APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2011, p. 2113–2121
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 6
General Suppression of Escherichia coli O157:H7 in Sand-Based
Dairy Livestock Bedding?†
Andreas Westphal,1,2Michele L. Williams,3Fulya Baysal-Gurel,2
Jeffrey T. LeJeune,3and Brian B. McSpadden Gardener2*
Julius Ku ¨hn-Institut, Federal Research Centre for Cultivated Plants, Mu ¨nster, Germany,1and Department of
Plant Pathology2and Food and Animal Health Research Program,3Ohio Agriculture Research and
Development Center (OARDC), The Ohio State University, Wooster, Ohio
Received 11 July 2010/Accepted 11 January 2011
Sand bedding material is frequently used in dairy operations to reduce the occurrence of mastitis and
enhance cow comfort. One objective of this work was to determine if sand-based bedding also supported the
microbiologically based suppression of an introduced bacterial pathogen. Bedding samples were collected in
summer, fall, and winter from various locations within a dairy operation and tested for their ability to suppress
introduced populations of Escherichia coli O157:H7. All sources of bedding displayed a heat-sensitive suppres-
siveness to the pathogen. Differences in suppressiveness were also noted between different samples at room
temperature. At just 1 day postinoculation (dpi), the recycled sand bedding catalyzed up to a 1,000-fold
reduction in E. coli counts, typically 10-fold greater than the reduction achieved with other substrates,
depending on the sampling date. All bedding substrates were able to reduce E. coli populations by over
10,000-fold within 7 to 15 dpi, regardless of sampling date. Terminal restriction fragment length polymorphism
(T-RFLP) analysis was used to identify bacterial populations potentially associated with the noted suppression
of E. coli O157:H7 in sand bedding. Eleven terminal restriction fragments (TRFs) were overrepresented in
paired comparisons of suppressive and nonsuppressive specimens at multiple sampling points, indicating that
they may represent environmentally stable populations of pathogen-suppressing bacteria. Cloning and se-
quencing of these TRFs indicated that they represent a diverse subset of bacteria, belonging to the Cytophaga-
Flexibacter-Bacteroidetes, Gammaproteobacteria, and Firmicutes, only a few of which have previously been iden-
tified in livestock manure. Such data indicate that microbial suppression may be harnessed to develop new
options for mitigating the risk and dispersal of zoonotic bacterial pathogens on dairy farms.
Escherichia coli O157:H7 is a food-borne pathogen of global
public health significance (39), so understanding the factors
affecting its survival in the environment is critical to minimize
its impact on human health. Cattle manure is a major reservoir
of Escherichia coli O157:H7. However, the factors contributing
to bovine colonization and shedding of E. coli O157:H7 are
poorly understood, and there are still very few tools available
to control these zoonotic bacteria in cattle populations (23).
Early epidemiological studies in livestock populations showed
that individual farms often tended to maintain either a high or
a low prevalence of E. coli O157:H7 over time (18, 45). This
relative stability of pathogen prevalence on individual farms
has been interpreted to be an indication for a role of stable
farm management factors in governing the relative abundance
of E. coli O157:H7 in cattle populations. In addition, the prev-
alence of E. coli O157:H7 is seasonally modulated. Most stud-
ies indicate a peak in prevalence in cattle that occurs in sum-
mer and is coincident with the seasonal peak in human
illnesses (3, 5, 11, 12, 17).
Interestingly, there are several similarities in the patterns of
E. coli O157:H7 carriage by dairy cattle and cases of mastitis
caused by E. coli (and other coliforms) in cows. Stanford et al.
(46) demonstrated an association between somatic cell counts,
a mastitis indicator, and E. coli O157:H7 prevalence on dairy
farms. As with the E. coli O157:H7 prevalence in cattle, the
incidence of coliform mastitis also fluctuates in the same sea-
sonal manner (44). The primary source of mastitis-causing
coliforms is the bedding material, and the incidence of co-
liform mastitis is influenced by the type of bedding material
used (sand or sawdust) (19, 33, 36, 56). Moreover, LeJeune
and Kauffman (22) demonstrated that dairy herds bedded on
sawdust showed a significantly greater prevalence of E. coli
O157:H7 than herds bedded on sand. Thus, because contami-
nated livestock bedding can significantly increase E. coli O157:H7
carriage in cattle, successful interventions targeted at this stage
of production could reduce prevalence on the farm and have
positive downstream impacts on human health.
Pathogen suppression can be mediated by a variety of biotic
and abiotic soil factors. In solid-phase media, pathogens are
the target of inhibitory soil functions that prevent them from
expressing pathological effects to their full potential (3). Gen-
eral suppression of plant pathogens is mediated by the quantity
and quality of soil organic matter that supports the resident
microbial populations (20, 32). Indeed, judicious applications
of soil organic matter can result in the suppression of diverse
plant diseases in field and greenhouse systems (43, 47). How-
ever, complex interactions in the environment can significantly
affect the suppressiveness of such amendments (6, 41). It is
* Corresponding author. Mailing address: Department of Plant Pa-
thology, The Ohio State University, OARDC, 1680 Madison Avenue,
Wooster, OH 44691. Phone: (330) 202-3565. Fax: (330) 202-3841.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 21 January 2011.
supposed that competition for nutrients is the primary driver of
general suppression. In contrast, specific suppression is medi-
ated by one or just a few microbial populations that antagonize
resident pathogens (49). Production of antibiotics is consid-
ered a key feature of microbes associated with specific sup-
pression (37, 38). Studies of suppressive soils have led to the
identification of useful biological control strategies for sup-
pressing plant pathogens. For example, culture-dependent (52)
and culture-independent (54, 55) methods have been used to
identify and isolate multiple microbial antagonists that can
effectively suppress soilborne pathogens in field trials (34, 35).
As a result, studies of soil microbial community structure are
considered useful for developing innovative methods for
pathogen and disease suppression (26).
Using concepts garnered from microbially based disease
suppression in soils, we hypothesized that suppression of E.
coli O157:H7 would be mediated by the predominant micro-
organisms present in the bedding in and around the cattle. The
objectives of the current study were to determine (i) the degree
to which biologically based suppression of E. coli O157:H7
occurred in bedding samples taken from different points on the
farm and (ii) which bacteria present in such samples might
contribute to the noted suppression. We focused on bacteria
because previous studies had demonstrated that an over-
whelming proportion of the microbial communities of manure
were expected to be prokaryotic.
MATERIALS AND METHODS
Livestock bedding sources. Samples were taken on18 August 2008, 10 Novem-
ber 2008, and 23 March 2009 (subsequently referred to as August, November,
and March, respectively) from a dairy operation near Orrville, OH. At this
operation, about 2,500 head of cattle were kept for milk production under
free-stall housing conditions, where animals had free access to enter and leave
resting areas in the barn that were lined at the bottom with a sand mix as bedding
material. This dairy used a sand-manure separator (an auger 90 cm in diameter
by 9.9 m long; McLanahan Corporation, Hollidaysburg, PA). Three times per
week, a mix of fresh and recycled sand (1:1) was supplied as bedding material to
the resting boxes with a mechanical distribution wagon. Through the action of
the cows entering and exiting the stall, the sand was pushed from the front to the
back end of the stall and eventually into the gutter, where it was mechanically
collected and transferred to a washing facility for recycling. In this process, the
material was moved by a separator auger from a collection pit into a temporary
storage pile while it was being washed with water to separate the organic material
from the sand. After it was air dried for 2 or more days in an open stockpile, the
used sand was mixed with freshly purchased commercial clean sand and distrib-
uted in the livestock stalls to start the cycle anew. In this study, samples were
collected from four sources within the recycling process of the livestock bedding:
(i) fresh, consisting of recently applied bedding sand collected from the front of
the resting stall (bedding 1); (ii) in use, consisting of bedding from the rear of the
resting stall (bedding 2); (iii) washed, consisting of used livestock bedding col-
lected immediately after separation in the sand-manure separator (bedding 3);
and (iv) recycled, consisting of washed bedding material rested for 2 or more
days in a pile (bedding 4).
Samples of 1,200 ml were taken and placed in plastic bags for transport to the
laboratory; beddings 1 and 2 were collected from eight stall locations (four in the
August experiment) from opposite sides of the barns. Samples of bedding 3 were
collected as far distant as possible in a circular pattern from around a pile of
1.5 m in height and 2.5 m in diameter 90 cm off the ground. Samples of bedding
4 were collected from a pile 2.0 m in height and 5 m in diameter 90 cm off the
ground. Samples were transported to the laboratory in ice coolers.
Chemical analysis of the bedding material. A portion of each bedding sample
was submitted to the Service Testing and Research Laboratory (STAR lab) at the
Ohio State Agricultural Research and Development Center (OARDC), Wooster,
OH, for analysis of soil chemical parameters. The organic matter content was
determined by the loss-on-ignition method (13), the pH was determined by the
method by Thomas (48), nitrogen was quantified as described by Mulvaney (30),
and carbon was quantified by the ISO 10694:1995(E) method (2).
Heat treatment and inoculation of bedding samples with E. coli O157:H7.
After overnight storage in the laboratory, two 150-g portions of each of the
samples were weighed into 250-ml Erlenmeyer flasks (250-ml beakers for the
August experiment) and covered with aluminum foil. One set remained non-
heated, and a second set was heated for 30 min at 80°C. For the latter, glass
beakers with the contained bedding material were submerged in the preheated
water of a 20-liter-capacity water bath and kept there for 30 min after a moni-
toring sample had reached the target temperature (experiments 1 and 2) or were
kept in the water bath for 30 min total (experiment 3). This heat treatment was
chosen because most bacteria should be severely reduced in number if they were
not killed off at this temperature; such heat treatments had been used to disrupt
the microbial communities of container substrates and suppressive soils (4, 52).
All inoculations were done with a green fluorescent protein-labeled E. coli
O157:H7 strain (American Type Culture Collection strain ATCC 43888). Such
transformation was previously demonstrated to be stable for at least 24 days
when survival in fruit juices was monitored (16), and the nontoxigenic strain was
expected to act similar to the toxigenic equivalent (21). After cooling of the
heated bedding samples to room temperature, all samples were inoculated with
suspensions of overnight cultures of this strain that had been spun down to
remove spent medium, resuspended in 1? phosphate-buffered saline (PBS), and
adjusted to deliver concentrations of 107CFU per gram of bedding material.
This was determined spectrophotometrically at 0.16 to 0.35 optical density (OD)
units/ml. The containers with the inoculated bedding samples were closed with
their corresponding aluminum foil and incubated at room temperature (21°C) in
the laboratory in the dark. Samples were taken after vigorous mixing with a
spatula for enumeration of the CFU of E. coli O157:H7 and for DNA extraction
en route to terminal restriction fragment length polymorphism (T-RFLP) anal-
Enumeration of CFU of E. coli O157:H7. In all three experiments, samples
were taken 1 day, 1 week, and 2 weeks after inoculation (experiment 1, at 1, 8,
and 15 days postinoculation [dpi]; experiment 2, at 1, 7, and 15 dpi; and exper-
iment 3, at 1, 10, and 17 dpi). Depending on the expected numbers of CFU of
E. coli O157:H7, samples of 1 to 10 g were taken from the incubated bedding
sources and suspended in 20 to 50 ml of 1? PBS (8.00 g NaCl, 0.20 g KCl, 1.44 g
Na2HPO4, and 0.24 g KH2PO4adjusted to pH 7.4) by vortexing 4 times for 15 s
each time, processing in a sonicator (Aquasonic ultrasonic cleaner 75HT; VWR,
West Chester, PA) for 1 min at 35 kHz, and vortexing at room temperature for
15 s. These suspensions were either plated directly onto the medium plates or
entered into a 5-fold dilution series in 1? PBS in microtiter plates and then
plated. The medium plates were LB medium (10 g Bacto tryptone, 5 g Bacto
yeast extract, 5 g NaCl, and 1 liter double-distilled water adjusted to pH 7; 15 g
agar was added and the medium was autoclaved for 20 min) amended, after it
was cooled to 50°C, with ampicillin at 96 ?g/ml and cycloheximide at 50 ?g/ml on
10-cm-diameter agar plates.
After incubation at 37°C for 24 h, colonies were counted over UV light, and
the terminal dilution factor and frequency of the green fluorescent colonies were
recorded at the proper dilutions. The numbers of CFU per gram bedding ma-
terial were calculated by a most-probable-number approach using the original
terminal dilution factors. The terminal dilution factors for each sample source
were converted to log-transformed data [log10(x) CFU per gram] by the proper
formulas, considering the different dilution ratios, and the medians were pre-
sented in box plots; statistical groupings were derived from the analysis of the
terminal dilution factors of the 5-fold dilution series.
DNA isolation. On the days of dilution plating, 0.25-g subsamples (0.25- to
0.35-g subsamples in experiment 1) of the incubated material were weighed into
centrifuge tubes and frozen at ?80°C for at least 24 h before DNA was extracted
with a PowerSoil DNA extraction kit (MO BIO Laboratories, Carlsbad, CA).
DNA extraction was done according to the manufacturer’s protocol, with two
exceptions: first, centrifugation was at 9,000 ? g (10,000 rpm) whenever
10,000 ? g was suggested, and second, 450 ?l was collected from each of the
samples in the extraction tubes at the first collection step where the protocol
suggested 400 to 500 ?l to improve sample size uniformity. After extraction,
DNA was either used immediately (being stored at 4°C between daily oper-
ations) or stored at ?20°C.
DNA amplification and digestion and fragment analysis. The 16S rRNA genes
in the DNA extracts of the bedding material were amplified with primer 8F
(5?-AGAGTTTGATCCTGGCTCAG-3?) labeled with WellRED dye D4
(Sigma, Proligo, St. Louis, MO) and primer 1492R (5?-ACGGCTACCTTGTT
ACGACTT-3?), on the basis of a previous description (49). A 25-?l reaction
mixture with final concentrations of 1.8 mM MgCl2, 0.2 mM deoxynucleoside
triphosphate mix, 0.8 pmol ?l?1each of primer 8F-D4 and primer 1492R, 0.04
2114WESTPHAL ET AL.APPL. ENVIRON. MICROBIOL.
mg ?l?1of RNase (Novagen, EMD Chemicals, Darmstadt, Germany), 1? PCR
buffer of the GoTaq Flexi kit (Promega Co., Madison, WI), and 0.06 U ?l?1
GoTaq Flexi (Promega) in sterile water (Sigma, St. Louis, MO), including 2.5 ?l
of the DNA extract (1:20 dilution of the original DNA extract), was submitted to
Amplification reactions were conducted in a PTC-200 thermal cycler (Peltier
thermal cycler; MJ Research Inc., Waltham, MA) with the following program:
after an initial 5 min at 95°C, 28 cycles of 1 min at 94°C, 45 s at 54°C, and 60 s
at 70°C and then 8 min at 70°C. The resulting amplification product was cooled
to 4°C and digested either immediately or after it was frozen once at ?20°C.
For restriction digestion, reaction mixtures of a total of 10 ?l containing 0.3 ?l
of the enzyme MspI (10 U/?l), 0.5 ?l of the corresponding buffer B (Promega),
3.5 ?l of the PCR product, and 5.7 ?l of sterile purified water were incubated for
4 h at 37°C and 25 min at 65°C and were stored initially at 10°C and later at 4°C.
For purification, according to a modified protocol of the Molecular and Cel-
lular Imaging Center (MCIC), 10 ?l of the digest was amended with 1 ?l of 3 M
sodium acetate (pH 5.2) and 27.5 ?l of 95% ethanol (cooled at ?20°C), mixed,
and incubated at ?80°C for at least 10 min. After centrifugation at 6,130 ? g in
a S5700 swinging-bucket rotor (Allegra 25R centrifuge; Beckman Coulter, Ful-
lerton, CA) for 15 min at 4°C, the pellet was rinsed twice with 70% ethanol
(chilled at ?20°C). After each rinse and careful blotting on paper towels, the
plates were spun at 6,130 ? g for 2 min at 4°C. After the rinses, the plates were
reversed in the centrifuge holders, spun at 180 ? g for 1 min to remove excess
rinsate, and then dried in a laminar-flow hood. The pellets were resuspended in
15 ?l of sterile purified water. A 5-?l mix (1:1) of the purified digest with sterile
purified water was submitted for fragment size analysis at the MCIC. There, 0.1
?l of the sample was mixed with 0.5 ?l 600-nucleotide (nt) size standard (CEQ
DNA size standard kit 600) and 40 ?l formamide solution. Samples were ana-
lyzed with a CEQ 8800 genetic analysis system (Beckman Coulter).
Fragment length data handling. Data from the fragment analysis obtained
from MCIC were entered into CEQ8000 software and analyzed in the mode for
amplified fragment length polymorphism analysis of the D4 signal with a dye
mobility calibration and a size standard of 600 nt in a quartic model; the bin size
was 1 nt. Fluorescence signals at intensities of ?300 were excluded from the data
to reduce background signals.
After export to a tabular processing program (Excel, Microsoft Office), the
data were processed before statistical analysis, as follows. Binning of the terminal
restriction fragments (TRFs) was first conducted. For TRFs of ?300 nt in length,
TRF size classes (bins) of ?1 nt were combined, when several of the signals
appeared in multiple bins under consideration. For TRFs of ?300 nt, such
combining was done for ?2 nt. In a second step, bins with ?4 signals across the
entire data set were eliminated from consideration, as no significant differences
would be attributable to such rare events. TRFs with 4 to 8 observations were
kept if the majority of the fluorescence was associated with specific treatments
and were eliminated if the signals were randomly scattered among treatments.
A tiered method of screening the data was used to identify fragments associ-
ated with E. coli O157:H7 suppression. In the first step, data were mined for
differences between the fluorescence intensities of a specific TRF in nonheated
versus heat-treated bedding samples. In the second step, signal strengths were
interval plotted to allow comparison of the fluorescence of nontreated and
heated samples within the bedding sources. For these candidate TRFs, signal
strengths were compared. In a third step, the numbers of CFU were plotted over
the fluorescence in the nonheated bedding sample. Thus, TRFs that fulfilled two
parameters were recorded: (i) significantly (P ? 0.05) stronger fluorescence in
the suppressive than the conducive bedding sample and (ii) a negative regression
between the fluorescence of the TRFs and the numbers of CFU of E. coli
O157:H7 within a specific nonheated source. If both parameters were fulfilled,
the TRF was recorded as being potentially important in suppression of E. coli
O157:H7. When the numbers of CFU were below the detection limit, only the
first parameter was applied.
Cloning and sequencing of MspI-generated 16S rRNA gene TRFs. Terminal
restriction fragments were isolated as described above and then cloned and
sequenced according to the protocol of Benítez and McSpadden Gardener (7).
Briefly, samples were selected for identification on the basis of the high-peak
intensity of the TRF of interest. One microliter of a double-stranded asymmet-
rical adapter was ligated to 2 ?l MspI-digested fragment using 4.5 U T4 DNA
ligase and 1 ?l 10? ligase buffer (Promega). The reaction mixture was incubated
at 16°C for 12 h. Following incubation, the fragments were gel extracted (Ultra-
Clean GelSpin DNA purification kit; MO BIO) after separation by agarose gel
electrophoresis. Twenty-four samples were enriched for the 16S rRNA gene by
amplification with primer 8F (5?-AGAGTTTGATCCTGGCTCAG-3?) and the
MspI adapter primer (5?-GATGAGTCCTGAGTACCG-3?). Amplification was
performed in 25-?l reaction volumes containing 1? Mg-free buffer, 1.8 mM
MgCl2, 0.2 mM dideoxynucleoside triphosphates, 1 pmol ?l?1of each primer,
0.04 mg ml?1RNase A (Novagen), 0.06 U ?l?1GoTaq Flexi DNA polymerase
(Promega), and 2.5 ?l template. Cycling parameters were as follows: 95°C for 5
min; 27 cycles of 94°C for 1 min, 54°C for 45 s, and 70°C for 1 min; and 70°C for
8 min. The TRF-enriched samples were pooled by sample month, ligated into the
pGEM-T Easy vector (Promega), and transformed into E. coli JM109 competent
cells (Promega). A total of 52 transformants were selected, and plasmids were
isolated by a Pure Yield miniprep kit (Promega) and submitted for sequencing
(ABI Prism 3100xl genetic analyzer system; MCIC).
Statistics. Statistical analyses were conducted using Minitab software (version
15.1; Minitab Inc., State College, PA). Median values of the samples were
compared by Mood’s median test, and pair-wise comparisons were based on
nonoverlapping 95% confidence intervals for the medians.
Nucleotide sequence accession numbers. The sequences of the cloned TRF
sequences can be found in GenBank as accession numbers HM146088 through
Soil physical and chemical properties. At initiation of the
three laboratory assays, significant variation in the total or-
ganic carbon and nitrogen levels of the samples was noted
(Table 1). Overall, the range of values varied by no more than
a factor of 3 or 4 (i.e., 0.8 to 2.4% C and 0.04 to 0.14% N).
In-use samples had higher carbon and nitrogen contents than
the washed bedding material, and the other samples had in-
termediate levels of both C and N. In the three experiments,
the pH was alkaline, ranging from pH 8.8 to 10.0. However, no
significant differences in average pH were detected among the
sources (data not shown).
Detection of microbially based suppression of E. coli O157:H7
in different bedding samples. At all sampling times, counts of
the inoculated E. coli O157:H7 were numerically larger in all
samples that had been heated to 80°C and then cooled to room
temperature prior to inoculation than in the nonheated equiv-
alents of samples from the same bedding source. Such differ-
ences in population size were significant (P ? 0.05) in three of
eight contexts in the August experiment (Fig. 1A), five of eight
contexts in the November experiment (Fig. 1B), and eight of
eight contexts in the March experiment (Fig. 1C). These data
indicate that all samples harbored heat-sensitive factors that
TABLE 1. Soil chemical parameters of samples at initiation of
laboratory assays for survival of Escherichia coli O157:H7 in
livestock bedding in August 2008, November 2008,
and March 2009a
% organic matterc
aMedians are presented; medians within one column followed by the same
letter were not significantly different at P equal to 0.05 when they were tested
with Mood’s median test. n ? 4 for August 2008, n ? 8 for November 2008, and
n ? 8 for March 2009.
bLivestock bedding samples were collected from (i) front of the cow box
(fresh), (ii) back of the cow box (in use), (iii) used material from the pile
following sand-manure separation (washed), and (iv) rested (?2 days) in a pile
of washed material (recycled).
cOrganic matter content determined by loss of weight on ignition (LOI) (13).
dNitrogen was quantified as described by Mulvaney (30).
VOL. 77, 2011E. COLI SUPPRESSION2115
contribute to the suppression of E. coli O157:H7 in sand bed-
The median numbers of CFU of E. coli O157:H7 declined in
all contexts (nonheated or heated) over time (Fig. 1A to C),
suggesting that inhibitory factors in addition to the microbial
suppressiveness were not destroyed by the heat treatments.
The dynamics of the decay curves varied somewhat from ex-
periment to experiment. However, initial levels of suppressive-
ness (i.e., decay during the first 24 h) were most dramatic in the
recycled bedding samples (sample 4) in all three experiments.
FIG. 1. Suppression of E. coli O157:H7 in four livestock bedding sources. Bedding samples were acquired on August 2008 (n ? 4) (A),
November 2008 (n ? 8) (B), and March 2009 (n ? 8) (C). Sources were as follows: fresh, front of the cow box; in use, back of the cow box; washed,
used material from the pile following use of the sand-manure separator; and recycled, rested (?2 days) pile of washed bedding material. Heat
indicates that fresh livestock bedding samples were left either nonheated (n) or heated (h) at 80°C for 30 min to kill off the majority of endogenous
microbes before they were subsequently cooled to room temperature. E. coli O157:H7 was then added at 107CFU g?1to all samples and incubated
at room temperature. The numbers of CFU of E. coli O157:H7 were measured by dilution plating on LB medium amended with ampicillin (50
?g ml?1) and cycloheximide (96 ?g ml?1) at 1 dpi or at 7, 10, and 15 dpi. Within each time point, the values for bars indexed with the same letter
were not significantly different at P equal to 0.05 when the terminal dilution factors of a 5-fold dilution series were analyzed with Mood’s median
test. The log-transformed [log10(x)] numbers of CFU of E. coli O157:H7 are presented.
2116 WESTPHAL ET AL.APPL. ENVIRON. MICROBIOL.
Such differences were significant (P ? 0.05) in two of the three
In the August experiment (Fig. 1A) at 1 dpi, the numbers of
CFU of E. coli O157:H7 were lower for all nonheated bedding
samples than the equivalent heated samples; these differences
were statistically significant for the source fresh only. Within
the nonheated or heated sample group, the numbers of CFU
were similar for the different sources. At 15 dpi, most of the
CFU of E. coli O157:H7 had declined; more E. coli O157:H7
cells survived only in samples from the heated fresh and
recycled sources than in the nonheated equivalents; within
the nonheated or heated samples, the numbers of CFU were
In the November experiment (Fig. 1B) at 1 dpi, the numer-
ical values for the numbers of CFU of E. coli O157:H7 were
lower for all nonheated bedding samples than the heated
equivalents; these differences were statistically significant for
the fresh and recycled bedding. Recycled bedding had the
fewest CFU of all nonheated samples, whereas the numbers of
CFU were similar for all heated samples. At 7 dpi, the numbers
of CFU of the nonheated samples were lower than those of the
heated ones, except for the in-use source; CFU were not de-
tected in nonheated samples from the washed source, and this
source had the lowest numbers of CFU of all nonheated bed-
In the March experiment (Fig. 1C) at 1 dpi, there were
significantly fewer CFU of E. coli O157:H7 in all nonheated
bedding samples than in the heated equivalents; the numbers
of CFU for all of the latter samples were similar. The recycled
bedding had fewer CFU than the nonheated in-use and washed
samples. At 10 dpi, the numbers of CFU in all nonheated
samples were lower than the numbers in the heated ones; the
numbers of CFU for the nonheated washed and recycled sam-
ples were lower than the numbers for the fresh material,
whereas the number of CFU in the in-use material was inter-
T-RFLP analyses and association of heat-sensitive bacterial
TRFs with suppressiveness. In order to determine what com-
ponents of the bacterial communities present in the bedding
samples might contribute to the patterns of observed suppres-
siveness, we conducted T-RFLP analyses of amplified 16S
rRNA gene sequences from all samples. Interval plotting and
regression analyses of the relative abundance of different TRFs
were conducted to identify candidate TRFs that were more
abundant in more suppressive sample pairings in each exper-
iment. For example, in the nonheated versus heated sample
pairings of each bedding source, 31 of the 100 total TRFs in the
August experiment (see Table S1 in the supplemental mate-
rial), 67 of the 149 total TRFs in the November experiment
(see Table S2 in the supplemental material), and 78 of the 131
total TRFs in the March experiment (see Table S3 in the
supplemental material) were tentatively identified to differ in
abundance. Because the two mathematical approaches identi-
fied different subsets of TRFs of interest, we focused our ef-
forts on identifying those TRFs repeatedly observed to differ
using both approaches (boldface values in Tables S1 to S3 in
the supplemental material). Thus, while there was a high de-
gree of sample-to-sample variation in the profiles, some TRFs
were repeatedly observed to occur in comparisons of more
suppressive sample pairings than less suppressive sample pair-
ings in individual experiments.
In the August experiment (see Table S1 in the supplemental
material) at dpi 1, a total of 22 TRFs had greater fluorescence
in the nonheated than the heated portions of the bedding
sources; 27 TRFs had a negative relationship of the numbers of
CFU of E. coli O157:H7 over fluorescence, but only 4 TRFs in
fresh bedding fulfilled both selection requirements simultane-
ously. At 15 dpi, there were 25 TRFs with higher fluorescence
in nonheated than heated soil, and 51 exhibited a negative
relationship with the numbers of CFU of E. coli O157:H7;
TRFs M130, M139, and M156 fulfilled both selection criteria
and occurred in fresh and in-use bedding materials. Additional
TRFs fulfilled both selection criteria but occurred only in one
In the November experiment (see Table S2 in the supple-
mental material) at 1 dpi, the numbers of TRFs of the candi-
dates were higher in the nonheated samples 59 times, and
35 negative regressions with the numbers of CFU of E. coli
O157:H7 were detected; but TRFs fulfilled both selection cri-
teria only 12 times when the information for in-use bedding,
washed, and recycled bedding is summarized; none were
detected in fresh bedding. At 15 dpi, the numbers of TRFs
of the candidates were higher in nonheated portions than
heated portions 126 times, and a negative regression of
the TRF fluorescence with the numbers of CFU of E. coli
O157:H7 was detected 84 times. TRFs fulfilled both parame-
ters 65 times. Several TRFs occurred in different sources at the
same sampling time; some occurred repeatedly within one
sampling time and at both samplings.
In the March experiment (see Table S3 in the supplemental
material) at dpi 1, fluorescence was stronger in the nonheated
than in the heated equivalents 102 times and there was a
negative regression of the numbers of CFU and TRF fluo-
rescence 109 times. TRF fulfilled both requirements a total
of 50 times. At dpi 10, the fluorescence was higher in non-
heated than in heated equivalents 86 times; there was a
negative regression 83 times. Both parameters were fulfilled 21
Identification of bacterial TRFs enriched in unheated bed-
ding samples with the highest initial suppressiveness. A sim-
ilar screening approach was used to identify TRFs that were
more abundant in the unheated bedding samples which dis-
played the greatest initial decay of inoculated E. coli O157:H7
populations (Fig. 1A to C). Comparisons of the T-RFLP pro-
files of the washed and/or recycled bedding to those of the
other samples were conducted for the two experiments where
significant differences in suppressiveness were noted, i.e., in
the November experiment (see Table S4 in the supplemental
material) and the March experiment (see Table S5 in the
At dpi 1, fluorescence was stronger in the nonheated recy-
cled bedding than in the nonheated fresh, in-use, or washed
material 29 times; there were negative regressions 27 times
(see Table S4 in the supplemental material). Four to six TRFs
that fulfilled both requirements were detected. At dpi 7, TRFs
of nonheated washed samples had higher fluorescence than
nonheated fresh, in-use, or recycled material 32 times; no re-
gression analysis could be conducted for the washed samples
because no E. coli O157:H7 was detected. TRF M118 occurred
VOL. 77, 2011E. COLI SUPPRESSION2117
in all these comparisons, TRF M299 and TRF M508 occurred
at 1 dpi, and TRF M202 occurred only at 7 dpi.
When samples from the recycled source were compared to
those from the fresh, in-use, and washed sources, fluorescence
was higher in recycled samples 17 times and the regression was
negative 10 times. Both parameters were fulfilled 14 times (see
Table S5 in the supplemental material). The washed samples
had more fluorescent TRFs five times and a negative regres-
sion six times, but no TRFs fulfilled both requirements. Simi-
larly, recycled samples had more fluorescence than fresh or
in-use samples five times, but none had a negative regression.
Identification of bacterial populations most commonly as-
sociated with suppressiveness to E. coli O157:H7. In the total
of 37 comparisons of the August, November, and March ex-
periments outlined above, elevated levels of TRF M299 oc-
curred in 9 comparisons spread over different experiments and
sampling times (Table 2). Similarly, TRF M118 was elevated
eight times. TRF M142 occurred a total of seven times at
various sampling times in all three experiments. TRF M89 and
TRF M156 occurred six times: the former in the November
and March experiments and the latter in all three experiments
at late or early times. TRF M96, TRF M99, TRF M202, TRF
M488, TRF M492, and TRF M508 all occurred in five contexts,
typically restricted to two of the three experiments.
In order to identify the bacteria giving rise to these TRFs, we
size selected and sequenced clones amplified from the original
samples. The sequences were trimmed from the 8F primer to
the MspI site and subjected to BLAST analysis (1). Small to
medium-sized TRFs were more often successfully cloned than
larger TRFs. To assess the novelty of the bacterial species
identified, we evaluated the percent identity across the full
length of the fragment for both the best overall match and the
best published match with a named genus or species designa-
tion (Table 3). The smaller TRFs belong to a variety of genera
in the diverse Bacteroidetes (previously known as the Cy-
tophaga-Flexibacter-Bacteroidetes group), which have 99 to
100% identity to uncultured bacteria from agricultural envi-
ronments. The medium-sized TRFs were mainly associated
with two bacterial groups, the gammaproteobacteria and the
firmicutes. The larger TRFs were associated with the gamma-
and betaproteobacteria. No clones corresponding to either
TRF M118 or M202 were identified.
Here we present the first evidence that the suppression of
E. coli O157:H7 in the environment is mediated by heat-sen-
sitive microorganisms (Fig. 1). Other work has established the
use of such heat treatments for the determination of biologi-
cally based pathogen suppressiveness (42, 51, 52, 53). LeJeune
and Kauffman (22) tested the ability of E. coli O157:H7 to
survive in sawdust and sand bedding materials collected from
dairy operations. The authors showed that E. coli O157:H7
survived at higher densities and for longer periods of time in
used sawdust bedding than in used sand bedding. No mecha-
nism for that apparent suppression was elucidated; however,
they posited that the differences in survival were influenced by
the presence of toxic substances and the lack of water, organic
matter, and/or nutrients in sand bedding. While physical and
chemical properties of the matrices might affect the survival
of the inoculated pathogen, we found no associations be-
tween the survival of E. coli O157:H7 and the sample pH or the
amount of carbon or nitrogen in this study. Effects of soil
moisture differences, though not recorded in detail, were also
discounted as a possible source of variable survival because
water potential was previously demonstrated to have limited
effects on survival of E. coli O157:H7 (40). These findings
further expand the collective understanding of pathogen pop-
ulation dynamics on the farm by illustrating that zoonotic bac-
terial pathogens can be suppressed in situ by other bacterial
It has long been known that diverse oomycete, fungal, and
TABLE 2. Occurrence of TRFs associated with E. coli O157:H7 suppression across all three experiments (August 2008,
November 2008, and March 2009)a
Result for the following TRFs generated by MspI digestion of amplified 16S sequencesb
M89 M96M99M118 M142M156M202 M299M488M492M508
Sum parameter A ? parameter Bc
1, 2, 3
1, 2, 3
Abundance in bedding source:
4 ? 1, 2, 3e
3 ? 1, 2, 4f
2 2, 3
aLivestock bedding samples from four distinct sources (fresh, in use, washed, recycled) were split into one nonheated (NH) and one heated (HT) sample, TRFs were
recorded for higher fluorescence in nonheated than heat-treated samples (parameter A) and negative regression of the surviving numbers of CFU of E. coli O157:H7
over fluorescence strength or between nonheated samples of different levels of suppressiveness (parameter B).
bTRFs of ?300 nt were at an accuracy of ?1 nt; TRFs of ?300 nt were binned at ?2 nt.
cFrequency of occurrence of TRFs with both parameters A and B fulfilled in a total of 37 contexts.
dSampling times: early, 1 dpi; late, 15 dpi (experiment 1), 7 dpi (experiment 2), or 10 dpi (experiment 3). The numbers in the table body indicate the experiment(s)
in which the TRFs were detected.
eThese comparisons among nonheated bedding samples 1 through 4 were conducted only in experiments 2 and 3; in experiment 1, the samples from the recycled
source did not have significantly more CFU than those from the fresh source. The numbers in the table indicate the experiment(s) in which the TRFs were detected.
fThese comparisons among nonheated bedding samples 1 through 4 were conducted only in experiments 2 and 3. In experiment 2, samples from the washed source
(bedding 3) did not have significantly fewer CFU of E. coli O157:H7 than those from the recycled bedding source 4. Also, in experiment 3, samples from the washed
source (bedding 3) did not have significantly fewer CFU of E. coli O157:H7 than those from the in-use bedding source 2. The numbers in the table indicate the
experiment(s) in which the noted TRFs were detected.
2118 WESTPHAL ET AL.APPL. ENVIRON. MICROBIOL.
nematode plant pathogens can be suppressed in some agricul-
tural soils (9, 20, 50), and in some instances, agricultural soils
can be suppressive to a bacterial plant pathogen (e.g., 31). Such
examples of pathogen-suppressive phenomena are known to
be mediated by various numbers and types of microorganisms
present in agricultural soils. Because of this, we hypothesized
that the observed suppression of E coli O157:H7 in sand live-
stock bedding was mediated by a subset of the microorganisms
present in and around the cattle on the farm. The character-
ization of this hypothesized subset of microorganisms was car-
ried out using the microbial community profiling approach
described by Borneman et al. (10). From the results obtained
using this approach, we assert that bacteria marked by the 11
TRFs in Table 2 are potentially involved in the E. coli O157:H7
suppressiveness. While this study focused on bacterial popula-
tions, it is possible that other microorganisms (e.g., phage,
archeae, oomycetes, and fungi) could have also contributed to
the noted suppressiveness.
Because these noted TRFs were observed more fre-
quently and/or more abundantly in the suppressive than the
nonsuppressive bedding samples, the bacteria giving rise to
these TRFs were associated with pathogen suppressiveness. It
has previously been shown that there is a monotonic relation-
ship between TRF signal strength and the abundance of the
bacteria giving rise to such a signal (24, 27). Thus, the relative
abundance of any given TRF will be correlated to the relative
abundance of the bacteria giving rise to that signal, and be-
cause this numerical relationship is likely weakened by con-
founding factors, such as the stochastic occurrence of multiple
bacteria giving rise to the same-sized TRF signals, any noted
correlation between suppression and TRF abundance is likely
to be more significant than that calculated using this method-
ology. The validity of this TRF-based approach was further
supported by the initial identification (8) and subsequent re-
covery (7) of novel bacterial groups belonging to the Genera
incertae associated with the suppression of plant pathogens.
Some of the TRFs identified here may also come from novel
species, on the basis of the low sequence levels of identity of
some TRFs to the database sequences (e.g., M488 and M510;
Table 3). Similar community profiling approaches have also
been used successfully to first identify and subsequently re-
cover microbial antagonists of plant pathogenic nematodes (9,
54, 55). Still, the ultimate proof that the 11 TRFs mark bacteria
that actually suppress E. coli O157:H7 in situ will depend on
culturing representative bacteria harboring those sequences.
Unfortunately, the dairy farm from which the samples were
taken in this study is no longer operational (a victim of the
recent economic crisis), so sampling for recovery from the
same materials is not possible. However, it seems likely that
similar, if not clonal, strains exist on other farms, given the
cosmopolitan nature of most bacterial species. Subsequent ef-
forts to recover and phenotypically characterize bacteria har-
boring the identified TRF tags are under way and could lead to
the development of an inoculant-based strategy to suppress
E. coli O157:H7, in a manner analogous to applied biocontrol
in the field of plant pathology.
Previously, Benítez et al. (8) used two separate statistical
approaches to identify candidate TRFs associated with (plant)
pathogen suppression. Here, we improved on that screening
approach by using an additional statistical criterion (regression
analysis) to further restrict the number of candidate TRFs
considered to be associated with suppression, thereby reducing
the potential number of false-positive associations of phenom-
enological significance. Remarkably, the more restrictive se-
TABLE 3. Sequence-based identifications of bacterial TRFs associated with E. coli O157:H7 suppression across all three experiments
Highest BLAST hitb
Highest published BLAST hit with named
genus or genus and species designationb
Uncultured bacterium from manure,
potato plant root bacterium
Uncultured compost bacterium,
uncultured bacterium from beetle
hindgut, uncultured bacterium from
cattle feedlot, uncultured Fluviicola
Uncultured bacterium from cattle feedlot
Cellulophaga tyrosinoxydans, Cytophaga
Proteiniphilum acetatigenes, Fluviicola spp.
Uncultured bacterium from wetland soil,
Uncultured bacterium from cattle
feedlot, uncultured bacterium from
Marinomonas spp., Cellulosimicrobium
Lysobacter spp., Caryophanon spp.
M156 Gammaproteobacteria, Firmicutes
M299Uncultured bacterium from bovine feces,
uncultured bacterium from cattle
feedlot, Marinobacter spp., Halomonas
Uncultured bacterium from biogas
Uncultured bacterium from wastewater
Uncultured compost bacterium
Clostridium spp., Faecalibacterium
prausnitzii, Marinobacter excellens,
M488Oenothera phytoplasma Mollicutes
aTRF is from the 8F primer site to the first MspI site.
bBLAST analysis (1) was used to compare TRF sequences to sequences in GenBank. Putative identifications are highlighted if they possess a ?99% (boldface)
or ?89% (underlined) sequence identity across the cloned TRFs.
cNo clones containing the target fragment length were identified.
VOL. 77, 2011E. COLI SUPPRESSION2119
lection criteria applied in this study resulted in an even greater
number of candidate TRFs than that previous study examining
root disease suppression (i.e., 11 candidates here [Table 2]
versus 8 previously ). The significance of this is not clear, but
it might indicate that bacterium-mediated suppression of strain
O157:H7 is less specific than the general suppressiveness to
soilborne plant pathogens noted in the earlier study. Such a
conclusion is further supported by the observation that many
more TRFs were associated with E. coli suppression at differ-
ent sampling times (Table 2), but only 11 TRFs were observed
repeatedly in different samplings (Table 2). Also, the results
presented do not preclude other organisms from being in-
volved in suppressiveness.
Because they were found in most all of the samples to var-
ious degrees, it is likely that the bacteria giving rise to the 11
noted TRFs were likely present in the feces of the cattle them-
selves. However, because the greatest suppressiveness was ob-
served in the recycled sand pile (bedding source 4, 1 dpi; Fig.
1), such populations are probably subdominant members of
the community until some curing of the bedding takes place,
allowing them to become more abundant and/or more active.
Interestingly, changes in the relative abundance of specific
antagonists, such as phlD-positive Pseudomonas species, have
also been associated with the suppression of some plant
pathogens (28, 29).
The identified subset of 11 TRFs appears to have been ampli-
fied from a variety of bacterial groups (Table 3). These include
the diverse Cytophaga-Flexibacter-Bacteroidetes group, the well-
known Gammaproteobacteria, and the primarily Gram-positive,
spore-forming Firmicutes. Although some 16S rRNA TRF se-
quences showed greater than 99% identity to sequences in the
database, there were some sequences whose best match
showed less than 90% identity across the length of the TRF. In
these cases of lower sequence identity to database entries, we
may have found markers for previously unidentified species of
bacteria that are particularly suppressive to E. coli O157:H7 in
situ. This hypothesis is supported by previous work where ex-
ploitation of similar correlations (8) led to the marker-assisted
recovery of two bacterial species that displayed the predicted
function of suppressiveness to the plant pathogens present in
the tested soils (7).
The ecology of E. coli O157:H7 in the farm environment
impacts the prevalence of O157:H7 on the farm and beyond.
Franz and van Bruggen (15) recently reviewed the ecology of
enteric pathogens and described a number of strategies to
reduce the risk of spread of the pathogen on and off farms.
Among these were strategies related to altering the manure
composition by altering feeding regimes and composting the
waste. Indeed, it has long been known that composting can
reduce enteric pathogen populations (14, 19, 25). This work
adds to previous studies by highlighting how the use of recycled
sand bedding might lead to the sustenance of microbially based
suppression, an approach that would likely reduce the preva-
lence of E. coli O157:H7 and possibly other enteric pathogens
in and around dairy cattle. By identifying management strate-
gies for dairy farms that select for microbial populations that
suppress E. coli O157:H7 in the environment, the contribution
of the farm environment to E. coli O157:H7 persistence is
effectively reduced and the risk of food-borne contamination is
This project was supported by an OARDC Interdisciplinary SEEDS
grant to J.T.L. and B.B.M.G.
The first author especially thanks S. Be ´nitez, C. Cao, and M. Kauff-
mann for help with conducting this research.
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