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Applied and Environmental Microbiology, April 2000, p. 1493-1498, Vol. 66, No. 4
0099-2240/00/$04.00+0
Effects of Acid Adaptation of Escherichia coli O157:H7
on Efficacy of Acetic Acid Spray Washes To Decontaminate Beef
Carcass Tissue
Elaine D.
Berry* and
Catherine N.
Cutter
Roman L. Hruska U.S. Meat Animal Research
Center, Agricultural Research Service, United States Department of
Agriculture, Clay Center, Nebraska 68933-0166
Received 27 September 1999/Accepted 27 January 2000
 |
ABSTRACT |
Exposure to low pH and organic acids in the bovine gastrointestinal
tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently
contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to
reduce populations of this organism on beef carcass tissue was
examined. Stationary-phase acid resistance and the ability to induce
acid tolerance were determined for a collection of E. coli
O157:H7 strains by testing the survival of acid-adapted and unadapted
cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant
(ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated
inducible acid tolerance (ATCC 43889) were used in spray wash studies.
Prerigor beef carcass surface tissue was inoculated with bovine feces
containing either acid-adapted or unadapted E. coli
O157:H7. The beef tissue was subjected to spray washing treatments with
water or 2% acetic acid or left untreated. For strains ATCC 43895 and
43889, larger populations of acid-adapted cells than of unadapted cells
remained on beef tissue following 2% acetic acid treatments and these
differences remained throughout 14 days of 4°C storage. For both
strains, numbers of acid-adapted cells remaining on tissue following
2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For
strain ATCC 43890, there was no difference between populations of
acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can
negatively influence the effectiveness of 2% acetic acid spray washing
in reducing the numbers of this organism on carcasses.
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INTRODUCTION |
The involvement of E. coli O157:H7 in food-borne illness outbreaks associated with the
consumption of acidic foods such as apple cider, fermented sausage,
yogurt, and mayonnaise (3, 9, 34) has drawn attention to the
acid resistance properties of this pathogen, and many subsequent
studies have demonstrated that this bacterium can survive in a variety
of acidic foods (8, 25, 32, 33, 37, 39). In addition, other
studies have shown that adaptation to acidic conditions can further
improve the survival of E. coli O157:H7 in foods that are
preserved by low pH and acids (30, 38). Leyer et al.
(30) found that acid-adapted E. coli O157:H7
survived better than unadapted cells during sausage fermentation and
exhibited enhanced survival in dry salami and apple cider. Tsai and
Ingham (38) reported that adaptation to acid enhanced
survival of E. coli O157:H7 in ketchup but not in mustard or
pickle relish. In addition to promoting survival in low-pH foods, the
development of acid resistance by E. coli O157:H7 may
provide cross-protection against heat, salt, and irradiation
preservation of foods (7, 11, 23, 36). Furthermore, several
works have indicated that acid tolerance of E. coli O157:H7
is enhanced or sustained longer upon refrigerated storage (10, 12,
22, 31, 33). Finally, it is thought that acid resistance and/or
induction of acid tolerance may better enable pathogens to survive
gastrointestinal acidity and ultimately cause disease and that it may
enhance virulence (1, 15, 26, 35).
Clearly, acid resistance and the development of acid tolerance by
food-borne pathogenic bacteria may be significant at several points
along the farm-to-table continuum of food production. It is important
that we understand how previous environment and processing conditions
can affect the acid tolerance status of food-borne E. coli
O157:H7 in order to devise strategies for better control of the
occurrence, growth, or survival of this organism in foods. Cattle are a
reservoir of E. coli O157:H7, and raw or undercooked beef
and milk, as well as food products likely contaminated with bovine
feces containing this organism, have been incriminated in many
food-borne illness episodes (21). Solutions of lactic and
acetic acid are commonly used by the beef slaughter industry as
antimicrobial spray wash interventions to reduce the microbial load on
freshly slaughtered beef carcasses. Because of the potential for acid
adaptation in the bovine gastrointestinal tract due to exposure to low
pH and organic acids (10, 15, 29), and because bovine feces
are a common source of bacterial contamination of carcasses, our
objective was to determine if acid adaptation can affect the ability of
2% (vol/vol) acetic acid (2% AA) spray washes to reduce populations
of E. coli O157:H7 on prerigor beef carcass surface tissue
(BCT). Initial experiments involved the assessment of acid resistance
characteristics of a selection of E. coli O157:H7 strains
available for these experiments.
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MATERIALS AND METHODS |
Microorganisms and inoculum preparation.
The E. coli O157:H7 strains used in this study are listed in Table
1. Acid-adapted (A) and unadapted (NA,
not adapted) stationary-phase cells of each strain were prepared by
cultivation in Trypticase soy broth with 1% glucose (TSB+G; BBL,
Becton Dickinson Microbiology Systems, Cockeysville, Md.) and without
glucose (TSB
G), respectively, according to the method described by
Buchanan and Edelson (6). To prepare inocula for both acid
challenge and spray wash experiments, 0.1-ml volumes from frozen
(20°C) 25% glycerol stock cultures were inoculated into 10 ml of
TSB+G and TSBG and incubated for 18 h at 37°C prior to use.
Determination of acid resistance characteristics of E. coli O157:H7 strains.
Initial screening of the E. coli O157:H7 strains to assess their stationary-phase acid
resistance and acid adaptation characteristics was done by examining
the survival of A and NA cells inoculated into pH-adjusted broth medium
(6). Brain heart infusion broth was adjusted to pH 2.5 with
concentrated HCl (BHI-2.5; Difco Laboratories, Detroit, Mich.). The
BHI-2.5 was dispensed in 10-ml volumes to test tubes and sterilized by
autoclaving, and the test tubes were preequilibrated to 37°C prior to experiments.
For the experiments, BHI-2.5 tubes were inoculated with a 0.1-ml volume
of an 18-h culture of either A or NA
E. coli O157:H7
cells.
Each tube was sampled immediately following inoculation
and mixing by
vortexing and then returned to 37°C to incubate
statically for 6 h, when the tube was sampled again. At each sampling
time, the BHI-2.5
samples were diluted if necessary in buffered
peptone water (BPW) and
spiral-plated in duplicate on both tryptic
soy agar (TSA) and MacConkey
sorbitol agar (SMAC) plates, using
a model D spiral plater (Spiral
Systems Instruments, Bethesda,
Md.). All plates were incubated at
37°C for 24 h prior to enumeration.
Using spiral-plating
procedures, the minimum detection level was
1.30 log
10
CFU/ml. Experiments were duplicated on separate days
for A and NA cells
of each
E. coli O157:H7
strain.
Spray wash experiments.
On each day of a spray wash
experiment, fresh bovine feces were collected from three different cows
on a corn silage ration. For each individual A and NA E. coli O157:H7 strain, 75-g samples of the three fecal specimens
were pooled in a sterile beaker and mixed well with 75 ml of sterile
0.85% NaCl. To reduce interference by indigenous E. coli
when we enumerated E. coli O157:H7, fecal slurries were
autoclaved at 121°C for 2 min and rapidly cooled on ice, with
occasional stirring using a sterile tongue depressor. An additional 35 ml of 0.85% NaCl was added to the cooled feces, and the mixtures were
held at room temperature until beef tissue inoculation. Immediately
prior to tissue inoculation, 2 ml of an 18-h A or NA E. coli
O157:H7 culture was added to the fecal slurry and mixed well. Prepared
in this fashion, the fecal slurries contained 6 log10 CFU
of E. coli O157:H7 per g and yielded ca. 5 log10
CFU of E. coli O157:H7 per cm2 when inoculated
with a paintbrush onto the external surface of a 15- by 20-cm piece of
lean BCT (17).
Lean BCT was obtained from the cutaneous trunci of prerigor carcasses
immediately after slaughter at a local cow and bull
processing
facility. The BCT was placed in plastic bags in an
insulated container
to minimize cooling and transported to the
laboratory at the Roman L. Hruska U.S. Meat Animal Research Center
for immediate use in
experiments. The BCT was aseptically trimmed
to 15- by 20-cm pieces.
The entire external surface of each piece
was inoculated with the
appropriate fecal slurry, prepared as
described above, with a sterile
5.1-cm-wide paintbrush. The inoculated
tissues were allowed to stand
for 15 min prior to spray washing;
inoculated untreated control tissues
were allowed to stand for
15 min prior to sampling for
enumeration.
An insertable pod of a commercial carcass washer, modified for use in a
biological safety hood, was used to apply the spray
washing treatments
to inoculated BCT (
19). Individual BCT samples
were mounted
on the surface of a stainless steel plate and, as
appropriate to
treatment, were spray washed with 25 ± 2°C sterile
tap water
(W) or 25 ± 2°C 2% AA prepared with W. The spray washes
were
delivered at 125 lb/in
2 for 15 s, with a spray nozzle
oscillation rate of 60 cycles/min.
Immediately following the spray wash treatments, BCT was placed on
sterile trays. A 5- by 5-cm sample was aseptically excised
from each
tissue piece and placed in sterile side-filter sample
bags (Spiral
Biotech, Bethesda, Md.) for bacterial enumeration.
The BCT surface pH
was measured using a flat-surface combination
probe (Corning, Inc.,
Corning, N.Y.). Each tray was then covered
with an inverted sterile
tray and stored at 4°C for 48 h. At 48
h, BCT was again
sampled for enumeration and pH determination.
At least two additional
5- by 5-cm areas were excised at this
time, placed in vacuum packaging
bags (3.2-mil nylon-copolymer
bags with an oxygen transmission rate at
23°C of 52 cm
3/m
2; Hollymatic, Inc.,
Countryside, Ill.), and vacuum sealed (model
LV10G; Hollymatic, Inc.).
The vacuum-packaged BCT was stored at
4°C and removed for sampling at
7 and 14
days.
For
E. coli O157:H7 enumeration, the excised 5- by 5-cm BCT
samples were pummeled for 2 min with 25 ml of BPW containing 0.1%
(vol/vol) Tween 20 using a Stomacher lab blender (model 400; Tekmar,
Inc., Cincinnati, Ohio). Following pummeling, the filtered samples
were
serially diluted in BPW as necessary and spiral plated or
spread plated
in duplicate onto SMAC plates containing 0.05 mg
of cefixime per liter
and 2.5 mg of potassium tellurite (CT-SMAC)
per liter. CT-SMAC plates
were incubated for 24 h at 37°C and
enumerated.
Statistical analyses.
Six replications of each spray wash
experiment were done, with three replications being done on each of two
separate days for each A and NA E. coli O157:H7 strain.
Numbers of bacteria from duplicate plates of spray wash experiments
were averaged and converted to log10 CFU per square
centimeter. Least squares means of bacterial populations were analyzed
as a completely randomized factorial design (six organisms [three A
and three NA] by three treatments by five sampling periods) using the
general linear model procedure of SAS (version 6.12; SAS Institute
Inc., Cary, N.C.). Statistical significance is defined as a
P of 
0.01 unless otherwise noted.
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RESULTS AND DISCUSSION |
To identify strains for use in spray wash experiments,
stationary-phase acid resistance and the ability to adapt to acidic conditions were determined for a selection of E. coli
O157:H7 strains utilizing the method described by Buchanan and Edelson (6). After 18 h of growth in TSBG, the final pHs of
the various cultures ranged from 6.7 to 7.2. Alternatively, after
18 h of growth in TSB+G, glucose fermentation by the cultures
resulted in final pHs ranging from 4.3 to 4.8, which is within the pH
range reported to induce acid tolerance in E. coli (5,
23, 30, 31). Final pHs of the TSB+G and TSBG cultures were
similar to those previously reported for E. coli O157:H7 in
the same media (6, 36). As other researchers have observed,
there is a range of responses to acidic conditions among different
strains of this organism (1, 6, 14, 33). In addition,
comparison of levels of recovery of the acid-challenged cells on both
nonselective TSA and the selective SMAC was useful for assessing the
degrees of both the acid resistance and the acid injury of the strains. Based on these survival and injury patterns following exposure to pH
2.5 for 6 h, and for our particular objectives, the E. coli O157:H7 strains tested were grouped into three broad
categories as either acid resistant, acid adaptable, or acid sensitive
(Table 1). Initial numbers and survival of A and NA E. coli
O157:H7 cells following a 6-h exposure to pH 2.5 are shown in Fig.
1 for strains ATCC 43895, ATCC 43889, and
ATCC 43890, which were categorized as acid resistant, adaptable, and
sensitive, respectively. These results are representative of those seen
with the other E. coli O157:H7 strains in the same acid
resistance categories (Table 1). For all acid-resistant strains,
adaptation to acidic conditions did improve survival at pH 2.5;
however, high numbers of NA cells remained viable after the 6-h
exposure. Members of this acid-resistant group may be similar to
E. coli strains that have been described as exhibiting
pH-independent acid tolerance or being constitutively acid tolerant
(6). For strains categorized as acid adaptable, growth in
TSB+G and the resultant adaptation to acidic conditions were important
to the survival of these strains upon exposure to pH 2.5. For adaptable
strains, A cell counts recovered on TSA after the 6-h exposure
approximated initial counts, although recovery on SMAC indicated ca.
90% cell injury. Alternatively, NA cell counts of the adaptable
strains approached or were below detectable levels on both TSA and SMAC
following the exposure to pH 2.5. For strains in the acid-sensitive
group, adaptation to acidic conditions did not enhance cell survival to
the pH 2.5 acid challenge. Following the 6-h exposure, both A and NA
cells of acid-sensitive strains typically were below detectable levels
on both TSA and SMAC.
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FIG. 1.
Survival of A and NA E. coli O157:H7 cells
initially (0 h) and after 6 h of exposure in BHI-2.5, as
enumerated on TSA and SMAC plates. E. coli O157:H7 strains
ATCC 43895, ATCC 43889, and ATCC 43890 were categorized as acid
resistant, acid adaptable, and acid sensitive, respectively. The
horizontal dotted line at 1.30 log10 CFU/ml denotes the
minimum detection level. Error bars indicate standard deviations.
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E. coli O157:H7 strains ATCC 43895, ATCC 43889, and ATCC
43890, representing each of the three acid resistance categories, were
used to examine the effects of acid adaptation on the ability of acetic
acid spray washes to reduce levels of E. coli O157:H7 from
beef carcasses. The initial levels of A and NA cells of all three
strains on BCT prior to spray washing were the same, at ca. 5 log10 CFU/cm2 (P 0.01). The
pH values of BCT surfaces following treatments and during 4°C storage
are shown in Table 2.
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TABLE 2.
Average pHs of beef tissue surfaces following the various
treatments and during 14 days of storage at 4°C
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Results of spray washing experiments with A and NA E. coli
O157:H7 ATCC 43895 cells are shown in Fig.
2. The acid adaptation of the resistant
strain ATCC 43895 was demonstrated to affect the ability of 2% AA
spray washes to reduce populations of this strain from BCT.
Significantly larger populations of ATCC 43895 A cells than NA cells
remained on BCT immediately following 2% AA treatments, and these
differences remained throughout the 14 days of refrigerated storage.
Instead, populations of A cells immediately following 2% AA washes
were the same as those of both A and NA cells remaining on BCT
following W washes. Similar results were seen with A and NA cells of
the adaptable E. coli O157:H7 strain ATCC 43889 (Fig.
3). Higher levels of A cells than of NA cells remained on the BCT following 2% AA treatments. As seen with
strain ATCC 43895, remaining A cell populations of strain ATCC 43889 immediately after 2% AA treatments were similar to those of A and NA
cells populations after W treatments. Sizes of populations of A and NA
cells on 2% AA-treated tissues were different through the 14 days of
storage. Figure 4 shows the effects of
2% AA and W washes on populations of A and NA E. coli
O157:H7 ATCC 43890 cells on BCT. Unlike with the resistant and
adaptable strains, no differences were seen between levels of A and NA
cells of this acid-sensitive strain immediately following 2% AA spray wash treatments. However, during the course of the refrigerated storage, NA cell populations declined, and cell levels of A and NA
cells on 2% AA-treated BCT were different at 2, 7, and 14 days.
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FIG. 2.
Initial reductions of cell numbers and growth or
survival of A and NA E. coli O157:H7 ATCC 43895 (acid-resistant strain) cells on lean BCT stored at 4°C following
spray washing treatment with W or 2% AA or after no treatment
(n = 6). The standard error of the least squares means
was equal to 0.14.
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FIG. 3.
Initial reductions of cell numbers and growth or
survival of A and NA E. coli O157:H7 ATCC 43889 (acid-adaptable strain) cells on lean BCT stored at 4°C following
spray washing treatment with W or 2% AA or following no treatment
(n = 6). The standard error of the least squares means
was equal to 0.14.
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FIG. 4.
Initial reductions of cell numbers and growth or
survival of A and NA E. coli O157:H7 ATCC 43890 (acid-sensitive strain) cells on lean BCT stored at 4°C following
spray washing treatment with W or 2% AA or following no treatment
(n = 6). The standard error of the least squares means
was equal to 0.14.
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For both A and NA cells of all strains examined, there was a trend that
indicated a gradual decline in E. coli O157:H7 populations on W-treated and untreated BCT over the 14 days of refrigerated storage
(Fig. 2 to 4). This same trend was not as apparent for populations on
2% AA-treated BCT, which, in comparison, remained constant during the
4°C storage. There are at least two possible reasons for this
observation. One possibility is that there were increasing populations
of a competing microflora present on W-treated and untreated BCT,
compared to those on 2% AA-treated BCT, resulting in a depression of
E. coli O157:H7 populations on these samples during storage.
General bacterial microflora populations were not monitored in this
study; however, previous studies have demonstrated that while both
organic acid and water spray washes reduce populations of mesophilic
aerobic bacteria, organic acids can contribute the added residual
effect of slowing or suppressing the growth of this group of bacteria
on beef tissue during storage, compared to their more rapid growth on
untreated or water-washed beef (18, 20). A second reason for
this observation may be the cross-protective relationship between
E. coli O157:H7 acid adaptation and cold temperature. Conner
and Kotrola (13) reported that the presence of organic
acids, including acetic, citric, and lactic acids, in broth medium held
at 4°C enhanced the survival of E. coli O157:H7, compared
to its survival in unacidified control medium held at the same
temperature. Likewise, other works have demonstrated that once induced,
the acid tolerance of this pathogen is enhanced or maintained longer
when the organism is held at colder temperatures (10, 12, 22, 31,
33). To determine if A cells of E. coli O157:H7
sustained acid tolerance on BCT during refrigerated storage, an
additional experiment was incorporated during the course of spray wash
experiments with the acid-adaptable E. coli O157:H7 strain
ATCC 43889. When BCTs were sampled for enumeration at 2, 7, and 14 days, 200 µl of the filtered samples, following pummeling, were
placed into 3 ml of BHI adjusted to pHs 2.5, 3.5, and 4.0 with HCl
(n = 3). The BHI media were incubated for 1 h at
37°C, surviving cells were enumerated on CT-SMAC, and these cell
numbers were compared to the initial counts. Because of the low numbers
of cells available for this assay and the small volume of inoculum that
could be applied without changing the pH of the BHI, the surviving cell
populations could only be estimated (data not shown). However, these
limited observations suggested that at 2, 7, and 14 days, A cells of
ATCC 43889 did not maintain the degree of acid resistance that they had
when they were initially inoculated onto the meat, following growth in
TSB+G. Counts of surviving cells after 1 h of exposure to pHs 3.5 and 4.0 suggested that there were possible differences in survival
between A and NA cells. Further studies are planned to confirm these observations.
Log reductions in viable cell counts were compared in order to
ascertain if differences in degrees of acid resistance between the
three E. coli O157:H7 strains affected immediate reductions on BCT by 2% AA spray washes. For NA cells of all three strains, there
were no differences in log reductions due to 2% AA spray washes.
However, when cells were adapted to acidic conditions, there was a
significant difference in log reductions by 2% AA treatments between
the acid-resistant strain ATCC 43895 and acid-sensitive strain ATCC
43890 (log reductions of 1.61 and 2.38, respectively; P 0.01). These results indicate that differences in acid resistance between strains can affect the efficacy of organic acid spray washes to
reduce the number of these organisms from BCT. Cutter and Siragusa
(14) previously reported E. coli O157:H7 strain differences in resistance to 1, 3, or 5% acetic, lactic, or citric acid washes of lean BCT, when bacteria were enumerated after a 24-h
incubation of the tissue at 4°C following washing. Among the strains
examined in that study, E. coli ATCC 43895 was observed to
exhibit the greatest resistance to the organic acid spray treatments, as was noted in the present study. The present study and other reports
have noted the high level of acid resistance of this E. coli
O157:H7 isolate (1, 30). In fact, differences in levels of
acid resistance or abilities to adapt to acidic conditions of different
isolates may account for conflicting reports of the efficacies of
organic acid spray washing treatments to reduce populations of E. coli O157:H7 on beef (4, 16, 18). This possibility
further emphasizes the importance of determining the resistance
characteristics of candidate bacterial strains as relevant to the
process under examination, prior to their use in studies to validate
food preservation processes.
A recent report concerning the effects of feed ration composition on
the development of acid resistance by E. coli in cattle has
sparked discussion about the relevance of this possible event to the
survival of E. coli O157:H7 in the human gastric environment (15, 28, 29). The pertinence of the development of acid tolerance in the bovine gut to public health has been questioned because of the length of time that typically occurs between the time
the pathogen is shed in feces and the time the pathogen may be consumed
in food or water. During this interval, the bacteria will experience
changes in environment and therefore subsequent changes in adaptive
state. In the present work, we have demonstrated that acid adaptation
of E. coli O157:H7 can negatively affect the ability of
organic acid spray washing to reduce the numbers of this organism from
prerigor BCT. Pathogens contaminating freshly slaughtered beef
carcasses typically are recent residents of the bovine gastrointestinal
tract, arriving in feces or ingesta from intestinal organs that are
accidentally damaged during removal from the carcass or from feces or
environmental soil from the hide or hooves (2, 24). Organic
acid spray washes are used in the early steps of beef carcass
processing. They may be applied to carcasses after hide removal and
before or after evisceration but prior to chilling of the carcasses.
Therefore, the acid tolerance status of E. coli O157:H7 as
shed from cattle is significant to the microbial safety of meat
products. Work is planned to determine the relative acid tolerance of
enterohemorrhagic E. coli as naturally shed from cattle, and
current work is focused on determining the variation in levels of acid
resistance and the ability to adapt to low pH and acidic conditions
among recent livestock isolates of this pathogen.
| |
ACKNOWLEDGMENTS |
We thank Rebecca Hartford and Jane Long for technical assistance,
Ken Ostdiek and Patty Beska for sample collection, and James Wray for
consultation on statistical analyses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: USDA, ARS, Roman
L. Hruska U.S. Meat Animal Research Center, P.O. Box 166, Clay
Center, NE 68933-0166. Phone: (402) 762-4225. Fax: (402)
762-4149. E-mail: berry{at}emailmarc.usda.gov.
Present address: Department of Food Science, The Pennsylvania State
University, University Park, PA 16802.
| |
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Applied and Environmental Microbiology, April 2000, p. 1493-1498, Vol. 66, No. 4
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Abstract
Introduction
