Escherichia coli O157:H7 attachment and 
persistence within root biofilm of common 
treatment wetlands plants
Rachel J. VanKempen-Fryling & Anne K. Camper
NOTICE: this is the author’s version of a work that was accepted for publication in Ecological 
Engineering. Changes resulting from the publishing process, such as peer review, editing, 
corrections, structural formatting, and other quality control mechanisms may not be reflected in 
this document. Changes may have been made to this work since it was submitted for publication. 
A definitive version was subsequently published in Ecological Engineering, [Vol. 98, January 
2017], DOI#:10.1016/j.ecoleng.2016.10.018.
VanKempen-Fryling RJ, Camper AK “Escherichia coli O157:H7 attachment and persistence within 
root biofilm of common treatment wetlands plants,” Ecological Engineering, 2017 Jan;98:64–69.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
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Fig. 1. Representative imagesof the fourexperimental surface types takenat2, 24and168h. Fluorescencewasobservedunder558nmwavelength.Due tonaturalfluorescence
of photosystem II chlorophyll in the plant tissue, plant surface is shown in C. utriculata and S. acutus images. No stains or dyes were used in Teflon nylon imaging under the
assumption stationary cells were attached to a surface. Scale bar represents 50m.
Another serious limitation is the focus on influent to efflu-
ent measurements, assuming that E. coli O157:H7 is not surviving
within the TW. Long-term lab studies in river and reservoir water
found E. coli O157:H7 can persist up to 6 months (Liu et al., 2009).
E. coli O157:H7 may also be surviving within the biofilm matrix or
incorporated into the plants, as seen in vegetable production (Fu
et al., 2005; Dinu and Bach 2011) E. coli O157:H7 can invade plant
tissuesof leafygreensandstalks (Deeringet al., 2012).Although this
is not a health concern in TW as with edible plants, E. coli O157:H7
within vascular openings of plants could provide means of survival
and potential release.
The hypothesis was that indigenous biofilm and living plant tis-
sue (with an established biofilm) impact the persistence of E. coli
O157:H7 in a simulated TW. Two plant species, C. utriculata and S.
acutus, were used in hydroponic reactors to determine interactions
with the roots. In previous research these two obligate wetland
species were the top performers for removal efficiency of carbon,
nitrogen and sulfate in pilot-scale wetland systems (Taylor et al.,
2011). Inert Teflon nylon material was used as a control both with
and without a pre-established biofilm. Pathogen persistence on
these surfaces was assessed using qPCR and microscopy.
2. Materials and methods
2.1. Growth of E. coli O157:H7
The E. coli O157:H7 was isolated from an outbreak in 1993 (Pyle
et al., 1995). A constitutively expressed DsRed plasmid with a car-
benicillin resistance selective gene (Clonetech) was incorporated
(Klayman et al., 2009) for detection by fluorescent microscopy.
Tryptic Soy Broth (TSB) was made using non-casein enzymatic
digest of protein (Klayman et al., 2009) for plasmid retention and
expression. E. coli O157:H7 was grown overnight to approximately
108 cellsmL−1 as determined by plate count. Survival and plas-
mid stability in simulated wastewater (SW; Supplemental Table 1)
was verified by plate counts, qPCR, and epifluorescencemicroscopy
(VanKempen-Fryling et al., 2015).
2.2. Plant growth and strain collection
Shoots with substantial root mass of C. utriculata and S. acutus
grown in a controlled greenhouse environmentwereharvested and
washed with nonsterile H2O to remove excess planting medium
and loosely associated bacteria while keeping the attached biofilm.
The rinsed plants (49.3mL±26.4mL, determined by the mass
displacement) were transferred to 300mL hydroponic chemostat
reactors under 24h light at 25 ◦C and grown for 3–7days in SW.
Comparisons were made between living roots to an inert sur-
face of Teflon nylon strings (D’Addario; GHS strings) similar in
dimensions to the roots (0.79, 0.81 and 1.01mm diameter). Clean
nylon strings were added to chemostats with SW immediately
prior to E. coli O157:H7 inoculation. Alternatively, strings were
added to effluent of the established plant mesocosms for 24h
in batch, followed by the addition of SW (flow conditions) for 1
week to encourage biofilm growth prior to pathogen inoculation.
Total weight of the nylon strings, cut into 17.5 cm lengths (half
immersed), was 2.94g.
2.3. Determination of surface area
To estimate the E. coli O157:H7 surface area attachment, stan-
dard curves were generated relating wet mass to root surface area.
Representative roots were excised, imaged, and analyzed with the
Rootscan® program to determine the surface area. The wet mass
was plotted against surface area to generate standard curves (Sup-
plemental Fig. 1). C. utriculata’s two distinct root types (taproot and
branched) formed unique standard curves; DNA extraction for C.
utriculata samples were separated by root type. Teflon surface area
was estimated by treating the strings as open ended cylinders. The
0%
20%
40%
60%
80%
100%
0 50 100 150 200
per
cen
t cl
um
ps 
per
 
tot
al s
ize
 ra
nge
s
0%
20%
40%
60%
80%
100%
0 20 40 60 80 100 120 140 160 180
Per
cen
t cl
um
ps 
per
 
tot
al s
ize
 ra
nge
s Single Cels
2-10 cels
11-100 cels
>100 cels
0%
10%
20%
30%
40%
50%
60%
70%
80%
0 50 100 150 200
per
cen
t cl
um
ps 
per
 
tot
al s
ize
 ra
nge
s
0%
20%
40%
60%
80%
100%
0 20 40 60 80 100 120 140 160 180Pe
rce
nt c
lu
mps
 pe
r 
tot
al s
ize
 ra
nge
s
Time (hours)
A.
D.
C.
B.
Fig. 2.Distribution of cel clumping over total counts per time point for each surface: (A)C. utriculata, (B)S. acutus, (C) clean Teflon nylon, and (D) biofilm Teflon nylon. Root
locations were combined for a total mean. Bars represent standard deviation from al experimental replicates.
total surface area of the 24 sample strings, 8 of each diameter was
53.32 cm2.
2.4. Reactor series
20 mL of overnightE. coliO157:H7 culture was spun at 4816 x g
in triplicate, washed in nanopure water, and concentrated into
1 mL. For each triplicate experimental condition (C. utriculata,S.
acutus,clean nylon, and established biofilm nylon), 1 mL of inocu-
lum was added and mixed via pipetting and rotating the plant or
nylon. One additional non-inoculated reactor served as a control.
Flow was at 3 mL min−1(100 min hydraulic residence times (HRT))
for one week. A time zero sample was taken from each reactor
immediately after inoculation. Effluent samples were colected at
45-min intervals for 6 h. One replicate reactor was destructively
sampled at 135 min, approximately 1 HRT (initial attachment).
Another effluent sample was taken at 24 h and the second replicate
reactor destructively sampled. The third replicate was destructively
sampled at one week. The uninoculated control reactor effluent was
sampled at 0 and 6 h and destructively sampled at one week. Al
water samples were frozen for further analysis.
Submerged roots from destructively sampled reactors were
divided in half by length for differentiation of basal and root tip,
rinsed with nanopure water to remove unattached cels, placed
into 2 mL microcentrifuge tubes and frozen for DNA extraction.
The nylon samples were taken in entirety, placed into 50 mL Falcon
tubes, and frozen. A representative root or nylon sample was puled
through a 2 mm square glass capilary approximately 90–110 mm
in length (Wale Apparatus, PA) using a wire hook (Aircraft Spruce),
the interior of the capilary rinsed with nanopure water to slough
off unattached cels, and attachment observed by epifluorescence
microscopy.
2.5. Epifluorescence microscopy
The glass capilaries were viewed under 600×magnification
(Nikon; Melvile, NY) and 558 nm wavelength for DsRed plasmid
excitation on a Nikon Eclipse E-800 microscope with a cooled CCD
camera (Photometrics). The root tip, mid-root, and top root were
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
2 24 168 2 24 168 2 24 168 2 24 168 2 24 168 2 24 168
Branched Thick Inactive Active Biofilm Clean
C. utriculata S. acutus Nylon
rel
ativ
e a
bun
da
nce
 (g
ene
 co
pie
s/c
m2 )
Fig. 3.Relative abundance ofE. coliO157:H7 per surface area normalized to back-
ground fluorescence. The data is differentiated over time and by root type or nylon
condition for each respective surface.
Table 1
DNA extraction quantification.
Sample Type Concentration
(ng/uL)
A260/230
C. utriculatawater
C. utriculataroot
S. acutuswater
S. acutusroot
Nylon clean water
Nylon clean surface
Nylon biofilm water
Nylon biofilm surface
80.08±40.56
96.38±30.02
57.34±51.16
72.09±37.17
59.81±29.91
48.37±36.63
72.22±45.80
40.03±24.86
2.01±0.21
2.02±0.15
1.49±0.61
1.88±0.25
2.04±0.21
1.94±0.70
1.95±0.48
1.99±0.34
scanned and 20 images taken in each area for samples colected at
2, 24 and 168 h. Natural photosystem I chlorophyl fluorescence
alowed the plant surface to be viewed without stain. The images
were semi quantitatively analyzed (MetaMorph®Image Analysis
Software) to determine number of cels and colony clump size using
the average pixel area forE. coliO157:H7 single cel fluorescence
(Wilson et al., 2004; Behnke et al., 2011). Thresholding was used to
subtract background autofluorescence and remaining objects were
counted.
2.6. DNA extraction and qPCR analysis
Thawed water samples were spun down at 4816×gfor 20 min
at 4◦C. The peleted sample was resuspended and transferred to
a PowerBead tube from the PowerSoil®DNA Isolation Kit (MoBio
Laboratories; Carlsbad, CA, USA). Mass of the roots was determined
prior to transfer to a PowerBead tube. DNA extraction folowed the
kit protocol except a Fastprep®-24 bead beater (M.P. Biomedicals;
Santa Ana, CA, USA) was used at 5.5 m sec−1for 45 s. Cel mate-
rial on the nylon strings was removed by adding glass beads to
the 5 mL mark of the 50 mL conical Falcon tube with 10 mL auto-
claved nanopure H2O, and vortexed for 60 s. The liquid fraction was
removed and centrifuged at 4816×gfor 5 min. The supernatant
was removed and the pelet resuspended and added to a PowerBead
tube for extraction as previously described. DNA was isolated in
100 L of DEPC water (Ambion®; Grand Island, NY, USA) and quan-
tified using a Nanodrop® ND-1000 spectrophotometer (Thermo
Scientific; Wilmington, DE, USA). Average quantification and purity
per sample type is listed inTable 1.
qPCR primers targeted the Shiga toxin 2 gene unique to the
O157:H7 serotype (Jothikumar and Griffiths, 2002). Control exper-
iments using plate counts verified that the cels had one copy of
the fStx1andStx2genes (Shaikh and Tarr, 2003). A 25 L reaction
mixture was prepared using SYBR Green Master Mix (Kapa Biosys-
tems; Wilmington, MA, USA), 0.5 mM primer, and 3.1×10−3mM
DNA diluted from the extraction. Programs for qPCR can be found
VanKempen-Fryling et al., 2015.
2.7. Data analysis
Multifactor analysis of variance (ANOVA) was performed to
determine statistical significance between surface types and time.
Comparisons were analyzed between plants and Teflon nylon
strings, the root tip or basal root, established biofilm compared
to clean nylon, and time. General linear models (GLM) were used
to compare interactions using Minitab 17®. Statistical significance
between specific factors for each variable were identified by pair-
wise Tukey’spost hocmultiple comparison test using a 0.05p-value
cut-off. The experimental replicates were input as random variables
nested within time.
3. Results
Initial washout ofE. coliO157:H7 during the first four HRT was
similar for al systems (Supplemental Fig. 2). A twenty-four hour
sample showed the continued release ofE. coliO157:H7 from the
inoculated reactors, which would result from detachment of pre-
viously attached cels.
E. coliO157:H7 could be imaged on the surfaces throughout the
time course (Fig. 1). Initial attachment was mainly single cels and
there was no observable difference in abundance by location or sur-
face type. At 24 h, microcolony formation was seen. The final time
point at 168 h showed a lower, sustainedE. coliO157:H7 cel pres-
ence and more variable microcolony formation. These observations
were substantiated with the MetaMorph®Image Analysis software
(Fig. 2) and reported as a percentage of the total events for al size
ranges from smal and medium clusters to large, >100 cel forma-
tions. The number of clumps greater than single cels increased on
al surfaces except the biofilm-colonized nylon, where increases
were limited to clumps >100 cels. This is more dramatic inS. acutus
(23 to 36 events) and clean nylon experiments (96 to 109 events).
Single cels were more varied between replicates, as seen through
the large error bars. The total clump counts decreased from 2 to
168 h (22.6 average events to 13.3 average events), however the
largest formations increased (average increase of 1 event for >100
cel clump size).
qPCR determined relative target cel abundance on inoculated
vs non-inoculated surfaces over time. For the root experiments,
location (root tip or basal root) and condition (branched or thick
forC. utriculata; red/live or black/dead coloration forS. acutus)
of the root were analyzed. For nylon experiments, the biofilm
presence/absence was compared. For al experimental surfaces
(Fig. 3), the population decreased during the period between 2
and 168 h, averaging a reduction from 704 gene copies cm−2to 55
gene copies cm−2(P < 0.001). Attachment onS. acutuswas signifi-
cantly higher (average 493 gene copies cm−2) thanC. utriculataby
209 gene copies cm=2(P = 0.002), as wel as both nylon replicates
(P = 0.021) in al time points. Nylon replicates andC. utriculatawere
statisticaly equivalent with the lowestE. coliO157:H7 attachment
across al time points. ForC. utriculata,E. coliO157:H7 preferred
branched roots to uniform taproots with an average difference of
82.1 gene copies cm−2to 281 gene copies cm−2(P < 0.001) across
al time points. InS. acutus, no meaningful effect of live roots was
observed. Localization was taken into account between root tip and
the basal roots, but there was no preference.
The two abiotic surfaces were also compared to identify any
difference with respect to preexisting biofilm alone. There was no
significant difference inE. coliO157:H7 attachment. A slight inter-
action between surface and time was seen from initial attachment
to 24h in clean nylon over biofilm-colonized nylon (Supplemental
Fig. 3).
4. Discussion
Epifluorescence microscopy and qPCR showed that E. coli
O157:H7 attached and persisted on all surfaces under flow con-
ditions for the duration of the experiment. Attachment was
significantly lower on abiotic nylon controls compared to S. acu-
tus roots. This supports the hypothesis that there is a benefit for
survival or potential growth for bacterial pathogens within an
established root biofilm (Tyler and Triplett 2008). Many plants
have been shown to foster biofilm growth along the root struc-
tures for reasons ranging from protection against plant pathogens
to nitrogen fixation (Gruyer et al., 2013). Wetland plants provide
labile organic carbon exudates during active periods and by slow
release during decay (García et al., 2010; Iannelli et al., 2011), and
also contribute ions and oxygen (Hawkes et al., 2007). Given previ-
ous extensive research on interactions between plants and bacteria
(DanhornandFuqua, 2007) it is possible thatE. coliO157:H7attach-
ment and persistencemay benefit from these interactions (Deering
et al., 2012). Less attachment to C. utriculata roots suggests that this
plant may have a lower potential for pathogen retention in TW.
Interestingly, there was no statistical difference in pathogen
attachment and persistence between the clean nylon surface and
nylon with an established biofilm. It was hypothesized that com-
petition with the natural consortia of bacteria in an environment
would protect against invading E. coli O157:H7 cells since this is
commonwith other organisms (Hibbing et al., 2010) andpathogens
in aquatic biofilms (Nocker et al., 2013). The mechanisms in this
system remain unknown and will warrant further research.
The fateof theundetectedE. coliO157:H7cells andwhether they
were sloughed into the effluent feed and still viable or inactivated
by predation or competition was not determined. Other studies
have indicated these factors play a large role in lowering human
pathogenretention in theenvironment (Boutilier et al., 2009; Jasper
et al., 2013; Li et al., 2014), however for strains with a low infection
rate such as E. coliO157:H7, thesemaynot be enough to completely
eradicate subsequent release and the potential infection. If E. coli
O157:H7 is able to attach to roots, it may have prolonged ability to
survive in aTW. Further research is needed for optimizingpathogen
removal in TW.
Acknowledgements
Betsey Pitts at the Center for BiofilmEngineering provided assis-
tance with microscopy and the Land Resources and Environmental
Sciences and Plant Sciences departments provided assistance with
the plant work. This research was supported by the Molecular Bio-
sciencesProgramFellowship, theMontana InstituteonEcosystems,
and the Center for Biofilm Engineering.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ecoleng.2016.10.
018.
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