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Applied and Environmental Microbiology, February 2000, p. 844-849, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Selective and Sensitive Method for PCR
Amplification of Escherichia coli 16S rRNA Genes in
Soil
G.
Sabat,1
P.
Rose,2
W. J.
Hickey,2,3,* and
J. M.
Harkin2,3
Department of
Bacteriology,1 Department of Soil
Science,3 and Environmental Toxicology
Center, University of Wisconsin
Madison, Madison, Wisconsin
53706-12992
Received 7 September 1999/Accepted 22 November 1999
 |
ABSTRACT |
A set of PCR primers targeting 16S rRNA gene sequences was
designed, and PCR parameters were optimized to develop a robust and
reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest
relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was
elevated to 72°C, which is 10°C higher than the optimum for the
primers. Sensitivity was retained by modifying the length of steps in
the PCR, by increasing the number of cycles, and most importantly by
optimizing the MgCl2 concentration. The PCR protocol
developed can be completed in less then 2 h and, by using Southern
hybridization, has a detection limit of ca. 10 genomic equivalents per
reaction. The method was demonstrated to be effective for detecting
E. coli DNA in heterogeneous DNA samples, such as those
extracted from soil.
| |
TEXT |
Bacteria of the
Enterobacteriaceae are important pathogens causing
intestinal and systemic illness of humans and other animals. Recent
outbreaks of gastrointestinal diseases focused public attention on one
of the more widely known members, Escherichia coli, and the
potential problems with strains of this organism as food-borne pathogens. Consumption of water polluted with fecal material is an
important exposure pathway, and while monitoring of total coliforms is
the standard technique, some research suggests that analysis for
E. coli specifically may be a better indicator
(3).
Traditional approaches for analysis of E. coli have relied
on cultural techniques, and many selective-differential media have been
developed. Generally, lactose fermentation is used for differentiation, sodium lauryl sulfate or bile salts are used as a selective agent, and
a fluorogenic reaction is used for confirmation. 
-Glucuronidase is
the target enzyme for confirmation of E. coli, which
mediates hydrolysis of
4-methylumbelliferyl--D-glucuronide (MUG) to a fluorescent product. However, other Enterobacteriaceae
produce -glucuronidase (e.g., Shigella, Salmonella, and Yersinia),
not all strains of E. coli express the uiaA gene
that encodes -glucuronidase, and some Staphylococcus spp.
hydrolyze MUG (6, 25). Biochemical analysis for an enzyme
associated with a particular pathogenic trait and immunodiagnostic
assays for O antigens associated with pathogenic strains have also been
developed (11, 16). Again, cross-reactivity limits the
utility of these techniques for identification of E. coli.
The emergence of DNA technology has opened new possibilities for
development of methods with improved selectivity for E. coli. Relevant techniques include use of PCR and/or hybridization
probes to detect E. coli-specific genes encoding invasion
proteins (12, 13), toxins (17, 23, 24), catabolic
enzymes (4, 8), or structural (lipo)proteins (10,
12). A drawback to these approaches has been that when
cross-reactivity tests were done, target sites were often detected not
only in E. coli but also in closely related organisms
(14, 17, 18). Cross-reaction is particularly problematic
with Shigella. This is not surprising insofar as 16S rRNA
and other genome-level similarities suggest that Escherichia
and Shigella are sufficiently similar for placement in a
single genus (5, 19). Nevertheless, for clinical,
epidemiological, and historical reasons they are regarded as different
genera. Thus, for development of molecular methods, the challenge is to identify sequences conserved within E. coli that may be
targeted to minimize false negatives yet can be distinguished from
similar sequences likely to be present in Shigella.
The goal of this study was to design a selective and sensitive PCR
method for amplification of a 16S rRNA gene region from E. coli. The key performance criterion for the method was to reliably amplify the targeted region from template levels equivalent to 100 or
fewer E. coli cells without cross-reaction with similar sequences present in Shigella. An additional consideration
was that the method be sufficiently robust for analysis of genomic DNA
extracted from water or soil samples as well as that prepared from
clinical isolates.
Cultures and DNA template preparation.
Cultures used in this
study are listed in Table 1. Genomic DNA
for PCR experiments was extracted from the liquid cultures as described
by Ausubel et al. (2). DNA concentration and quality were
determined by UV light absorbance at 260 and 280 nm and by band
intensity densitometry using NIH Image version 1.55 (National Institutes of Health, Bethesda, Md.).
Primers and probes.
Primers targeting hypervariable regions of
the E. coli 16S rRNA gene were developed by using
PrimerSelect (DNAStar, Madison, Wis.). Three sets of primer pairs were
designed and tested: ECP79F (forward, targeting bases 79 to 96;
5'-GAAGCTTGCTTCTTTGCT-3')-ECR620R (reverse, targeting bases
602 to 620; 5'-GAGCCCGGGGATTTCACAT-3'); ECB75F (forward,
targeting bases 75 to 97; 5'-GGAAGAAGCTTGCTTCTTTGCTG-3'-ECR620R (reverse, described above); and ECA75F (forward, targeting
bases 75 to 99; 5'-GGAAGAAGCTTGCTTCTTTGCTGAC-3')-ECR619R
(reverse, targeting bases 594 to 619;
5'-AGCCCGGGGATTTCACATCTGACTTA-3'). The optimal melting
temperature and expected PCR product sizes for the primer pairs
were as follows: ECP79F-ECR620R, 55°C and 541 bp;
ECB75F-ECR620R, 59°C and 545 bp; and ECA75F-ECR619R,
60°C and 544 bp. The probe used in Southern hybridization
experiments was S-D-Bact-0338-a-A-1 (previously referred to as EUB338
[1]). This probe targeted a 16S rRNA gene sequence
conserved in the domain Bacteria and occurring near the
center of the PCR products generated by all primer pairs. The
oligonucleotide was 5' labeled with digoxygenin by the supplier
(Sigma-Genosys, The Woodlands, Tex.).
PCR and hybridization protocols.
PCR protocols were developed
empirically for each primer set to obtain maximum selectivity (E. coli versus Shigella and other enteric bacteria) while
retaining sensitivity (desired detection level of 10 fg to 1 pg, ca. 1 to 100 cell equivalents). Optimization focused on levels of primers,
DNA polymerase, and MgCl2 in the reaction mixture as well
as thermal cycling programs. For ECP79F-ECR620R, the reaction mixture
(50 µl, total volume) contained 1:10 dilution of 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3], 15 mM MgCl2, 0.01%
[wt/vol] gelatin), 200 µM each deoxynucleoside triphosphate, 0.6 µM primers, and an appropriate amount of template. The thermal cycling program consisted of a hot start (5 min, 94°C) before 1.25 U
of AmpliTaq DNA polymerase (PE Biosystems, Foster City, Calif.) was
added per reaction. The reaction was run for 40 cycles with 45-s
denaturation (94°C), 45-s annealing (50°C), 1.5-min extension
(72°C), and a final extension (5 min, 72°C). For ECA75F-ECR619R and
ECB75F-ECR620R, reaction mixtures (50 µl, total volume) contained 1:10 dilution of 10× PCR buffer (100 mM Tris-HCl [pH 9.0], 500 mM
KCl, 0.1% Triton X-100), 200 µM each deoxynucleoside triphosphate, 2 mM MgCl2, 0.4 µM primers, bovine serum albumin (40 µg/reaction), and an appropriate amount of template. The thermal
cycling program consisted of a hot start (30 s, 94°C) followed by
addition of 1.5 U of native Taq DNA polymerase (Promega,
Madison, Wis.) per reaction. The thermal cycling program was run for 40 cycles of denaturation (45 s, 94°C) and annealing-extension (45 s,
72°C) and then a final extension (10 min, 72°C).
All of the reactions were done in thin-walled 0.5-ml Eppendorf tubes
(USA/Scientific Plastics, Ocala, Fla.) and were assembled
in a
CloneZone unit (USA/Scientific Plastics) to prevent airborne
contamination or template carryover from previous experiments.
Thermal
cycling was done with a DeltaCycler I unit (Ericomp, San
Diego,
Calif.). After amplification, 10 µl of each PCR mixture
was analyzed
by electrophoresis (8 mV/cm, 1 h) in ethidium bromide-stained
agarose (2%, wt/vol) gels. A 100-bp ladder (Promega) was included
for
molecular weight estimation. Gels were placed on an UV
transillumination
unit (Fotodyne, Hartland, Wis.) for band
visualization and photography.
Southern hybridization to DNA
immobilized on Hybond N
+ charged nylon membranes (Amersham,
Arlington Heights, Il.) was
done according to the manufacturer's
directions. Bound probe was
detected by chemiluminescence using the
Genius system and CPD-Star
substrate solution (Boehringer Mannheim,
Indianapolis, Ind.).
The membrane was then exposed to X-ray film
(Eastman Kodak, Rochester,
N.Y.), and the film was developed according
to the manufacturer's
instructions.
Detecting E. coli in soil by PCR.
Soil was sampled
from experimental plots located at the Arlington Research Station
(Arlington, Wis.) and at the Lakeland Agricultural Complex (Lakeland,
Wis.). The Arlington soil was a Plano silt loam (typic argiudoll; pH
6.9, 48 g of organic matter kg
1), while that from
Lakeland was a Griswold silt loam (aquic argiudoll; pH 7.0, 42 g
of organic matter kg1). At both sites, the plots were
established as continuous corn and had not been amended with animal
manure for at least 12 years. The samples collected were kept on ice
for transport to the laboratory and then stored frozen at 
20°C.
Prior to use, the soils were sieved through 2-mm mesh screen and their
moisture contents were determined. DNA was extracted by a freeze-thaw
method essentially as described by Tsai and Olson (22)
except that CaCl2 (final concentration of Ca2+,
90 mM) was substituted for NaCl in the lysis solution. The extracts were further purified by a Wizard kit (Promega). Use of
Ca2+ in the lysis solution precipitates humic materials and
makes cleanup by the Wizard kit more effective. Concentrations of
Ca2+ in extracts were 690 µM or less as determined by
atomic absorption spectroscopy and shown not to interfere with the PCR
(21). DNA purity and concentrations were determined by UV
absorbance at 260 and 280 nm and by gel image analysis with NIH Image
version 1.55 software (National Institutes of Health).
Two groups of experiments were done to examine the efficiency of the
PCR method for amplification of
E. coli 16S rRNA gene
sequences from soil. In the first, DNA was extracted from the
Arlington
and Lakeland soils, and an aliquot of each purified
extract (containing
approximately 100 ng of DNA) was then spiked
with either 1 ng or 1 pg
of
E. coli genomic DNA and used for PCR.
In the second
experiment, Lakeland soil (4 g, wet weight) was
inoculated with
E. coli at densities ranging from 7 × 10
4
to 7 × 10
7 cells g
1 (each inoculum
level established in triplicate) and then incubated
for 1 h. The
incubation time in soil was kept relatively short
to allow interaction
between cells and soil particles while minimizing
the extent to which
inoculum densities were altered by population
growth or death. Plate
counts were made on Levine eosin-methylene
blue (Difco Laboratories,
Detroit, Mich.) agar and
Shigella-Salmonella agar (Difco).
Plate counts on the two media were not significantly
different and
confirmed that numbers of cells inoculated into
and recovered from soil
after 1 h incubation were similar. DNA
was extracted from the
soils as described above. The amount of
DNA recovered from the
noninoculated soil was approximately 7
µg g
1, while
that extracted from the inoculated soils ranged from ca.
7 to 10 µg
g
1. A total of 10 ng of DNA from each extract was used in
the PCR
tests.
Primer design and PCR development.
The primary criterion for
selection of PCR primers was the identification of 16S rRNA gene
sequences that could be used to distinguish E. coli from its
closest relative, Shigella. The 16S rRNA gene of E. coli differs from that of Shigella flexneri, S. sonnei, S. boydii, and S. dysenteriae by 4, 5, 8, and 17 bases, respectively (7, 26). When the E. coli and Shigella 16S rRNA genes are aligned, only two
areas (both within hypervariable regions) have more than two mismatches
over 20 contiguous nucleotides, the length of a typical PCR primer. One
region spans bases 75 to 100 and was chosen as the target site for the
forward primer because of its relatively high number of mismatches with
Shigella (Table 2). The other
region has substantial disagreement between reported
Shigella sequences and was deemed unsuitable. Within the
region from bp 75 to 100, a number of primers targeting various stretches of nucleotides were considered, and one (ECP79F) that presented the best balance between the desired selectivity and potential unfavorable secondary structure characteristics was chosen
for further study. The reverse primer was ultimately targeted to bases
594 to 620, as mismatches in this region provide selection against
other Proteobacteria that may cross-react with the forward primer.
The first set of tests used the primer pair ECP79F-ECR620R and the
40-cycle protocol. Amplification was very effective, and
the amount of
product accumulated from as little as 50 fg of
E. coli
genomic DNA (five genomic equivalents) was sufficient to
allow easy
visualization in ethidium bromide-stained agarose gels.
However, in
these tests a product of the expected size was also
amplified from the
water blanks. We subsequently determined that
the latter originated
from
E. coli DNA present in the recombinant
polymerase
AmpliTaq and found that this problem could be eliminated
by treatment
of AmpliTaq (DNase digestion, UV light irradiation),
use of purified
recombinant polymerase (AmpliTaq LD), or using
Taq DNA
polymerase isolated from
Thermus aquaticus. The latter
was
the most cost- and time-efficient and was used as standard
practice.
Use of ECP79F-ECR609 combined with native
Taq polymerase in
the extended PCR program achieved the desired sensitivity. However,
subsequent experiments demonstrated that the PCR protocol was
not
adequately selective for
E. coli. In these specificity
tests,
there was amplification of the expected 541-bp product
from other
Enterobacteriaceae (
Citrobacter
freundii,
Proteus vulgaris,
Enterobacter aerogenes, and
Enterobacter nimipressuralis) and even
from
Pseudomonas aeruginosa. Amplification could have been
driven by the reverse
primer alone, but this would result in linear
amplification and
probably not yield the amount of product observed.
The annealing
conditions used were optimal for the primers, so it was
unlikely
that amplification resulted from nonspecific annealing to and
extension of regions outside the 16S rRNA gene. Selectivity was
not
improved by reducing template concentrations from 100 to 1
ng, although
at lower levels the amount of PCR product amplified
from
E. coli template was greater than that from nontarget organisms.
A
variety of modifications to the PCR program's annealing temperature
and the reaction mixture composition (e.g., inclusion of dimethyl
sulfoxide or modulation of MgCl
2 level) were also tested,
but
none eliminated the cross-reactivations.
The main constraint for improving PCR selectivity was restriction to a
single region within the 16S rRNA gene for which
E. coli-specific primers could be targeted. The strategy adopted
was
to increase stringency by increasing the annealing temperature
and by
designing longer forward primers (ECA75F and ECB75F) expected
to have
greater stability at elevated temperatures. Initial PCR
experiments
with ECA75F-ECR619R and ECB75F-ECR620R were run using
the optimal
annealing temperatures for each primer pair. Both
sets showed good
sensitivity, giving detectable amplification
from
E. coli
DNA template levels of at least 500 fg, but cross-reaction
with
Shigella and other enteric bacteria persisted. Selective
amplification of
E. coli was attained when the annealing
temperature
was increased to 72°C, but only with ECA75F-ECR619R.
However,
while the increase in annealing temperature gave the desired
selectivity,
it had a serious negative impact on the sensitivity as
E. coli template levels needed to be in the nanogram range
for
detection.
Subsequent tests focused on increasing sensitivity of the PCR by
altering amounts of reaction mixture components. First, the
MgCl
2 concentration was reexamined for its effects on the
efficiency
of amplification from low template levels (all reactions
done
in a total volume of 50 µl). Amplification from high amounts of
template (100 ng, ca. 10
8 copies) was efficient with
MgCl
2 levels ranging from 1.0 to 3.0
mM. However, with low
amounts of template (500 fg), amplification
was efficient only between
2.0 and 2.5 mM MgCl
2. Next, the combination
of
Taq DNA polymerase and MgCl
2 levels that gave
the best balance
in sensitivity and selectivity (
E. coli
versus
S. sonnei) at low
template levels (500 fg, ca. 500 copies) was examined and identified
as 1.5 U of
Taq and 2.0 mM MgCl
2. The selectivity of the optimized
PCR protocol was
then verified with a battery of different
E. coli,
Shigella, and
Salmonella isolates (Fig.
1). The detection
limit of the optimized
PCR was established by Southern hybridization
as 100 fg of template,
which is equivalent to ca. 10 cells of
E. coli (Fig.
2).
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FIG. 1.
Specificity of primer pair ECA75F-ECR619R in the
optimized PCR for amplification of E. coli 16S rRNA. (A)
Agarose gel separation of PCR mixtures; (B) Southern hybridization of
the gel in panel A to EUB338. Lanes: 1, 100-bp DNA ladder; 2, no
template; 3 to 5, 1 ng, 100 pg, and 10 pg of E. coli UW8101
template; 6 to 8, 1 ng, 100 pg, and 10 pg of E. coli UW8002
template; 9 to 11, 1 ng, 100 pg, and 10 pg of E. coli UW8204
template; 12 to 14, 1 ng, 100 pg, and 10 pg of E. coli
UW8009 template; 15 and 16, 1 ng and 100 pg of Salmonella
serovar Typhimurium UW8P14 template; 17 and 18, 1 ng and 100 pg of
Salmonella serovar Typhimurium UW8P40 template; 19 to 21, 1 ng, 100 pg, and 10 pg of E. coli UW8410 template; 22 to 24, 1 ng, 100 pg, and 10 pg of E. coli UW8P39 template; 25 to 27, 1 ng,
100 pg, and 10 pg of E. coli UW8001D template; 28 and 29, 1 ng and 100 pg of Shigella dysenteriae UW8P01 template; 30 and 31, 1 ng and 100 pg of Shigella dysenteriae WSLH
template; 32 and 33, 1 ng and 100 pg of Shigella flexnerii
UW8P02 template; 34 and 35, 1 ng and 100 pg of Shigella
sonnei UW8P15 template. Strain designations refer to those given
in Table 1.
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FIG. 2.
Sensitivity of primer pair ECA75F-ECR619R in the
optimized PCR for amplification of E. coli 16S rRNA DNA
template. (A) Agarose gel separation of PCR mixtures; (B) Southern
hybridization of the gel in panel A to EUB338. Lanes: 1, 100-bp DNA
ladder; 2, no template; 3 to 12, 100 pg, 50 pg, 10 pg, 5 pg, 1 pg, 500 fg, 100 fg, 50 fg, 10 fg, and 5 fg of E. coli genomic DNA.
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Specificity was also examined by using BLAST to search GenBank for
sequences similar to ECA75F and ECR619R.
Vibrio gazogenes and
Enterobacter gergoviae have single mismatches to the 5'
end
of ECA75F but several mismatches to ECR619R. There were 15 exact
matches to ECA75F, 13 of these were bacteria of the
Pasteurellaceae family (various species or subspecies of
Haemophilus,
Mannheimia,
and
Pasteurella), and two were unknown
-
Proteobacteria. All of
these organisms, however,
had a number of mismatches to ECR619R.
The greatest similarity to
our primers was with
Erwinia psidii,
Pseudomonas
flectens,
Salmonella enterica serovar Waycross, and
S. enterica serovar Chingola, all of which had either a
single
mismatch or single point deletion with ECA75F and were exact
matches
to ECR619R. This last group would theoretically pose the
greatest
potential for cross-reaction. However, these organisms are
probably
not typical water or soil inhabitants (
E. psidii
and
P. flectens are described as originating from Brazilian
guava fruit and an
Australian bean, respectively), nor are they common
residents
of the human digestive tract. Thus, based on both empirical
and
theoretical testing, the method is expected to be reliable for
selective detection of
E. coli in soil or water contaminated
by
human
wastes.
We evaluated the optimized PCR protocol for detection of
E. coli in environmental samples. Soil was selected as it is an
important
reservoir for
E. coli, and its chemical complexity
provides a
good test of the method's robustness. Initial tests to
examine
matrix effects on the PCR method were done with DNA extracted
from soil and spiked with
E. coli genomic DNA. These
experiments
showed that method performance with a heterogeneous mixture
was
similar to that obtained with pure
E. coli genomic DNA:
as little
as 1 pg of
E. coli template added to the PCR
mixture gave sufficient
product to detect by gel electrophoresis (Fig.
3). The sensitivity
was comparable to
that obtained using pure
E. coli genomic DNA
(Fig.
2). In
tests to evaluate detection of
E. coli DNA extracted
from
soil inoculated with various densities of
E. coli cells,
amplification was positive from all DNA extracts, and the expected
product was detectable using agarose gels or Southern hybridization
(Fig.
4). Furthermore, the lack of
amplification from noninoculated
soil, which likely had an indigenous
population of enteric bacteria
(e.g.,
Klebsiella), suggests
that the method provided the necessary
selectivity. The main difference
between treatments was that with
extracts prepared from the two
lowest-inoculum densities, PCR
products were sometimes detected only by
hybridization. This probably
reflects variability in
E. coli
DNA recoveries. Because many variables
affect DNA extraction from
bacterial cells in soil (e.g., soil
type, residence time of cells in
the soil, and soil biomass level),
the detection limits achievable by
this method will vary on a
case-by-case basis.
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FIG. 3.
Amplification of E. coli 16S rRNA from DNA
extracted from Arlington (A) and Lakeland (L) soil and spiked with
either 100 ng or 1 pg of E. coli genomic DNA. Lanes: 1, 100-bp DNA ladder; 2, A, nonspiked; 3, L, nonspiked; 4, A, 1-ng spike;
5, A, 1-pg spike; 6, L, 1-ng spike; 7, L, 1-pg spike; 8, 1 ng of
E. coli genomic DNA; 9, 1 pg of E. coli genomic
DNA; 10, no template.
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FIG. 4.
Amplification of E. coli 16S rRNA from
nonsterile soil inoculated with various densities of E. coli. (A) Ethidium bromide-stained agarose gel; (B) Southern
hybridization of gel from panel A to EUB338. Lanes: 1, 100-bp DNA
ladder; 2, no template; 3, 10 ng of DNA extract template from
nonsterile, nonseeded soil sample; 4 to 18, 10 ng of DNA extract
template from nonsterile soil samples seeded with stationary-phase
E. coli at 7 × 104, 7 × 105, 2 × 106, 7 × 106,
and 7 × 107 CFU g1, respectively.
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In summary, a sensitive and robust PCR protocol was developed that
discriminated
E. coli from
Shigella and other
enteric bacteria
based on selective amplification of 16S rRNA gene
sequences. To
the best of our knowledge, this is the first report of a
PCR protocol
based on amplification of a 16S rRNA that effectively
distinguishes
E. coli from these closely related bacteria.
For environmental
analysis, the extent to which the method's
sensitivity of ca.
10 genomic equivalents can be exploited is
controlled primarily
by the efficiency with which DNA is extracted from
the target
organism, concentrated, and purified for the samples used in
the
PCR. For high-volume sample analysis, the protocol could be coupled
with rapid methods for PCR product detection such as fluorogen
labeling
or enzyme-linked immunosorbent
assay.
| |
ACKNOWLEDGMENTS |
This work was supported by funding from the U.S. Department of the
Interior, U.S. Geological Survey, Regional Competitive Grants Program
(award 1434-HQ-GR-02707) and from the Wisconsin Department of Commerce
(award DDG70000228).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Soil Science, University of WisconsinMadison, Madison, WI 53706-1299. Phone: (608) 262-9018. Fax: (608) 265-2595. E-mail:
wjhickey{at}facstaff.wisc.edu.
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Applied and Environmental Microbiology, February 2000, p. 844-849, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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