment E. coli, it is important to under- bactericidal antibiotics use a secondary pathway
stand the virulence factors, antibiotic resistance, and that induces ROS production in bacterial cells and
other physiological characteristics of these bacteria. inflicts damages to the bacteria (Belenky & Col-
Characteristics such as virulence gene possession, lins, 2011; Dwyer et al., 2014). Kohanski et al.
biofilm forming capacity, antibiotic resistance, and (2007) demonstrated that oxidative stress may play
the cellular level of reactive oxygen species (ROS) a role as a secondary killing mechanism of bacte-
can contribute to the viability of E. coli in the envi- ricidal antibiotics. With bactericidal antibiotics, a
ronment. Although some E. coli virulence genes play cell will undergo drug induced stress. This stress
an important role in the pathogenic mechanisms of will increase its level of aerobic respiration thereby
the bacteria to cause diseases to humans, most patho- generating excess superoxide anions, hydrogen per-
genic strains require a combination of several viru- oxide, and hydroxyl radicals (Wang et al., 2010).
lence genes to express their pathogenicity (Chapman Along with increased aerobic respiration, the desta-
et al., 2006; Gyles et al., 1998). Furthermore, some bilization of iron-sulfur clusters in dehydratase
of these virulence genes, such as the iron sequester- enzymes occurs providing an opportunity for the
ing gene, can be useful for the bacteria to survive iron molecule to be released as a ferrous ion that
and establish in the environment (Neilands, 1995). will eventually generate hydroxyl radicals through
However, little is known about the kind of virulence the Fenton reaction. During this process, the dis-
genes of naturalized E. coli in freshwater sediments. mutation of hydrogen peroxide will generate addi-
The ability of the bacteria to form biofilms, organ- tional hydroxyl radicals that are toxic to the cells
ized bacterial communities inside of an extracellular (Van Acker et al., 2014; Wang et al., 2010). This
polymeric matrix that can adhere to each other, on rise in the level of hydroxyl radicals will in turn
biotic or abiotic surfaces can aide in their survival lower the number of cells that survive antibiotic
in the environment (Costerton, 1995). It is believed treatment (Wang et al., 2010). By altering the cen-
that these complex structures evolved as an adaptive tral metabolism of the cell, bactericidal antibiotics
behavior in order to protect themselves and survive can cause drug-induced killing through respiration
in hostile environments (Beloin et al., 2008; Macia and iron metabolism (Dwyer et al., 2014; Van Acker
et al., 2014). This structure allows for different bac- et al., 2014; Vatansever et al., 2013). Furthermore,
teria to function and live in harmony allowing each (Dwyer et al., 2014) suggested that an increase in
other to survive in different conditions such as with resistance to ROS can help bacteria to become more
exposure to antibiotics (Costerton, 1995). tolerant or even resistant to antibiotics. Pelicano
With more than 25 million courses of antibiotics et al. (2004) showed that cancer cells exhibited a
prescribed each year in Canada, the effects of these high level of ROS and hence promoted their drug
antibiotics once they leave our bodies and enter the and antibiotic tolerance. Therefore, cellular ROS

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concentration might be an indicator for oxidative 2.2 Sample Collection and E. coli Isolation
or antibiotic tolerance of cells. Currently, little is
known about the cellular levels of ROS in environ- 2.2.1 Sediment Samples
mental E. coli. With this new secondary mechanism
of antibiotic killing being increasingly studied, it In this study, sediment samples were collected from
is of interest to determine if there is a correlation locations in both Southern and Northwestern Ontario.
between the cellular ROS level and the antibiotic- The Southern Ontario samples were taken from four
resistant ability of the E. coli isolates in this study. locations, areas surrounding Lake Simcoe, and the
In this study, we examined the virulence genes, southeastern shore of Georgian Bay. These samples
biofilm forming capacities, antibiotic-resistance were collected from Holland River in the Township
patterns, and cellular ROS levels of E. coli iso- of Innisfil [44° 9′ 45.83″ N 79° 31′ 15.77″ W], Kettle
lated from sediment and sewage, and a collection Lake, Awenda Park [44° 50′ 40.59″ N 79° 58′ 22.58″
of Shiga toxin producing E. coli O157 (Gyles et al., W], Farlain Lake Township [44° 49′ 46.13″ N 79° 58′
1998). Sediment and sewage samples were obtained 26.07″ W], and Ben’s Ditch [44° 35′ 30.59″ N 79° 25′
from various locations in Orillia and Thunder Bay, 15.29″ W]. The Northwestern Ontario samples were
Ontario, Canada, to allow for a diverse range of E. collected from around Boulevard Lake in Thunder
coli isolates to be examined. This study also exam- Bay, Ontario [48° 27′ 39″ N, 89° 11′ 53″ W].
ined the correlation between antibiotic resistance of Briefly, sediment samples were collected using
and cellular levels of ROS in the E. coli isolates. a sterile stainless-steel sediment corer that was sub-
merged in the sediment to a depth of approximately
15 cm. Three separate samples were taken from each
sampling site and all samples were processed within
2 Materials and Methods 24 h. All samples were processed as follows. In ster-
ile 160 mL milk dilution bottles, 10 g of sediment
2.1 Bacterial Strains was shaken with 90 mL of 0.85% sterile saline for
5 min to release bound bacteria from the sediment.
ATCC 25,922 E. coli strain obtained from Cedar- After letting settle for 45 min, 10 mL of the superna-
lane Corporation (Burlington, Ontario) was used as tant was filtered through a sterile 0.45 μm membrane
positive control for uropathogenic virulence genes. filter (Thermo Fisher Scientific, Ottawa, Ontario).
Positive controls of diarrheagenic virulence genes The filter was then placed onto a Differential Coli-
were detected in Enterotoxigenic E. coli (ETEC) form Agar plate (DC Agar, Oxoid Limited, Nepean,
strains ETEC 505 and ETEC 07, an Enteroinvasive Ontario). This product has been renamed to Bril-
E. coli (EIEC) strain 0136, and an Enteropatho- liance E. coli/Coliform Agar) and incubated at 37 °C.
genic E. coli (EPEC) strain 055 provided by Dr. After 24 h, blue colonies were individually streaked
B. Ciebin, Ontario Ministry of Health, Etobicoke, onto new DC agar plates to obtain pure cultures of
Ontario. Shiga toxins I and II genes in Hemorrhagic the isolates. The isolates were confirmed to be E.
E. coli were detected using a Shiga toxin-producing coli through the standard Indole, Methyl Red, Voges-
O157 E. coli positive control strain EC 920,004 (a Proskauer, and Citrate utilization (IMViC) test (John-
bovine isolate) provided by Dr. C. Gyles at the Uni- son & Case, 2013). The isolates were cultured in
versity of Guelph, Guelph, ON. Tryptic Soy Broth (TSB) and stored in a sterile 25%
Six additional samples belonging to the O157 glycerol solution (v/v final concentration) at − 80 °C
serogroup were also provided by C. Gyles and used for long-term storage.
to compare with E. coli isolated from the sediment
and sewage for their biofilm forming capacities, anti- 2.2.2 Sewage Samples
biotic resistance, and level of cellular ROS. Strains
EC960959, EC920026, and EC920037 were isolated The Thunder Bay sewage E. coli were isolated from
from bovine, whereas strains EC970112, EC961020, samples collected from the primary clarifier (before
and B0965105 were isolated from human sources. the secondary treatment) of the Thunder Bay Waste

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Water Treatment Plant (Thunder Bay, ON) in 2010 reaction (PCR). Multiplex PCR tests were performed
and 2011 (between July and September). Sewage on the positive controls to optimize the reaction con-
samples were also obtained from the City of Orillia ditions. Nine genetic biomarkers were targeted by
Wastewater Treatment Centre (Orillia, ON) in Sep- the PCR assays in this study: hs (heat stable toxin
tember 2017. E. coli bacteria were isolated using the gene), hl (heat labile toxin gene), ial (invasion asso-
same procedure described in the sediment E. coli ciated loci gene), bfpA (bundle-forming pili gene),
isolation process with slight modifications. Samples stx1 (Shiga toxin 1 gene), stx2 (Shiga toxin 2 gene),
were diluted ­103–105 times using sterile 0.85% saline iroNE. coli (catechol siderophore receptor gene), hylA
and 10 mL of each diluted sample was filter through a (hemolysin gene), and papA (pyelonephritis-associ-
sterile 0.45 μm membrane filter (Thermo Fisher Sci- ated pili gene) (Table 1).
entific). The filter was then placed onto a Differential Multiplex PCR reactions were performed to detect
Coliform Agar plate (Oxoid Limited) and the E. coli the presence of nine virulence genes in three reac-
samples were isolated and stored as described for the tions. Each multiplex reaction contained three sets
sediment samples. of primers. The PCR primers were put into groups
based on their amplicon size and interactions to
2.3 Detection of Virulence Genes in E. coli isolates ensure that nonspecific amplification was eliminated
while also ensuring that the three amplicons in each
2.3.1 DNA Extraction and Purification grouping were well separated. All three primer pairs
were added with 1 μL each in both the forward and
A total of 145 E. coli isolates from sediment reverse direction (each primer had a final concentra-
(n = 72), sewage (n = 66), and O157 (n = 7) were tion of 0.2 μM). Each multiplex PCR reaction tube
screened in the detection of virulence genes. DNA contained the following reagents from Fermentas: 27
was extracted from each isolate using the XS buffer μL of UV treated autoclaved double distilled water,
solution (1% w/v potassium ethyl xanthogenate, 5 μL of 2 mM dNTP mix, 5 μL of 25 mM ­MgCl2, 5
100 mM Tris–HCl, 20 mM EDTA, 1% w/v SDS, and μL of 10X Taq Polymerase buffer, and 1 μL of Taq
800 mM ammonium acetate) as described by Tillett DNA polymerase (1 U/μL). The template DNA added
et al. (2000). Two microliters of RNase (10 mg/mL) at a volume of 1 μL (approximately 150 ηg) to give
(Fermentas, Waltham, Massachusetts) was added to a total reaction volume of 50 μL. The reaction mix-
the XS buffer-treated sample before being incubated ture was mixed and placed into a MJ Mini Thermocy-
at 37 °C in a water bath (Fisher Scientific, Ottawa, cler (BioRad, Mississauga, Ontario). The PCR reac-
Ontario) for 1 h and transferred to an additional tion involved the following steps: the samples were
water bath at 70 °C. The sample was placed on ice initially heated at 95 °C for 5 min. The PCR cycling
for 30 min and centrifuged for 10 min at 18,800 × g, then began at 95 °C for 1 min to denature the DNA,
after which, 750 μL of the supernatant was collected 55 °C for 1 min for annealing, and 72 °C for 1 min
and 750 μL of 100% isopropyl alcohol was added to for extension. After 35 cycles, the samples were kept
precipitate the DNA. The samples were then cooled at 72 °C for 10 min and left at 4 °C until the samples
in a − 30 °C freezer for 12–20 h. The DNA was then were retrieved from the PCR machine. The PCR sam-
pelleted by centrifuging the samples at 18,800 × g for ples were analyzed by agarose gel electrophoresis and
10 min before the DNA was washed twice with 750 visualized with the Chemi Genius Bio Imaging Sys-
μL of 70% ethanol. Lastly, the samples were left to air tem (Syngene, Cambridge, England).
dry in a Biosafety Cabinet (BSC) for approximately
30 min before the DNA was dissolved in 100 μL of 2.4 Biofilm‑Forming Capacity
sterile UV-treated double distilled water.
The biofilm-forming capacities of the sediment, sew-
2.3.2 Multiplex PCR Assays age, and O157 E. coli were compared. To initiate
biofilm growth within a 96-well flat-bottomed poly-
The virulence genes of the E. coli isolated from the styrene microplate (Costar, Corning, New York, New
sediment and sewage samples and the O157 group York), E. coli inoculum was prepared as described by
were determined by multiplex polymerase chain Moreira et al. (2012) with minor modifications. This

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Table 1  Virotypes and strains used as positive controls to detect uropathogenic and diarrheagenic E. coli genes with their respective
primers arranged in the multiplex PCR groups
Multiplex Gene Primer sequence Ampli- Virotype (strain ID) Primers’ reference
PCR con size used as positive control
group (bp)

1 hs F:5′-TCT​TTC​CCC​TCT​TTT​AGT​ 170 ETEC (ETEC 505) Rappelli et al. (2001)

CAGTC-3′
R:5′-CCA​GCA​CAG​GCA​GGA​TTA​C-3′
1 hl F:5′-TCT​CTA​TGT​GCA​CAC​GGA​GC-3′ 322 ETEC (ETEC 07) Rappelli et al. (2001)
R:5′-CCA​TAC​TGA​TTG​CCG​CAA​T-3′
1 iroNE. coli F:5′-AAG​TCA​AAG​CAG​GGG​TTG​ 665 UPEC (UPEC 25,922) Chapman et al. (2006)
CCCG-3′
R:5′-GAC​GCC​GAC​ATT​AAG​ACG​
CAG-3′
2 ial F:5′-GGT​ATG​ATG​ATG​ATG​AGT​ 650 EIEC (EIEC 0136) López-Saucedo et al. (2003)
CCA-3′
R:5′-GGA​GGC​CAA​CAA​TTA​TTT​CC-3′
2 bfpA F:5′-GCC​GCT​TTA​TCC​AAC​CTG​GTA-3′ 324 EPEC (EPEC 055) López-Saucedo et al. (2003)
R:5′-TGC​TGG​ACC​TAC​ATT​TAA​TTCC-
3′
2 hlyA F:5′-CAT​CTC​TGG​TTG​GTG​CAC​ 1000 UPEC (UPEC 25,922) Chapman et al. (2006)
CGGTA-3′
R:5′-AAC​TTG​TCG​GCA​CGC​GTG​
GTC-3′
3 stx1 F:5′-CTG​GAT​TTA ATG​TCG​CAT​AGT​ 150 EHEC (EHEC 920,004) López-Saucedo et al. (2003)
G-3′
R:5′-AGA​ACG​CCC​ACT​GAG​ATC​
ATC-3′
3 stx2 F:5′-GGC​ACT​GTC​TGA​ AAC​TGC​ 255 EHEC (EHEC 920,004) López-Saucedo et al. (2003)
TCC-3′
R:5′-TCG CCA​GTT​ATC​TGA​ CAT​TCT​
G-3′
3 papA F:5′-ATG​GCA​GTG​GTG​TCT​TTT​ 720 UPEC (UPEC 25,922) Chapman et al. (2006)
GGTG-3′
R:5′-CGT​CCC​ACC​ATA​CGT​GCT​
CTTC-3′

group of 78 samples included 47 sediment samples ninety microliters of minimal salt medium (1.249 mM
(20 from Lake Simcoe, 15 from Georgian Bay, and ­KH2PO4, 3.73 mM K ­2HPO4, 0.4 mM M ­ gSO4,
12 from Boulevard Lake), 24 sewage samples (9 from 0.02 mM ­FeSO4, and 1.4 mM N ­ H4Cl) with 0.04%
Orillia and 15 from Thunder Bay), and 7 O157 iso- glucose (MSMG) was transferred to each well to give
lates. This reduction in number of samples allowed a final total volume of 200 µl. The 96-well plate was
for a more manageable sample size that included then incubated for 48 h at 22 °C with gently shaking
representation of all sample locations. Three inde- at 25 rpm. After incubation, the 96 well plate was
pendently grown cell samples were prepared for each removed, and three separate washings of the plate
isolate. Each cell suspension optical density was were performed with sterile double distilled water
adjusted with a NovaSpec spectrophotometer (Bio- before the plate was air dried. The biofilm cells were
chrom LTD, Cambridge, UK) to an ­ OD600 of 1.0 stained with 150 µl of a 0.1% crystal violet solu-
which was approximately 1 × ­109 CFU. Ten microlit- tion for 10-min. Excess crystal violet solution was
ers aliquots of the cell suspension were transferred removed from the plate and the plate was washed
into four separate wells of a sterile 96-well flat-bot- three times with sterile double-distilled water before
tomed polystyrene plate (Costar). One hundred and being dried. To release the crystal violet from the

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biofilm cells, 200 µl of a de-staining solution made sterile syringe, the antibiotic solution was filter steri-
up of 80% acetone and 20% ethanol (v/v) was added lized with a sterile 0.2-μm polycarbonate hydrophilic
to each well of the plate and left for 10 min. One hun- membrane filter (EMD Millipore). The desired anti-
dred and fifty microliters of the de-staining solution biotic dose for each disk for ampicillin, colistin, gen-
was transferred into a new 96-well plate for absorb- tamycin, vancomycin, tetracycline, ciprofloxacin,
ance measurement at 595 ηm using a Fluostar Optima sulfanilamide, and erythromycin were 10, 10, 10, 15,
automated plate reader (BMG Labtech, Cary, North 30, 30, 5, and 5 μg respectively. Ten microliters of
Carolina). The background staining of the crystal vio- antibiotic solution was added to each individual disk
let was subtracted from each measurement to quantify to achieve the desired dose of antibiotic per disk. The
the amount of biofilm formed by each sample. The disks were left to dry in the BSC for 2 h. Blank disks
average of the three independently prepared cell sam- (i.e., disks without antibiotics) were prepared and
ples, with four replicates for each sample, was used to used as controls.
represent the biofilm forming capacity for each of the The E. coli samples cultured in TSB overnight at
sediment, sewage, and O157 E. coli isolates. 37 °C were washed with sterile PBS, and the opti-
cal density of the final cell suspension was adjusted
2.5 Antibiotic‑Resistance Testing to an ­OD600 of 0.10 which is equal to the McFarlane
standard for turbidity of 0.5. Two hundred microlit-
2.5.1 Antibiotic‑Resistance Testing Under CLSI ers of each of the ­OD600 of 0.10 cultures were spread
Conditions evenly on the Mueller–Hinton Agar plates and left to
dry for approximately 30 min in the BSC before the
To use a more manageable number of samples when antibiotic disks were placed onto the agar. Six rep-
testing for antibiotic resistance, 45 isolates were lications of each antibiotic were performed on each
selected from the three groups of E. coli tested in the E. coli sample, in addition to a blank disk to act as
biofilm assay. The isolates were chosen covering the the control. The disks were gently pushed onto the
full range of biofilm forming capacities from each agar to ensure proper contact before incubation. The
sample group. The isolates selected included 19 sedi- sample plates were incubated for 18 h in an Isotemp
ment isolates (9 from Lake Simcoe, 6 from Georgian Incubator (Thermo Fisher Scientific) at 37 °C and
Bay, and 4 from Boulevard Lake), 19 sewage isolates after which the diameters of the inhibition zones were
(4 from Orillia and 15 from Thunder Bay), and the 7 measured.
O157 isolates. Eight antibiotics were chosen based on
numerous factors including their presence in the envi-
ronment, frequency in human prescriptions filled, and 2.5.2 Comparing Antibiotic Resistance of Biofilm
to ensure variation in the antibiotic target on the bac- and Non‑Biofilm Cells
teria (Kümmerer, 2009; Kohanski et al., 2010; Public
Health Agency of Canada, 2016). The eight antibiot- A subsample of 21 isolates used in the standard CLSI
ics used in this experiment were ampicillin, colistin, antibiotic-resistance testing was used in the following
gentamycin, erythromycin, vancomycin, sulfanila- experiments where there were 10 sediment, 8 sew-
mide, tetracycline, and ciprofloxacin (Sigma-Aldrich age, and 3 O157 isolates. To examine the antibiotic
Canada, Oakville, ON). Sterile filter paper disks resistance of the E. coli isolates under optimal biofilm
(6 mm in diameter) were prepared from Whatman forming conditions, adjustments to the standard CLSI
filter paper #3 (General Electric Healthcare, Mis- protocol were implemented. The incubation tempera-
sissauga, ON) that was cut using a standard 1-hole ture was adjusted to 22 °C instead of 37 °C to resem-
punch and autoclaved for 15 min. To comply with the ble the optimal temperature for biofilm formation
CLSI (Clinical and Laboratory Standards Institute) (Moreira et al., 2012). The cultures were grown in
standard (CLSI: Clinical and Laboratory Standards MSMG broth to resemble the biofilm assay protocol
Institute 2015) for antibiotic testing with the Kirby- established by Moreira et al. (2012). Lastly, Mueller
Bauer method, Mueller Hinton agar (Oxoid) plates Hinton agar was substituted by agar plates made with
were used. Antibiotic solutions were freshly pre- MSMG in order to keep the media conditions compa-
pared on the day of the experiments. Using a 10-mL rable to biofilm growth.

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Planktonic cultures were prepared in 30 mL of 22 °C with shaking at 25 rpm. To harvest the biofilm
sterile MSMG incubated overnight at 22 °C with cells from the glass fiber filters, the planktonic cells
shaking at 150 rpm (Moreira et al., 2012). One mil- were removed aseptically and discarded using a ster-
liliter of each overnight culture was transferred into ile glass pipette. The filters were washed three times
30 mL of fresh sterile MSMG, vortexed, and incu- inside the glass bottles by adding 10 mL of sterile
bated for an additional 6 h at 22 °C with shaking at PBS and swirling gently before removing the liquid
150 rpm. This allowed for the bacterial culture to be from the bottle. The filter disks were transferred asep-
in the exponential phase of growth, rather than in sta- tically to a sterile 100-mL glass bottle containing 5 g
tionary phase. When the optical density (at 600 ηm) of sterile glass beads (450–600 μm, Sigma-Aldrich).
reached approximately 0.20, the cells were harvested Eight milliliters of sterile double distilled water was
and washed with sterile PBS twice. The planktonic then added to the glass bottle, and the sample was
cells were then re-suspended to an optical density of vortexed vigorously for 3 min. The biofilm cell sam-
0.20 with sterile double distilled water. Two hundred ples were then transferred into individual sterile stom-
microliters of planktonic culture was transferred onto acher bags to remove excess glass fibers and the bio-
an MSMG plate and spread evenly on a plate and film cell samples were collected in sterile tubes. The
left to dry for 1 h. Antibiotic disks were then placed biofilm cell samples were then adjusted to an ­OD600
onto the respective plates as described previously and of 0.20 with sterile double distilled water and kept
plates were incubated at 22 °C for 18 h. After incuba- on ice until ready to use. Antibiotic disks were pre-
tion, zones of inhibition were measured and recorded. pared as described above. Two hundred microliters of
In order to determine the antibiotic resistance of biofilm culture was transferred onto a sterile MSMG-
biofilm cells under biofilm conditions, poloxamer 30% poloxamer plate and left to dry for 1 h. Antibi-
407 (Sigma Aldrich) plates were used. The proto- otic disks were then placed onto respective plates as
col to prepare these biofilm plates was adopted from described previously and the plates were incubated at
Yamada et al. (2011) with modifications. Polox- 22 °C for 18 h. After incubation, zones of inhibition
amer 407 was incorporated into MSMG media at a were measured and recorded.
30% concentration. The poloxamer-MSMG growth
medium was refrigerated at 4 °C for 48 h in order for 2.6 Cellular ROS of E. coli
the poloxamer to fully dissolve into the solution. The
poloxamer growth solution was autoclaved for 20 min The same group of 21 isolates used in the biofilm
and returned to the refrigerator for 48 h to allow the antibiotic-resistance testing was used in the reactive
medium to liquefy. The liquid poloxamer growth oxygen species (ROS) assay. The cellular ROS con-
medium was poured into sterile petri plates inside the centrations of planktonic and biofilm E. coli cells
BSC in volumes of approximately 30 mL. The polox- from the sediment, sewage, and O157 samples were
amer-MSMG plates were solidified and maintained at determined with the addition of DCF-DA (2′,7′-di-
room temperature until the experiment. chlorofluoresceine diacetate). The planktonic and bio-
To determine the antibiotic resistance of the E. film E. coli cells were prepared using the same proce-
coli biofilm cells on MSMG-poloxamer plates, bio- dures described in the modified antibiotic-resistance
film cell suspensions were prepared as described by testing protocol whereby both planktonic and biofilm
Magajna and Schraft (2015). E. coli samples were cells were grown at 22 °C and in MSMG with biofilm
grown in MSMG broth as described for the plank- cells being grown on glass fiber filters.
tonic cultures, except eliminating the additional A 1000 μM DCF-DA solution was prepared and
transfer of culture and the 6 h of additional incuba- 20 μL portions of the solution were added to sterile
tion. The cells were washed three times with sterile 15-mL tubes containing 1980 μL of either the plank-
PBS and re-suspended with sterile double distilled tonic or biofilm E. coli cell samples suspended in
water to an ­OD600ηm of about 0.05. One milliliter of sterile double distilled water. The cell samples were
this cell suspension was then transferred into 20 mL incubated in the dark at 25 °C for 30 min to allow the
of MSMG in a sterile 250 mL Pyrex glass bottle con- DCF-DA to interact with the cells. In a dark room,
taining 0.1 g of sterile glass fiber filters (pore size 100 μL of each sample was then transferred into a
0.7 μm, Whatman GF/F) and incubated for 48 h at Costar 96-well flat-bottomed plate (Corning). Each

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sample was loaded into six individual wells where the depending on individual experiments. The patterns of
fluorescence was measured at 495 ηm excitation and antibiotic resistance of the three E. coli groups were
525 ηm emission using a BMG Labtech FLUOrstar compared using discriminant function analysis in the
OPTIMA plate reader. The average of the six fluores- SPSS statistic program (IBM, Markham, ON). Linear
cence measurements was determined for each sam- regression assays were performed (SigmaPlot12) to
ple where the fluorescence was proportional to the determine the correlations between the cellular ROS
amount of ROS in the sample. concentrations and the antibiotic resistances of the
One milliliter aliquots of the planktonic and bio- isolates in planktonic state.
film E. coli cultures were collected, and their protein
concentrations were determined. Protein concentra-
tions of the cell samples were determined with the 3 Results and Discussion
use of the B-PER Bacterial Protein Extraction Kit
(Thermo Fisher Scientific) and the Coomassie (Brad- 3.1 Virulence Gene Detection
ford) Protein Assay Kit (Thermo Fisher Scientific).
One milliliter of each cell suspension was washed Seventy-two sediment (27 from Lake Simcoe, 23
with PBS and pelleted in a sterile 1.5-mL Eppendorf from Georgian Bay, and 22 from Boulevard Lake),
tube. One milliliter of the B-Per Reagent was added 66 sewage (57 from Thunder Bay and 9 from Oril-
to the cell pellet and vortexed until the pellet was lia), and seven O157 E. coli isolates were examined
homogenized before it was incubated at room temper- by the multiplex PCR assay. The sediment isolates
ature for 15 min. The lysate was then centrifuged for did not contain any of the six genetic markers belong-
5 min at 15,000 × g to remove the cell debris, and the ing to either the diarrheagenic or hemorrhagic E. coli
protein concentration of sample was determined by (Table 2). Some isolates from the sediment (12.5%)
the Bradford assay. The Coomassie (Bradford) Pro- contained the iroN gene that is a virulence gene of
tein Assay (Thermo Fisher Scientific) was performed uropathogenic E. coli. The iroN gene has clinical
as outlined in the manufacturer’s manual for the importance for uropathogenic E. coli as it allows them
microplate protocol. Protein standards were prepared to elicit infections. By forming an iron siderophore-
using BSA at concentrations of 0, 2.5, 5, 10, 15, 20, based acquisition system, it gives bacteria the ability
and 25 μg/mL. Each E. coli protein extract (150 μL) to use iron from the host (Anastasi et al., 2012; Wiles
was added to the microplate in triplicate where 150 et al., 2008). Neilands (1995) suggested that the iroN
μL of the Coomassie (Bradford) reagent was added offers bacteria the ability to sequester iron from the
to each well containing samples or standards. The environment, where the amount of iron available
plate was allowed to incubate at room temperature for to the bacteria is low. The possession of this gene
10 min. The absorbance of each well was measured at within sediment E. coli populations does not neces-
595 ηm. A standard curve was constructed using the sarily translate into negative effects on human health
BSA standards and the protein concentrations of the as this gene may have been retained or acquired for
E. coli samples were determined by comparing their better survival in the environment. Anastasi et al.
absorbance to the BSA standard calibration curve. (2012) found similar results to this study when they
examined sediment E. coli and found that environ-
2.7 Statistical Analysis mental samples harbored only the hylA or iroNE.coli
genes. With an understanding that the iroN gene may
All statistical analyses were performed by the Sig- be present in bacteria for survival in the environment,
maPlot 12 Software integrated with SigmaStat (Sys- it is reasonable that the sediment E. coli were found
tat Software Inc., San Jose, USA) unless otherwise to have this virulence gene.
stated. Analysis of variance (ANOVA) was performed Luna et al. (2010) showed that genetic heteroge-
in various sections of this study (biofilm forming neity between sediment E. coli could be influenced
capacities, antibiotic resistance, and ROS concentra- by fecal contamination from waste waters, the geo-
tions). ANOVAs were performed on data obtained in graphical and climate of the sampling site, and from
three independently prepared samples and the num- horizontal gene transfer that is facilitated by the sedi-
ber of replications for each sample varied from 4–6 ment bacterial community. If the sediment E. coli

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Water Air Soil Pollut (2021) 232: 296 Page 9 of 19 296

Table 2  Detection of uropathogenic and diarrheagenic genes in sediment, sewage, and O157 E. coli samples by multiplex PCR
Number of + ve isolates (%)
Sediment Sewage O157
(n = 72) (n = 66) (n = 7)

Uropathogenic Only iroN 9 (12.5%) 5 (7.6%) 0 (0%)

E. coli (UPEC) Only hylA 0 (0%) 4 (6.1%) 0 (0%)
genes
Only papA 0 (0%) 12 (18.2%) 0 (0%)
iroN & hylA 0 (0%) 2 (3%) 0 (0%)
iroN & papA 0 (0%) 3 (4.5%) 0 (0%)
iroN & hylA & papA 0 (0%) 2 (3%) 0 (0%)
Total number of isolates containing one or more virulence 9 (12.5%) 28 (42.4%) 0 (0%)
gene
Diarrheagenic E. ETEC, EIEC, and EPEC genes Only hs 0 (0%) 0 (0%) 0 (0%)
coli genes Only hl 0 (0%) 0 (0%) 0 (0%)
Only ial 0 (0%) 1 (1.5%) 0 (0%)
Only bfpA 0 (0%) 0 (0%) 0 (0%)
Hemorrhagic E. coli (EHEC) Genes Only stx1 0 (0%) 1 (1.5%) 5 (71%)
Only stx2 0 (0%) 0 (0%) 2 (29%)
stx1 & stx2 0 (0%) 0 (0%) 2 (29%)
Total number of isolates containing one or more virulence 0 (0%) 2 (3%) 7 (100%)
gene

n indicates the number of E. coli isolates tested in each group

are collected from an area frequently contaminated This low level of contamination from sewage may
by sewage, this would alter the amount of virulence explain the observations from this study where very
genes possessed by these bacteria. Likewise, the tem- low percentages of the sediment E. coli were found to
perature and geographical region that the sediment contain virulence genes.
exists in also plays a role in the characteristics of the In contrast to sediment, sewage E. coli have been
E. coli population. Luna et al. (2010) analyzed sedi- widely studied (Anastasi et al., 2012; Reinthaler et al.,
ments from marine waters for virulence genes using 2003). Overall, the sewage E. coli isolates were found
multiplex PCR to reveal that between 65 and 90% of to contain significantly more virulence genes than the
sediment isolates tested contained at least one E. coli sediment samples for both uropathogenic and diar-
virulence gene that has the potential to cause disease rheagenic genes. In this study, 42.4% of the sewage
in humans. Their study included adhesion genes, an samples were positive for uropathogenic genes iroN,
intimin gene, membrane protein genes, hemolysin hylA, papA, or combinations of the three (Table 2).
gene, Shiga toxin genes, and siderophore genes of There were only 1.5% of the sewage isolates that con-
pathogenic E. coli. They revealed that their sampling tained the ial gene, and only 1.5% contained the stx1
areas were potentially contaminated with sewage gene. Mokracka et al. (2011) used multiplex PCR to
and that the sediment was a potential reservoir for detect virulence genes (eae, bfpA, ST[hs], LT[hl],
pathogenic bacteria (Luna et al., 2010). These bacte- ipaH, stx1, and stx2) in municipal waste water and
ria could be released into the water column from the found that 50.5% of the E. coli isolates were positive
sediment by water turbulence (Sherer et al., 1992; for these virulence genes. Diarrheagenic pathotypes
Fevre and Lewis, 2003). The two Southern Ontario made up 21% of all isolates tested and 17.4% of the
locations in this study (Georgian Bay and Lake Sim- isolates had three or more virulence genes in their
coe) had minimal urban influence with less than 10% study (Mokracka et al., 2011). Sabaté et al. (2008)
urban cover. Boulevard Lake in Northwestern Ontario also found that human wastewater contained a num-
also had minimal exposure to sewage contamination. ber of uropathogenic genes. Despite the fact that all

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sewage studies show high levels of virulence genes in the sediment environment including nutrient limi-
contained within the E. coli, the frequency and com- tation and stress (Hall-Stoodley et al., 2004). Sew-
position of the various virulence genes ranges widely age E. coli illustrated heterogeneity between isolates,
for each study. The large variations between the per- with some being moderate biofilm formers and oth-
centages of virulence genes present in sewage sam- ers being poor biofilm formers; however, the overall
ples from different studies indicate that the popula- finding was that sewage isolates were poorer biofilm
tions feeding into each sewage treatment plant are not formers than sediment E. coli. Similar observations
uniform. were shown by (Reisner et al., 2006) where they
found that mammalian sources had varying capaci-
3.2 Biofilm‑Forming Capacity ties of forming biofilms across all their isolates tested
from healthy individuals. The sewage isolates’ bio-
E. coli have evolved to survive and establish in non- film forming capabilities differ greatly from the path-
enteric habitats due to a high degree of versatility ogenic O157 isolates, which exhibited low biofilm-
and genetic diversity (Quero et al., 2015; Touchon forming capabilities across all samples tested with
et al., 2009). One of these factors includes forming minimal variation among the isolates. This lack of
biofilms as it allows bacteria to survive and establish variation among the O157 samples may be a reflec-
in an environment where there are fluctuating harsh tion of the relatively short evolutionary history of this
conditions (Hall-Stoodley et al., 2004). The ability of serogroup of Shiga toxin producing E. coli (Percival
bacteria to form biofilms has been studied extensively et al., 2004).
(Costerton, 1995; Hall-Stoodley et al., 2004; Prakash
et al., 2003). However, few studies have directly com- 3.3 Antibiotic Resistance
pared the biofilm-forming capabilities of sediment
and sewage E. coli. Bacterial resistance to antibiotics has greatly
In this study, the biofilm forming capacities of the increased due to the large amounts of antibiotics
sediment, sewage, and O157 E. coli were found to be being prescribed today (Kümmerer, 2009). Cipro-
significantly different among one another (p < 0.001) floxacin, tetracycline, erythromycin, and sulfanila-
(Fig. 1). Comparisons between the three groups illus- mide have all been commonly prescribed and found
trated that sediment samples were the best biofilm in the environment (Kummerer, 2009; Public Health
formers. This has physiological relevance, as they Agency of Canada, 2016). Ampicillin, vancomycin,
would have to survive harsh, sub-optimal conditions and gentamycin are also commonly used antibiotics

Fig. 1  Biofilm forming

capacity of the sediment
(n = 47), sewage (n = 24),
and O157 E. coli (n = 7).
n indicates the number
of E. coli isolates tested
in each group. Biofilm
measurement of each isolate
was the mean of three
independently prepared
cell samples with four
replicates for each sample.
ANOVA showed that the
biofilm-forming capacities
of the three E. coli groups
were significantly different
(p < 0.001)

13
Water Air Soil Pollut (2021) 232: 296 Page 11 of 19 296

and bacteria have been found to be resistant to them the least amount of multi-resistant isolates. All of the
in either clinical or environmental settings (Kum- sediment (100%) were resistant to vancomycin and
merer, 2009; Sáenz et al., 2004; Boles & Singh, erythromycin with only 5% of the isolates showing
2008). Colistin is a polymyxin antibiotic and cur- an additional resistance to sulfanilamide. All of the
rently being used as a last resort antibiotic due to its O157 isolates (100%) were resistant to erythromy-
effectiveness against multidrug resistant Gram-nega- cin, and only 86% were also resistant to vancomycin
tive bacteria (Falagas & Kasiakou, 2006; Pamp et al., while the other 14% of the isolates were resistant to
2008). Although some antibiotics used in this study ampicillin. The sewage isolates displayed the greatest
(namely, erythromycin and vancomycin) are known multiple antibiotic resistance, with 100% were resist-
to be ineffective against E. coli (Antonoplis et al., ant to a combination of three antibiotics and 32% to
2019; Kappell et al., 2015), the degrees of response to four antibiotics. Sediment E. coli in this study were
these antibiotics (i.e., diameters of the zone of inhibi- isolated from freshwater lakes that are not frequently
tion) varied for the different E. coli isolates. A study exposed to antibiotic contaminations, which may
conducted by Begum et al. (2005) found that 79% of explain the lower levels of antibiotic resistance found
the pathogenic E. coli isolated from the water column within the sediment E. coli group. However, with the
was resistant to at least one antibiotic. The presence continuous rise of antibiotic contamination in aquatic
of this antibiotic-resistant pathogenic E. coli within environments, the risk of the increase of antibiotic
the water column indicates the need for further stud- resistance will only be worsened (Kummerer, 2009).
ies on bacteria living in the environment. Ishii et al. A canonical plot was constructed through discri-
(2006) noted that established sediment E. coli popu- minant function analysis to compare the antibiotic
lations have not been previously examined in detail; resistance-patterns (zone of inhibition distances)
however, this study offers new information into how of the sediment, sewage, and O157 E. coli. It illus-
susceptible these bacteria are to antibiotics. The sew- trates a clear separation between the three sample
age E. coli were overall the most resistant group fol- groups indicating that the antibiotic-resistance pat-
lowed by the sediment isolates (Fig. 2). All isolates, terns of the E. coli between these groups are very
regardless of sample type, displayed resistance/inter- different from each other (χ2 = 187.61 and p < 0.001)
mediate resistance, according to the CLSI guide- but within the same group, the isolates are very
lines, to more than one antibiotic tested in this study similar (Fig. 3). Classification from the discrimi-
(Table 3). The sediment and O157 samples illustrated nant function analysis showed that overall 98% of

Fig. 2  Antibiotic resistance

of the sediment (n = 19),
sewage (n = 19), and O157
(n = 7) E. coli. n indicates
the number of E. coli iso-
lates tested in each E. coli
group. Antibiotic resistance
of each group was the aver-
age zone of inhibition of the
eight antibiotics. Antibiotic-
resistance measurement of
each isolate was the mean
of three independently pre-
pared cell samples with six
replicates for each sample.
*
p < 0.05

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296 Page 12 of 19 Water Air Soil Pollut (2021) 232: 296

Table 3  Comparison of the percentage of E. coli isolates classified as resistant or intermediate resistant to each of the eight antibiot-
ics tested in this study
Antibiotic(s) Percentage of isolates that are resistant/intermediate ­resistant†
Sediment (n = 19) Sewage (n = 19) O157 (n = 7)

Ampicillin 0% 32% 14%

Ciprofloxacin 0% 0% 0%
Colistin 0% 0% 0%
Erythromycin 100% 100% 100%
Gentamycin 0% 0% 0%
Sulfanilamide 5% 100% 0%
Tetracycline 0% 0% 0%
Vancomycin 100% 100% 86%
Ampicillin and erythromycin 0% 100% 14%
Erythromycin and vancomycin 100% 100% 86%
Erythromycin and sulfanilamide and vancomycin 5% 100% 0%
Ampicillin and erythromycin and sulfanilamide and van- 0% 32% 0%
comycin
†
According to CLSI Antibiotic Resistance Classification (Performance Standards for Antimicrobial Susceptibility Testing 2015)
n indicates the number of E. coli isolates tested in each group

the original grouped cases were correctly classified physiologically distinct. Minimal differences among
where 100% of the sediment, 100% of the sewage, the sediment E. coli were observed despite their dif-
and 85.7% of the O157 isolates were correctly clas- ferent geographic origins. Similar observations were
sified into their respective groups (Table 4). This found with the E. coli isolated from sewage, where
agrees with our findings in the biofilm and virulence there were no overall differences between samples
gene studies where the three groups of E. coli were isolated from the two geographic locations used in

Fig. 3  Canonical discri-

minant function plot to
differentiate the antibiotic-
resistance patterns of the
sediment (n = 19), sewage
(n = 19), and O157 (n = 7)
E. coli. n indicates the
number of E. coli samples
in each group (χ2 = 187.61
and p < 0.001)

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Water Air Soil Pollut (2021) 232: 296 Page 13 of 19 296

Table 4  Discriminant function classification of the sediment (n = 19), sewage (n = 19), and O157 (n = 7) E. coli based on their anti-
biotic-resistance patterns
Sample type Predicted group membership Total
Sediment Sewage O157

Count (%) Sediment 19 (100.0%) 0 (0%) 0 (0%) 19 (100.0%)

Sewage 0 (0%) 19 (100.0%) 0 (0%) 19 (100.0%)
O157 1 (14.3%) 0 (0%) 6 (85.7%) 7 (100.0%)

n indicates the number of E. coli isolates in each group

this study. However, these similarities in character- phenotype similar to the biofilm cells, thus conclud-
istics would likely be altered if the environmental ing the hydrogel method as a simple and reliable
conditions of some of these sites were changed to assay to produce and study biofilm cells. Yamada
have a higher level of contamination or greater lev- et al. (2011) advanced the use of hydrogels for
els of exposure to antibiotics. biofilm antibiotic-resistance testing by using 30%
The Kirby-Bauer disk diffusion method to study poloxamer 407 incorporated into Mueller Hinton
antibiotic resistance of planktonic cells has been broth to create biofilm growth plates. A protocol
well documented. However, recent studies have similar to Yamada et al. (2011) was employed in
shown that this method, with modifications, can this study to compare the antibiotic-resistance pat-
also be used to study the resistance of biofilm cells. terns of both planktonic and biofilm cells using the
Gilbert et al. (1998) assessed biofilm cells’ response Kirby-Bauer disk diffusion method.
to biocide treatment using 30% hydrogels composed To compare the antibiotic resistances of the
of poloxamer F127, a non-toxic, di-block copoly- planktonic and biofilm cells, a subgroup of 21
mer of polyoxyethylene and polyoxypropylene. isolates was chosen. Antibiotic-resistance testing
They compared the protein expression of plank- comparing the planktonic and biofilm cultures
tonic, biofilm, and poloxamer grown cultures using in this study illustrated clear differences, with
SDS-PAGE to determine the characteristics of the the biofilm cells being significantly more resist-
poloxamer grown cells. They found that poloxamer ant to antibiotics than their planktonic counter-
cultures exhibited a general protein pattern and parts (p < 0.001) (Fig. 4). This increased level of

Fig. 4  Comparing the

antibiotic resistance of
planktonic and biofilm E.
coli. The average antibiotic
resistances of the planktonic
and biofilm E. coli were
determined by averaging the
inhibition zones of the 21 E.
coli samples, including 10
sediment, 8 sewage, and 3
O157 isolates, tested on the
eight antibiotics. Each anti-
biotic resistance measure-
ment was the mean of three
independently prepared cell
samples with six replicates
for each sample. **p < 0.001

13
296 Page 14 of 19 Water Air Soil Pollut (2021) 232: 296

resistance to antibiotics has been attributed to phe- the biofilm cells were not significantly different
notypical differences among planktonic and bio- from their planktonic counterparts.
film cells including the exopolymeric matrix and
reduced cellular activity. When biofilm cells are 3.4 Reactive Oxygen Species
enclosed in the exopolymeric matrix, it reduces
the effectiveness for polar and charged antibiotics This study compared the cellular levels of ROS in E.
to reach the bacteria (Cloete, 2003; Kirby et al., coli obtained from different sources (sediment, sew-
2012). Cells in a biofilm also enter stress tolerant age, and O157). The planktonic ROS levels of the
phase, where they can reduce their rate of metab- three groups of E. coli (sediment, sewage, and O157)
olism and respiration, which in turn allows it to were not significantly different from each other. How-
become less susceptible to the antibiotics that are ever, the findings showed that the average ROS level
effective against metabolically active and readily of the planktonic E. coli was over eightfold higher
respiring bacteria (Lewis, 2001). Overall, the bio- than that of their biofilm counterparts (p < 0.001)
film cells were more resistant to antibiotics than (Fig. 6). Jakubowski and Bartosz (2000) explained
planktonic cells with ampicillin, vancomycin, tet- that biofilm cells have higher protection against oxi-
racycline, ciprofloxacin, and sulfanilamide yield- dative stress because they have reduced metabolic
ing significant results (p < 0.05) (Fig. 5). How- process, which can lead to lower concentrations of
ever, colistin is more effective on cells with low free radicals generated within the cells. An E. coli’s
metabolic activity and less effective on cells with response to a stressful stimulus will include activa-
higher levels of metabolic activity (Pamp et al., tion of stress response enzymes including catalase
2008). Our observation of planktonic and biofilm and superoxide dismutase that will reduce the amount
cultures with colistin agreed with the findings of of oxidative stress within the bacteria by scavenging
Pamp et al. (2008) where the planktonic cells were the free radicals within the bacterial cell (Jakubowski
significantly more resistant to colistin (p < 0.05) & Walkowiak, 2015). This response is exceptionally
than their biofilm counterparts (Fig. 5). Further- active in biofilm E. coli cells due to their possession
more, gentamycin and erythromycin resistance of of the RpoS (stationary phase sigma factor) that will
initiate the response to stress (Battesti et al., 2011;

Fig. 5  Antibiotic resistance of planktonic and biofilm E. coli against various antibiotics. The average antibiotic resistances of the 21
E. coli samples were determined by averaging the inhibition zones for each specific antibiotic. *p < 0.05 and **p < 0.001

13
Water Air Soil Pollut (2021) 232: 296 Page 15 of 19 296

Fig. 6  Comparing the aver-

age ROS concentrations of
planktonic and biofilm E.
coli, including the sediment
(n = 10), sewage (n = 8),
and O157 (n = 3) samples. n
indicates the number of iso-
lates in each E. coli group.
**
p < 0.001

Mah & O’Toole, 2001; Schellhorn, 2014). Actively higher antibiotic resistance for isolates with higher
growing planktonic cells do not possess this sigma cellular ROS concentrations. However, when the out-
factor, which corresponds with the data presented lier at 5.1 ROS/μg protein was removed, none of the
in this study demonstrating that the concentration of eight antibiotics showed any significant correlation
ROS in planktonic cells was significantly higher than between the cellular ROS concentrations of the E.
those in biofilm cells. coli strains and their antibiotic resistances. The cellu-
lar ROS levels of bacteria vary under different physi-
3.5 Correlation Between Cellular ROS and ological and environmental conditions. Therefore,
Antibiotic Resistance the E. coli ROS concentrations in this study may not
necessarily represent the true oxidative tolerances of
A secondary antibiotic mechanism occurs through the isolates. This may explain the lack of correlations
an interaction with the bacteria’s tricarboxylic acid between the cellular ROS concentrations and antibi-
(TCA) cycle and electron transport chain to over- otic resistances.
produce ROS molecules such as superoxide, hydro- This study characterized the complexity of E. coli
gen peroxide, and hydroxyl radicals (Kohanski et al., isolated from freshwater sediment and sewage in
2007). When these ROS molecules are produced in both the planktonic and biofilm states. Strong bio-
excess, the bacteria will go into an oxidative stress film-forming capabilities allowed for the sediment
state. Eventually, this increase in oxidative stress E. coli to establish in the aquatic environment while
will be overwhelming for the bacteria and cause also providing protection from antibiotics and oxi-
cell death. To examine if this correlation exists dative damage. They also contained very few viru-
among the isolates used in this study, linear regres- lence genes and were generally more susceptible to
sion analysis between the levels of cellular ROS and antibiotics in their planktonic state. Sewage E. coli
antibiotic resistance was performed (Fig. 7). Bacte- contains more uropathogenic and diarrheagenic E.
ricidal antibiotics gentamycin, ampicillin, vancomy- coli genes and acquired a greater level of resistance
cin, colistin, and ciprofloxacin were used in addition to common antibiotics. With the continuous rise of
to bacteriostatic antibiotics including tetracycline, antibiotic and fecal contamination into freshwater
sulfanilamide, and erythromycin. Only two antibiot- systems, it may alter the antibiotic resistance and
ics, tetracycline and ciprofloxacin, were found to be virulence of the sediment E. coli and could become
statistically significant in this correlation, showing a threat to human health.

13
296 Page 16 of 19 Water Air Soil Pollut (2021) 232: 296

Fig. 7  Linear regression analysis to determine the correlation between the cellular ROS concentration and antibiotic resistance (as
measured by the zone of inhibition) of the E. coli samples

13
Water Air Soil Pollut (2021) 232: 296 Page 17 of 19 296

Acknowledgements We thank Drs. C. Gyles (University of from healthy and diarrheic swine. Applied and Environ‑
Guelph) and B. Ciebin (Ontario Ministry of Health) for provid- mental Microbiology, 72(7), 4782–4795.
ing the pathogenic E. coli strains in this study. We also thank Cloete, T. E. (2003). Resistance mechanisms of bacteria to
B. Hicks and N. Ek for helping to run the multiplex PCR and antimicrobial compounds. International Biodeterioration
N. Fligg with collecting the sediment samples. & Biodegradation, 51(4), 277–282. https://​doi.​org/​10.​
1016/​S0964-​8305(03)​00042-8
Funding This work was funded by the National Science and Costerton, J. W. (1995). Overview of microbial biofilms. Jour‑
Engineering Research Council of Canada. nal of Industrial Microbiology, 15(3), 137–140.
Dwyer, D. J., Belenky, P. A., Yang, J. H., MacDonald, I. C.,
Martell, J. D., Takahashi, N., et al. (2014). Antibiotics
Data Availability The datasets generated during and/or ana-
induce redox-related physiological alterations as part of
lyzed during the current study are available from the corre-
their lethality. Proceedings of the National Academy of
sponding author on reasonable request.
Sciences, 111(20), E2100–E2109.
Falagas, M. E., & Kasiakou, S. K. (2006). Toxicity of poly-
Declarations
myxins: A systematic review of the evidence from old and
recent studies. Critical Care, 10(1), 1–13. https://​doi.​org/​
Conflict of Interest The authors declare no competing inter- 10.​1186/​cc3995
ests. Gilbert, P., Jones, M., Allison, D., Heys, S., Maira, T., & Wood,
P. (1998). The use of poloxamer hydrogels for the assess-
ment of biofilm susceptibility towards biocide treatments.
Journal of Applied Microbiology, 85(6), 985–990.
References Gyles, C., Johnson, R., Gao, A., Ziebell, K., Pierard, D., Alek-
sic, S., & Boerlin, P. (1998). Association of enterohem-
Anastasi, E., Matthews, B., Stratton, H., & Katouli, M. (2012). orrhagic Escherichia coli hemolysin with serotypes of
Pathogenic Escherichia coli found in sewage treatment Shiga-like-toxin-producing Escherichia coli of human and
plants and environmental waters. Applied and Environ‑ bovine origins. Applied and Environmental Microbiology,
mental Microbiology, 78(16), 5536–5541. 64(11), 4134–4141.
Badgley, B. D., Nayak, B. S., & Harwood, V. J. (2010). The Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Bac-
importance of sediment and submerged aquatic vegeta- terial biofilms: From the natural environment to infectious
tion as potential habitats for persistent strains of entero- diseases. Nature Reviews Microbiology, 2(2), 95–108.
cocci in a subtropical watershed. Water Research, 44(20), Hong, Y., Zeng, J., Wang, X., Drlica, K., & Zhao, X. (2019).
5857–5866. Post-stress bacterial cell death mediated by reactive oxy-
Battesti, A., Majdalani, N., & Gottesman, S. (2011). The RpoS- gen species. Proceedings of the National Academy of Sci‑
mediated general stress response in Escherichia coli. ences, 116(20), 10064–10071.
Annual Review of Microbiology, 65, 189–213. Hood, M. A., & Ness, G. E. (1982). Survival of Vibrio chol-
Begum, Y. A., Talukder, K. A., Nair, G. B., Qadri, F., Sack, erae and Escherichia coli in estuarine waters and sedi-
R. B., & Svennerholm, A.-M. (2005). Enterotoxigenic ments. Applied and Environmental Microbiology, 43(3),
Escherichia coli isolated from surface water in urban and 578–584.
rural areas of Bangladesh. Journal of Clinical Microbiol‑ Ishii, S., Ksoll, W. B., Hicks, R. E., & Sadowsky, M. J. (2006).
ogy, 43(7), 3582–3583. Presence and growth of naturalized Escherichia coli in
Belenky, P., & Collins, J. J. (2011). Antioxidant strategies to temperate soils from Lake Superior watersheds. Applied
tolerate antibiotics. Science, 334(6058), 915–916. and Environmental Microbiology, 72(1), 612–621.
Beloin, C., Roux, A., & Ghigo, J.-M. (2008). Escherichia coli Jakubowski, W., & Bartosz, G. (2000). 2, 7-Dichlorofluorescin
Biofilms. In Bacterial Biofilms (pp. 249–289). Springer. oxidation and reactive oxygen species: What does it meas-
Boles, B. R., & Singh, P. K. (2008). Endogenous oxidative ure? Cell Biology International, 24(10), 757–760.
stress produces diversity and adaptability in biofilm com- Jakubowski, W., & Walkowiak, B. (2015). Resistance of oxi-
munities. Proceedings of the National Academy of Sci‑ dative stress in biofilm and planktonic cells. Brazilian
ences, 105(34), 12503–12508. Archives of Biology and Technology, 58(2), 300–308.
Burton, G., Gunnison, D., & Lanza, G. R. (1987). Sur- Johnson, T. R., & Case, C. L. (2013). Laboratory Experiments
vival of pathogenic bacteria in various freshwater sedi- in Microbiology (10th ed.). Pearson.
ments. Applied and Environmental Microbiology, 53(4), Kirby, A. E., Garner, K., & Levin, B. R. (2012). The relative
633–638. contributions of physical structure and cell density to the
Canadian Institute for Health Informaiton. (2017). Guidelines antibiotic susceptibility of bacteria in biofilms. Antimicro‑
for Canadian Drinking Water Quality—Summary Table bial Agents and Chemotherapy, 56(6), 2967–2975.
(Health Canada). Water and Air Quality Bureau, Healthy Kohanski, M. A., Dwyer, D. J., & Collins, J. J. (2010). How
Environments and Consumer Safety Branch, Health Can- antibiotics kill bacteria: From targets to networks. Nature
ada, Ottawa, Ontario. Reviews Microbiology, 8(6), 423.
Chapman, T. A., Wu, X.-Y., Barchia, I., Bettelheim, K. A., Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, C. A.,
Driesen, S., Trott, D., et al. (2006). Comparison of viru- & Collins, J. J. (2007). A common mechanism of cellu-
lence gene profiles of Escherichia coli strains isolated lar death induced by bactericidal antibiotics. Cell, 130(5),
797–810.

13
296 Page 18 of 19 Water Air Soil Pollut (2021) 232: 296

Ksoll, W. B., Ishii, S., Sadowsky, M. J., & Hicks, R. E. (2007). Pelicano, H., Carney, D., & Huang, P. (2004). ROS stress in
Presence and sources of fecal coliform bacteria in epilithic cancer cells and therapeutic implications. Drug Resistance
periphyton communities of Lake Superior. Applied and Updates, 7(2), 97–110.
Environmental Microbiology, 73(12), 3771–3778. Percival, S. L., Chalmers, R., Embrey, M., Hunter, P. R., Sell-
Kümmerer, K., & Henninger, A. (2003). Promoting resistance wood, J., & Wyn-Jones, P. (2004). Microbiology of water‑
by the emission of antibiotics from hospitals and house- borne diseases (1st ed.). Academic Press.
holds into effluent. Clinical Microbiology and Infection, Prakash, B., Veeregowda, B. M., & Krishnappa, G. (2003).
9(12), 1203–1214. Biofilms: A survival strategy of bacteria. Current Sci‑
Kümmerer, K. (2009). Antibiotics in the aquatic environment– ence, 85(9), 1299–1307.
A review–part I. Chemosphere, 75(4), 417–434. https://​ Public Health Agency of Canada. (2016). Canadian Antimi‑
doi.​org/​10.​1016/j.​chemo​sphere.​2008.​11.​086 crobial Resistance Surveillance System Report.
Le Fevre, N., & Lewis, G. (2003). The role of resuspension in Quero, G. M., Fasolato, L., Vignaroli, C., & Luna, G. M.
enterococci distribution in water at an urban beach. Water (2015). Understanding the association of Escherichia
Science and Technology, 47(3), 205–210. coli with diverse macroalgae in the lagoon of Venice.
Lewis, K. (2001). Riddle of biofilm resistance. Antimicrobial Scientific Reports, 5, 10969. https://​doi.​org/​10.​1038/​
Agents and Chemotherapy, 45(4), 999–1007. srep1​0969
López-Saucedo, C., Cerna, J. F., Villegas-Sepulveda, N., Rappelli, P., Maddau, G., Mannu, F., Colombo, M., Fiori, P.,
Thompson, R., Velazquez, F. R., Torres, J., et al. (2003). & Cappuccinelli, P. (2001). Development of a set of mul-
Single multiplex polymerase chain reaction to detect tiplex PCR assays for the simultaneous identification of
diverse loci associated with diarrheagenic Escherichia enterotoxigenic, enteropathogenic, enterohemorrhagic and
coli. Emerging Infectious Diseases, 9(1), 127. enteroinvasive Escherichia coli. The New Microbiologica,
Luna, G., Vignaroli, C., Rinaldi, C., Pusceddu, A., Nicoletti, 24(1), 77–83.
L., Gabellini, M., et al. (2010). Extraintestinal Escheri- Reinthaler, F. F., Posch, J., Feierl, G., Wüst, G., Haas, D.,
chia coli carrying virulence genes in coastal marine sedi- Ruckenbauer, G., et al. (2003). Antibiotic resistance of E
ments. Applied and Environment Microbiology, 76(17), coli in sewage and sludge. Water Research, 37(8), 1685–
5659–5668. 1690. https://​doi.​org/​10.​1016/​S0043-​1354(02)​00569-9
Macia, M., Rojo-Molinero, E., & Oliver, A. (2014). Antimi- Reisner, A., Krogfelt, K. A., Klein, B. M., Zechner, E. L., &
crobial susceptibility testing in biofilm-growing bacteria. Molin, S. (2006). In vitro biofilm formation of commensal
Clinical Microbiology and Infection, 20(10), 981–990. and pathogenic Escherichia coli strains: Impact of envi-
Magajna, B. A., & Schraft, H. (2015). Campylobacter jejuni ronmental and genetic factors. Journal of Bacteriology,
biofilm cells become viable but non-culturable (VBNC) 188(10), 3572–3581.
in low nutrient conditions at 4 C more quickly than their Sabaté, M., Prats, G., Moreno, E., Ballesté, E., Blanch, A. R.,
planktonic counterparts. Food Control, 50, 45–50. & Andreu, A. (2008). Virulence and antimicrobial resist-
Mah, T.-F.C., & O’Toole, G. A. (2001). Mechanisms of biofilm ance profiles among Escherichia coli strains isolated from
resistance to antimicrobial agents. Trends in Microbiol‑ human and animal wastewater. Research in Microbiology,
ogy, 9(1), 34–39. 159(4), 288–293.
Merkx-Jacques, A., Coors, A., Brousseau, R., Masson, L., Sáenz, Y., Brinas, L., Domínguez, E., Ruiz, J., Zarazaga, M.,
Mazza, A., Tien, Y.-C., & Topp, E. (2013). Evaluating the Vila, J., & Torres, C. (2004). Mechanisms of resistance
pathogenic potential of environmental Escherichia coli by in multiple-antibiotic-resistant Escherichia coli strains of
using the Caenorhabditis elegans infection model. Applied human, animal, and food origins. Antimicrobial Agents
and Environment Microbiology, 79(7), 2435–2445. and Chemotherapy, 48(10), 3996–4001.
Mokracka, J., Koczura, R., Jabłońska, L., & Kaznowski, A. Schellhorn, H. E. (2014). Elucidating the function of the RpoS
(2011). Phylogenetic groups, virulence genes and qui- regulon. Future Microbiology, 9(4), 497–507.
nolone resistance of integron-bearing Escherichia coli Sheldon, J. R., Yim, M., Saliba, J. H., Chung, W., Wong, K.-Y.,
strains isolated from a wastewater treatment plant. Antonie & Leung, K. T. (2012). Role of rpoS in Escherichia coli
Van Leeuwenhoek, 99(4), 817–824. O157:H7 strain H32 biofilm development and survival.
Moreira, S., Brown, A., Ha, R., Iserhoff, K., Yim, M., Yang, Applied and Environmental Microbiology, 78(23), 8331–
J., et al. (2012). Persistence of Escherichia coli in fresh- 8339. https://​doi.​org/​10.​1128/​AEM.​02149-​12
water periphyton: Biofilm-forming capacity as a selective Sherer, B. M., Miner, J. R., Moore, J. A., & Buckhouse, J.
advantage. FEMS Microbiology Ecology, 79(3), 608–618. C. (1992). Indicator bacterial survival in stream sedi-
Neilands, J. (1995). Siderophores: Structure and function of ments. Journal of Environmental Quality, 21(4), 591–
microbial iron transport compounds. Journal of Biologi‑ 595. https://​doi.​org/​10.​2134/​jeq19​92.​00472​42500​21000​
cal Chemistry, 270(45), 26723–26726. 40011x
Pamp, S. J., Gjermansen, M., Johansen, H. K., & Tolker- Tillett, D., Dittmann, E., Erhard, M., Von Döhren, H., Börner,
Nielsen, T. (2008). Tolerance to the antimicrobial peptide T., & Neilan, B. A. (2000). Structural organization of
colistin in Pseudomonas aeruginosa biofilms is linked to microcystin biosynthesis in Microcystis aeruginosa
metabolically active cells, and depends on the pmr and PCC7806: An integrated peptide–polyketide synthetase
mexAB-oprM genes. Molecular Microbiology, 68(1), system. Chemistry & Biology, 7(10), 753–764.
223–240. Touchon, M., Hoede, C., Tenaillon, O., Barbe, V., Baeriswyl,
S., Bidet, P., et al. (2009). Organised genome dynamics in

13
Water Air Soil Pollut (2021) 232: 296 Page 19 of 19 296

the Escherichia coli species results in highly diverse adap- treatment: Implications for environmental discharge
tive paths. PLoS genetics, 5(1), e1000344. and wastewater recycling. Water Research, 41(18),
Van Acker, H., & Coenye, T. (2017). The role of reactive oxy- 4164–4176.
gen species in antibiotic-mediated killing of bacteria. Wiles, T. J., Kulesus, R. R., & Mulvey, M. A. (2008). Origins and
Trends in Microbiology, 25(6), 456–466. virulence mechanisms of uropathogenic Escherichia coli.
Van Acker, H., Van Dijck, P., & Coenye, T. (2014). Molecu- Experimental and Molecular Pathology, 85(1), 11–19.
lar mechanisms of antimicrobial tolerance and resist- Yamada, H., Koike, N., Ehara, T., & Matsumoto, T. (2011).
ance in bacterial and fungal biofilms. Trends in Micro‑ Measuring antimicrobial susceptibility of Pseudomonas
biology, 22(6), 326–333. https://​doi.​org/​10.​1016/j.​tim.​ aeruginosa using Poloxamer 407 gel. Journal of Infection
2014.​02.​001 and Chemotherapy, 17(2), 195–199.
Vatansever, F., de Melo, W. C., Avci, P., Vecchio, D., Sadasi- Antonoplis, A., Zang, X., Wegner, T., Wender, P. A., and
vam, M., Gupta, A., et al. (2013). Antimicrobial strategies Cegelski, L. (2019). A vancomycin-arginine conjugate
centered around reactive oxygen species–bactericidal anti- inhibits growth of carbapenemresistantE. coli and targets
biotics, photodynamic therapy, and beyond. FEMS Micro‑ cell-wall synthesis. Chemical Biology, 14(9), 2065–2070.
biology Reviews, 37(6), 955–989. Kappell, A.D., DeNies, M.S., Ahuja, N.H., Ledeboer, N.A.,
Vignaroli, C., Di Sante, L., Magi, G., Luna, G. M., Di Cesare, Newton, R.J., and Hristova, K.R. (2015). Detection of
A., Pasquaroli, S., et al. (2015). Adhesion of marine cryp- multi-drug resistant Escherichia coli inthe urban water-
tic Escherichia isolates to human intestinal epithelial cells. ways of Milwaukee, WI. Frontiers in Microbiology, 6,
The ISME Journal, 9(2), 508. 336. https://doi: 10.3389/fmicb.2015.00336
Wang, X., Zhao, X., Malik, M., & Drlica, K. (2010). Contribu-
tion of reactive oxygen species to pathways of quinolone- Publisher’s Note Springer Nature remains neutral with regard
mediated bacterial cell death. Journal of Antimicrobial to jurisdictional claims in published maps and institutional
Chemotherapy, 65(3), 520–524. affiliations.
Watkinson, A., Murby, E., & Costanzo, S. (2007). Removal
of antibiotics in conventional and advanced wastewater

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