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Applied and Environmental Microbiology, October 2000, p. 4571-4574, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A PCR Assay To Discriminate Human and Ruminant Feces on the Basis
of Host Differences in Bacteroides-Prevotella Genes
Encoding 16S rRNA
Anne E.
Bernhard and
Katharine G.
Field*
Department of Microbiology, Oregon State
University, Corvallis, Oregon 97330
Received 14 April 2000/Accepted 26 July 2000
 |
ABSTRACT |
Our purpose was to develop a rapid, inexpensive method of
diagnosing the source of fecal pollution in water. In previous
research, we identified Bacteroides-Prevotella ribosomal
DNA (rDNA) PCR markers based on analysis. These markers length
heterogeneity PCR and terminal restriction fragment length polymorphism
distinguish cow from human feces. Here, we recovered 16S rDNA clones
from natural waters that were close phylogenetic relatives of the
markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination.
| |
TEXT |
The inability to identify the source
of fecal contamination is partly to blame for the persistent problem of
fecal pollution in coastal and inland waters. Although methods exist to
quantify fecal pollution, none quickly and accurately identifies the
animal source. Antibiotic resistance patterns of fecal streptococci
(8, 16, 17) and Escherichia coli ribosomal DNA
(rDNA) tracking (14; D. Akre and J. Wilcox,
Northwest Algal Symp. Pacific Estuarine Res. Soc. Joint Meet.,
1998) have recently emerged as potentially useful, but
labor-intensive, solutions to the problem. Their reliability, however,
may be considerably less than 100% (16, 17).
Unlike these methods, which require culturing indicator organisms,
detection of host-specific molecular markers does not require culturing
and holds promise as a precise, rapid method for identifying sources of
fecal contamination. The Bacteroides-Prevotella group is one
of several noncoliform bacterial groups that has been proposed as an
alternative fecal pollution indicator (1, 5, 10), partly
because of its abundance in feces. The use of molecular methods makes
it more feasible to use anaerobic bacteria that are potentially
difficult to grow, such as members of the
Bacteroides-Prevotella group, as indicators.
We recently identified host-specific Bacteroides-Prevotella
16S rDNA markers for humans and cows by screening fecal DNAs by length
heterogeneity PCR (LH-PCR) (15) or terminal restriction fragment length polymorphism (T-RFLP) (11) analysis
(2). Cloning and sequencing experiments revealed that each
marker comprised multiple sequences forming host-specific gene
clusters. Here, we have identified additional clones, recovered from
water samples, that cluster with the fecal clones. Using the sequences
from fecal and water clones, we developed cluster-specific primers that
can discriminate between human and ruminant feces.
Clones recovered from water samples.
To identify fecal
Bacteroides-Prevotella rDNA markers in water, we collected
six 1-liter water samples from areas in Tillamook Bay, Oreg., that are
frequently contaminated with fecal pollution. We processed the samples
as previously described (2). DNAs from each water sample
were amplified with Bacteroides-Prevotella-specific primers
(Bac32F and Bac708R) as described previously (2). Equal portions of PCR products from all water samples were pooled and cloned
into pGEM T-Easy vectors according to the manufacturer's directions
(Promega, Madison, Wis.).
To locate marker clones, we screened 192 clones for LH-PCR and
T-RFLP host-specific patterns, and we found 7 unique clones that
corresponded to human or cow genetic markers previously identified (2). Clones with host-specific LH-PCR or T-RFLP patterns
were sequenced as described elsewhere (2). All
sequences were checked for chimeric structure with CHECK_CHIMERA of the
Ribosomal Database Project (12) and by comparisons to other
clones in our study. Similarities were calculated using the distance
function in GCG, version 10 (Genetics Computer Group, Madison, Wis.),
with the Kimura two-parameter correction. Sequence analysis of clones
recovered from water samples revealed that they were all very similar,
but not identical, to clones recovered from human and cow fecal samples (Fig. 1) (2).
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|
FIG. 1.
Phylogenetic relationships among partial 16S rDNA
sequences (558 positions) of clones recovered from Tillamook Bay water
samples (TB). HF and CF are host-specific genetic markers identified
from human and cow fecal clone libraries, respectively. The tree was
inferred by neighbor joining. Numbers above the internal branches are
percentages of bootstrap replicates that support the branching order.
Bootstrap values below 50% are not shown. Bootstrap values for
branches a and b dropped from 68 to 47 and 76 to
40, respectively, when TB147 was added to the analysis. The sequence
from Cytophaga fermentans was used to root the tree.
|
|
Although previous analyses confirmed that noncontaminated water does
not contain detectable
Bacteroides-Prevotella DNA
(
2),
we performed additional experiments to confirm that the
clones
recovered from water samples were fecal in origin. We designed
primers specific to two of the water clones, TB141 and TB147,
and
amplified 16S rRNA genes from cow fecal DNAs. The methods
for cow fecal
sample collection and processing are presented elsewhere
(
2). Sequence analysis of the PCR products confirmed that
the
sequences were the same as the sequences of the two
clones.
We aligned these clones with the fecal clones from our previous study
and inferred a phylogenetic tree with the neighbor-joining
algorithm
(
13) in PHYLIP, version 3.5c (
4). Six of the
seven
clones recovered from water samples clustered with human- or
cow-specific
sequences identified in our earlier study (Fig.
1). TB13
corresponded
to the human-specific cluster HF8 and was greater than
99% similar
to other clones in this cluster. The TB13 sequence
differed by
only one or two bases from HF8, HF117, and HF145; these
differences
could be attributed to PCR or sequencing errors. The
remaining
clones corresponded to the cow-specific markers. TB141 had
the
same T-RFLP pattern as CF46, CF68, and CF151 and was 84.7 to 90.4%
similar to the other CF151 clones. TB101, TB106, TB135, and TB146
had
the same T-RFLP pattern as the other clones in the CF123 cluster
and
were 93.3 to 96.1% similar. TB147 had the same T-RFLP pattern
as the
clones in the CF123 cluster, but the sequence grouped with
the CF151
cluster. Additionally, TB147 had the highest similarity
with CF17
(88.2%), which is in the CF123 cluster. Bootstrap values
for the CF151
cluster dropped considerably when TB147 was included
in the analysis,
suggesting that the branching order of TB147
is not strongly supported.
It is unlikely that TB147 is a chimeric
sequence since the same
sequence was recovered from fecal and
water samples
independently.
Primer design.
To develop a PCR assay for identifying sources
of fecal bacteria in water, we designed primers specific for each
cluster and for clone HF10 (Table 1). We
established specificity and optimal annealing temperatures for all
primer pairs by using plasmid DNAs from target and closely
related nontarget sequences as well as Bacteroides DNA from
cultures (B. distasonis, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis,
and B. vulgatus; all were gifts from A. Salyers). Additional
confirmation of specificity was obtained through PROBE_MATCH of the
Ribosomal Database Project. PCR mixtures were described by us
previously (2). A thermal minicycler (MJ Research,
Watertown, Mass.) was used for all reactions, with the following
conditions: 25 cycles of 94°C for 30 s, appropriate annealing
temperature (Table 1) for 30 s, and 72°C for 1 min followed by a
final 6-min extension at 72°C. To increase the sensitivity of
detection, 1 µl of each PCR product was reamplified using the same
conditions. PCR products were visualized in a 1% agarose gel stained
with 1 µg of ethidium bromide/ml.
Host-specific primers were further tested by amplifying fecal DNAs from
target hosts (Table
2). DNAs from human
and cow feces
and sewage were collected and processed according to
methods described
elsewhere (
2). We detected genes
corresponding to the HF8 cluster
in 11 of 13 human fecal samples, all
of the sewage samples, and
none of the cow fecal samples. Using the
HF10-targeted primers,
we detected PCR product in less than half of the
sewage and human
fecal samples and in one cow fecal sample. Because HF8
genes were
more widely distributed among the humans and primers for
HF10
were not as specific as desired, we tested only for HF8 genes
in
subsequent analyses. Genes from the CF151 and CF123 clusters
were
detected in all cow samples but in none of the human or sewage
samples.
To determine the host specificity of these primers, we tested fecal
samples collected from other animals (Table
3). Samples
were collected with sterile
utensils and placed in sterile 50-ml
tubes or plastic bags, kept on ice
for transport to the lab, and
immediately stored at
80°C. Fecal
DNAs were extracted using the
Fast DNA kit for soil (Bio 101, Vista,
Calif.), by following the
manufacturer's directions. Samples were
tested for marker genes
by PCR. HF8 sequences were not detected in any
samples (Table
3). CF123 and CF151 sequences, however, were
detected in all
ruminant animals and in llamas, which are members of
the same
order (
Artiodactyla) but are considered
pseudoruminants (
3).
A positive PCR result for CF123 or
CF151, therefore, does not
rule out wildlife sources, such as
deer and elk, but land use
evaluation could determine the likelihood of
an agricultural or
wildlife source.
PCR sensitivity.
Sensitivity of the PCRs was evaluated by
amplifying marker genes from serial dilutions of plasmid DNAs
from the clones CF123, CF68, and HF145. Detection limits were
approximately 10
12 g of DNA (105 gene copies)
for all three plasmid DNAs.
We also tested the sensitivity of our host-specific primers using
serial dilutions of cow feces or raw sewage. Sensitivity
assays were
carried out as described elsewhere (
2). DNAs from
each
dilution were tested for the markers by PCR. We measured
fecal
coliforms in each dilution according to standard methods
(
7).
Detection of CF123 genes was as sensitive as detection of fecal
coliforms (Table
4). Detection of fecal
coliforms, however,
was 10- to 100-fold more sensitive than detection
of CF151 and
HF8 genes. The sensitivity assay using cow fecal dilutions
was
repeated with feces from different cows, and similar results were
obtained (Table
4). Although the results varied slightly, we
believe
that these differences are not significant. Some of the
variability may
be due to uneven dispersion of cells during fecal
suspension and
dilution. In addition, because we are not currently
able to measure the
exact number of the marker genes in a fecal
sample and there may be
individual variability, these limits of
detection represent
approximations.
If the detection limit of 10
5 gene copies using plasmid
DNAs is extrapolated to the detection results from the serial dilutions
of feces, then we must assume that 2 × 10
6 g of cow
feces (the average sensitivity for cow feces samples
A and B in Table
4) contains at least 10
5 gene copies. This translates to
5 × 10
10 copies/g of feces. Assuming an average of
3 × 10
11 bacterial cells/g of feces (
6)
and an average of five 16S
rDNA operons per
Bacteroides cell
(rRNA Operon Copy Number Collection
[
http://rdp.cme.msu.edu/rrn/]),
then 5 × 10
10 copies/g of feces represents 3% of the
total bacteria. If
Bacteroides cells comprise 30% of the
total fecal bacteria (
9), we estimate
a density of
10
11 Bacteroides cells/g of feces; based on this
estimate, the host-specific
markers would represent 10% of the
Bacteroides cells. This estimate
seems reasonable,
especially considering potential errors associated
with pipetting fecal
slurries.
These detection limits are similar to other estimates of the
contribution of host-specific marker genes to total
Bacteroides cells. We calculated the relative abundance of
the host-specific
LH-PCR peak for the CF151 cluster (
2),
compared to the relative
abundance of total
Bacteroides PCR
amplicons. The relative fluorescence
of the host-specific peak (the
area under the peak relative to
the total area) was approximately 7%
of the total
Bacteroides PCR products (data not shown).
Additionally, marker sequences
recovered from the Tillamook Bay clone
library comprised 4% of
all
Bacteroides clones, which is
consistent with the percentages
of marker sequences found in our human
and cow fecal clone libraries
(3.1 and 6.3%, respectively)
(
2).
Although extensive field testing is required to determine the efficacy
of the assays and the geographic distribution of the
host-specific
markers before these markers can be used for routine
water quality
monitoring, we believe that these PCR assays provide
a promising
diagnostic tool for identifying nonpoint sources of
fecal pollution.
Additionally, our approach for the identification
of diagnostic markers
can be easily applied to find markers for
animals besides humans and
ruminants.
Nucleotide sequence accession numbers.
The sequences described
in this paper have been submitted to GenBank with accession
numbers AF294903, Af294904, AF294905, AF294906, AF294907,
AF294908, and AF294909.
| |
ACKNOWLEDGMENTS |
We are grateful for assistance from Weerathep Pongprasert, Mike
Rappé, Nancy Ritchie, and Kevin Vergin.
This work was partially supported by grant NA76RG0476 (project no.
R/ECO-04) from the National Oceanic and Atmospheric Administration to
the Oregon State University Sea Grant College Program, by
appropriations made by the Oregon State legislature, and by grant
R827639-01-0 from the U.S. Environmental Protection Agency. This work
was also supported by the Research Council of Oregon State University.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR
97331. Phone: (541) 737-1837. Fax: (541) 737-0496. E-mail:
fieldk{at}bcc.orst.edu.
| |
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Applied and Environmental Microbiology, October 2000, p. 4571-4574, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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