A Pcr Assay To Discriminate Human And Ruminant Feces On The Basis ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 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
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.
The inability to identify the source of fecal contamination is
samples were pooled and cloned into pGEM T-Easy vectors
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partly to blame for the persistent problem of fecal pollution in
according to the manufacturer’s directions (Promega, Madi-
coastal and inland waters. Although methods exist to quantify
son, Wis.).
fecal pollution, none quickly and accurately identifies the an-
To locate marker clones, we screened 192 clones for LH-
imal source. Antibiotic resistance patterns of fecal streptococci
PCR and T-RFLP host-specific patterns, and we found 7
(8, 16, 17) and Escherichia coli ribosomal DNA (rDNA) track-
unique clones that corresponded to human or cow genetic
ing (14; D. Akre and J. Wilcox, Northwest Algal Symp. Pacific
markers previously identified (2). Clones with host-specific
Estuarine Res. Soc. Joint Meet., 1998) have recently
LH-PCR or T-RFLP patterns were sequenced as described
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emerged as potentially useful, but labor-intensive, solutions to
elsewhere (2). All sequences were checked for chimeric struc-
the problem. Their reliability, however, may be considerably
ture with CHECK_CHIMERA of the Ribosomal Database
less than 100% (16, 17).
Project (12) and by comparisons to other clones in our study.
Unlike these methods, which require culturing indicator or-
Similarities were calculated using the distance function in
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ganisms, detection of host-specific molecular markers does not
GCG, version 10 (Genetics Computer Group, Madison, Wis.),
require culturing and holds promise as a precise, rapid method
with the Kimura two-parameter correction. Sequence analysis
for identifying sources of fecal contamination. The Bacteroides-
of clones recovered from water samples revealed that they
Prevotella group is one of several noncoliform bacterial groups
were all very similar, but not identical, to clones recovered
that has been proposed as an alternative fecal pollution indi-
from human and cow fecal samples (Fig. 1) (2).
cator (1, 5, 10), partly because of its abundance in feces. The
Although previous analyses confirmed that noncontami-
use of molecular methods makes it more feasible to use an-
nated water does not contain detectable Bacteroides-Prevotella
aerobic bacteria that are potentially difficult to grow, such as
DNA (2), we performed additional experiments to confirm
members of the Bacteroides-Prevotella group, as indicators.
that the clones recovered from water samples were fecal in
We recently identified host-specific Bacteroides-Prevotella
origin. We designed primers specific to two of the water clones,
16S rDNA markers for humans and cows by screening fecal
TB141 and TB147, and amplified 16S rRNA genes from cow
DNAs by length heterogeneity PCR (LH-PCR) (15) or termi-
fecal DNAs. The methods for cow fecal sample collection and
nal restriction fragment length polymorphism (T-RFLP) (11)
processing are presented elsewhere (2). Sequence analysis of
analysis (2). Cloning and sequencing experiments revealed that
the PCR products confirmed that the sequences were the same
each marker comprised multiple sequences forming host-spe-
as the sequences of the two clones.
cific gene clusters. Here, we have identified additional clones,
We aligned these clones with the fecal clones from our
recovered from water samples, that cluster with the fecal
previous study and inferred a phylogenetic tree with the neigh-
clones. Using the sequences from fecal and water clones, we
bor-joining algorithm (13) in PHYLIP, version 3.5c (4). Six of
developed cluster-specific primers that can discriminate be-
the seven clones recovered from water samples clustered with
tween human and ruminant feces.
human- or cow-specific sequences identified in our earlier
Clones recovered from water samples. To identify fecal Bac-
study (Fig. 1). TB13 corresponded to the human-specific clus-
teroides-Prevotella rDNA markers in water, we collected six
1-liter water samples from areas in Tillamook Bay, Oreg., that
ter HF8 and was greater than 99% similar to other clones in
are frequently contaminated with fecal pollution. We pro-
this cluster. The TB13 sequence differed by only one or two
cessed the samples as previously described (2). DNAs from
bases from HF8, HF117, and HF145; these differences could
each water sample were amplified with Bacteroides-Prevotella-
be attributed to PCR or sequencing errors. The remaining
specific primers (Bac32F and Bac708R) as described previ-
clones corresponded to the cow-specific markers. TB141 had
ously (2). Equal portions of PCR products from all water
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%
* Corresponding author. Mailing address: Dept. of Microbiology,
220 Nash Hall, Oregon State University, Corvallis, OR 97331. Phone:
similar. TB147 had the same T-RFLP pattern as the clones in
(541) 737-1837. Fax: (541) 737-0496. E-mail: fieldk@bcc.orst.edu.
the CF123 cluster, but the sequence grouped with the CF151
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BERNHARD AND FIELD
APPL. ENVIRON. MICROBIOL.
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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.
cluster. Additionally, TB147 had the highest similarity with
specificity and optimal annealing temperatures for all primer
CF17 (88.2%), which is in the CF123 cluster. Bootstrap values
pairs by using plasmid DNAs from target and closely related
for the CF151 cluster dropped considerably when TB147 was
nontarget sequences as well as Bacteroides DNA from cul-
included in the analysis, suggesting that the branching order of
tures (B. distasonis, B. fragilis, B. ovatus, B. thetaiotaomicron,
TB147 is not strongly supported. It is unlikely that TB147 is a
B. uniformis, and B. vulgatus; all were gifts from A. Salyers).
chimeric sequence since the same sequence was recovered
Additional confirmation of specificity was obtained through
from fecal and water samples independently.
PROBE_MATCH of the Ribosomal Database Project. PCR
Primer design. To develop a PCR assay for identifying
mixtures were described by us previously (2). A thermal
sources of fecal bacteria in water, we designed primers specific
minicycler (MJ Research, Watertown, Mass.) was used for all
for each cluster and for clone HF10 (Table 1). We established
reactions, with the following conditions: 25 cycles of 94°C for
TABLE 1. Primers used in this studya
Primer
Sequence (5 –3 )
Target
Annealing temp (°C)
Reference
Bac32F
AACGCTAGCTACAGGCTT
Bacteroides-Prevotella
53
2
Bac708R
CAATCGGAGTTCTTCGTG
Bacteroides-Prevotella
2
CF128F
CCAACYTTCCCGWTACTC
CF123 cluster
58
This study
CF193F
TATGAAAGCTCCGGCC
CF151 cluster
55
This study
HF134F
GCCGTCTACTCTTGGCC
HF10
61
This study
HF183F
ATCATGAGTTCACATGTCCG
HF8 cluster, HF74
59
This study
HF654R
CCTGCCTCTACTGTACTC
HF10
61
This study
a Bac, Bacteroides-Prevotella; HF, human-specific; CF, cow-specific. Numbers correspond to the numbers of the E. coli 16S rRNA gene. All forward primers except
HF134F were paired with Bac708R. HF134F was paired with HF654R. Annealing temperatures were empirically determined for each primer pair as described in the
text.
VOL. 66, 2000
IDENTIFICATION OF HUMAN AND RUMINANT FECES BY PCR
4573
TABLE 2. Distribution of host-specific genetic markers
TABLE 4. Detection limits of host-specific genetic
in feces from targeted hosts
markers and fecal coliformsa
No. of positive PCR resultsa
Detection limit (g of dry feces/liter)
No. of
Source of
Target
samples
Human markers
Cow markers
DNA
HF8
CF123
CF151
Fecal
tested
cluster
cluster
cluster
coliforms
HF8
HF10
CF123
CF151
cluster
cluster
cluster
cluster
Cow feces A
ND
2.8
10 7
2.8
10 5
2.8
10 7
Cow feces B
ND
3.6
10 6
3.6
10 5
3.6
10 6
Human
13
11
6
0
0
Sewage
1.4
10 6
ND
ND
1.4
10 7
Sewage
3
3
1
0
0
Cow
19
0
1
19
19
a Results are from dilution assays using either cow feces or raw sewage. Each
cow sample combined feces from four cows. The sensitivity of detection of cow
a PCR results are from two rounds of 25 cycles each.
feces was measured twice, with two independent samples (A and B). Sewage
dilutions were not replicated. Results for detection of the genetic markers are
from two rounds (25 cycles each) of PCR. ND, not determined.
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 l of each PCR product
were detected in all ruminant animals and in llamas, which
was reamplified using the same conditions. PCR products were
are members of the same order (Artiodactyla) but are con-
visualized in a 1% agarose gel stained with 1 g of ethidium
sidered pseudoruminants (3). A positive PCR result for CF123
bromide/ml.
or CF151, therefore, does not rule out wildlife sources, such as
Host-specific primers were further tested by amplifying fecal
deer and elk, but land use evaluation could determine the
DNAs from target hosts (Table 2). DNAs from human and cow
likelihood of an agricultural or wildlife source.
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feces and sewage were collected and processed according to
PCR sensitivity. Sensitivity of the PCRs was evaluated by
methods described elsewhere (2). We detected genes corre-
amplifying marker genes from serial dilutions of plasmid
sponding to the HF8 cluster in 11 of 13 human fecal samples,
DNAs from the clones CF123, CF68, and HF145. Detection
all of the sewage samples, and none of the cow fecal samples.
limits were approximately 10 12 g of DNA (105 gene copies)
Using the HF10-targeted primers, we detected PCR product in
for all three plasmid DNAs.
less than half of the sewage and human fecal samples and in
We also tested the sensitivity of our host-specific primers
one cow fecal sample. Because HF8 genes were more widely
using serial dilutions of cow feces or raw sewage. Sensitivity
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distributed among the humans and primers for HF10 were not
assays were carried out as described elsewhere (2). DNAs from
as specific as desired, we tested only for HF8 genes in subse-
each dilution were tested for the markers by PCR. We mea-
quent analyses. Genes from the CF151 and CF123 clusters
sured fecal coliforms in each dilution according to standard
were detected in all cow samples but in none of the human or
methods (7).
sewage samples.
Detection of CF123 genes was as sensitive as detection of
by on February 9, 2010
To determine the host specificity of these primers, we tested
fecal coliforms (Table 4). Detection of fecal coliforms, how-
fecal samples collected from other animals (Table 3). Samples
ever, was 10- to 100-fold more sensitive than detection of
were collected with sterile utensils and placed in sterile 50-ml
CF151 and HF8 genes. The sensitivity assay using cow fecal
tubes or plastic bags, kept on ice for transport to the lab, and
dilutions was repeated with feces from different cows, and
immediately stored at 80°C. Fecal DNAs were extracted us-
similar results were obtained (Table 4). Although the results
ing the Fast DNA kit for soil (Bio 101, Vista, Calif.), by fol-
varied slightly, we believe that these differences are not signif-
lowing the manufacturer’s directions. Samples were tested for
icant. Some of the variability may be due to uneven dispersion
marker genes by PCR. HF8 sequences were not detected in
of cells during fecal suspension and dilution. In addition, be-
any samples (Table 3). CF123 and CF151 sequences, however,
cause 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 105 gene copies using plasmid DNAs
TABLE 3. Distribution of host-specific genetic markers
is extrapolated to the detection results from the serial dilutions
in feces from nontarget animals
of feces, then we must assume that 2
10 6 g of cow feces (the
No. of positive PCR resultsa
average sensitivity for cow feces samples A and B in Table 4)
No. of
Human
contains at least 105 gene copies. This translates to 5
1010
Animal
samples
marker
Cow markers
copies/g of feces. Assuming an average of 3
1011 bacterial
tested
cells/g of feces (6) and an average of five 16S rDNA operons
HF8
CF123
CF151
per Bacteroides cell (rRNA Operon Copy Number Collection
cluster
cluster
cluster
[http://rdp.cme.msu.edu/rrn/]), then 5
1010 copies/g of feces
Cat
3
0
0
0
represents 3% of the total bacteria. If Bacteroides cells com-
Deerb
3
0
2
3
prise 30% of the total fecal bacteria (9), we estimate a density
Dog
3
0
0
0
of 1011 Bacteroides cells/g of feces; based on this estimate, the
Duck
3
0
0
0
host-specific markers would represent 10% of the Bacteroides
Elkb
3
0
3
3
cells. This estimate seems reasonable, especially considering
Goatb
1
0
1
1
Llamac
1
0
1
1
potential errors associated with pipetting fecal slurries.
Pig
3
0
0
0
These detection limits are similar to other estimates of the
Seagull
3
0
0
0
contribution of host-specific marker genes to total Bacteroides
Sheepb
4
0
4
4
cells. We calculated the relative abundance of the host-specific
a
LH-PCR peak for the CF151 cluster (2), compared to the
PCR results are from two rounds of 25 cycles each.
b Ruminant.
relative abundance of total Bacteroides PCR amplicons. The
c Pseudoruminant.
relative fluorescence of the host-specific peak (the area under
4574
BERNHARD AND FIELD
APPL. ENVIRON. MICROBIOL.
the peak relative to the total area) was approximately 7% of
4. Felsenstein, J. 1989. PHYLIP—phylogeny inference package (v3.5). Cladis-
the total Bacteroides PCR products (data not shown). Addi-
tics 5:164–166.
tionally, marker sequences recovered from the Tillamook Bay
5. Fiksdal, L., J. S. Maki, S. J. LaCroix, and J. T. Staley. 1985. Survival and
clone library comprised 4% of all Bacteroides clones, which is
detection of Bacteroides spp., prospective indicator bacteria. Appl. Environ.
Microbiol. 49:148–150.
consistent with the percentages of marker sequences found in
6. Franks, A. H., H. J. M. Harmsen, G. C. Raangs, G. J. Jansen, F. Schut, and
our human and cow fecal clone libraries (3.1 and 6.3%, respec-
G. W. Welling. 1998. Variations of bacterial populations in human feces
tively) (2).
measured by fluorescent in situ hybridization with group-specific 16S rRNA-
Although extensive field testing is required to determine the
targeted oligonucleotide probes. Appl. Environ. Microbiol. 64:3336–3345.
efficacy of the assays and the geographic distribution of the
7. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1992. Standard
methods for the examination of water and wastewater, 18th ed. American
host-specific markers before these markers can be used for
Public Health Association, Washington, D.C.
routine water quality monitoring, we believe that these PCR
8. Hagedorn, C., S. L. Robinson, J. R. Filtz, S. M. Grubbs, T. A. Angier, and
assays provide a promising diagnostic tool for identifying non-
R. B. Reneau, Jr. 1999. Determining sources of fecal pollution in a rural
point sources of fecal pollution. Additionally, our approach for
Virginia watershed with antibiotic resistance patterns in fecal streptococci.
the identification of diagnostic markers can be easily applied to
Appl. Environ. Microbiol. 65:5522–5531.
9. Holdeman, L. V., I. J. Good, and W. E. C. Moore. 1976. Human fecal flora:
find markers for animals besides humans and ruminants.
variation in bacterial composition within individuals and a possible effect of
Nucleotide sequence accession numbers. The sequences de-
emotional stress. Appl. Environ. Microbiol. 31:359–375.
scribed in this paper have been submitted to GenBank with
10. Kreader, C. A. 1995. Design and evaluation of Bacteroides DNA probes for
accession numbers AF294903, Af294904, AF294905, AF294906,
the specific detection of human fecal pollution. Appl. Environ. Microbiol.
AF294907, AF294908, and AF294909.
61:1171–1179.
11. Liu, W.-T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization
of microbial diversity by determining terminal restriction fragment length
We are grateful for assistance from Weerathep Pongprasert, Mike
polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol.
Rappe´, Nancy Ritchie, and Kevin Vergin.
63:4516–4522.
This work was partially supported by grant NA76RG0476 (project
12. Maidak, B. L., N. Larsen, M. J. McCaughey, R. Overbeek, G. J. Olsen, K.
Downloaded from
no. R/ECO-04) from the National Oceanic and Atmospheric Admin-
Fogel, J. Blandy, and C. R. Woese. 1994. The Ribosomal Database Project.
istration to the Oregon State University Sea Grant College Program,
Nucleic Acids Res. 22:3485–3487.
by appropriations made by the Oregon State legislature, and by grant
13. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method
R827639-01-0 from the U.S. Environmental Protection Agency. This
for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425.
work was also supported by the Research Council of Oregon State
14. Samadpour, M., and N. Chechowitz. 1995. Little Soos Creek microbial
source tracking: a survey. Department of Environmental Health, University
University.
of Washington, Seattle.
15. Suzuki, M., M. S. Rappe´, and S. J. Giovannoni. 1998. Kinetic bias in esti-
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