Appl Environ Microbiol. 1998 March; 64(3): 907-913.
PMCID: PMC106345
Copyright © 1998, American Society for Microbiology
Detection of Clostridium proteoclasticum and Closely Related Strains in
the Rumen by Competitive PCR
K. Reilly and G. T. Attwood*
AgResearch Grasslands, Palmerston North, New Zealand
*Corresponding author. Mailing address: AgResearch, Ruminant Digestion
and Metabolism, Grasslands Research Centre, Tennent Drive, Private Bag
11008, Palmerston North, New Zealand. Phone: 64 6 356 8019. Fax: 64 6
351 8003. E-mail: attwoodg/at/agresearch.cri.nz.
Received August 7, 1997; Accepted December 4, 1997.
Small right arrow pointing to: This article has been cited by other
articles in PMC.
Abstract
A competitive PCR technique was used to enumerate the proteolytic
bacterium Clostridium proteoclasticum from the rumen. A PCR primer,
which circumscribes this organism and several closely related strains,
was designed for a variable region within their 16S rRNA genes and was
used in conjunction with a universal forward primer. This primer pair
was tested for specificity against 85 ruminal bacterial strains. An
internal control DNA was constructed for use in competitive PCRs and
was shown to amplify under the same reaction conditions and with the
same amplification efficiency as the target DNA. DNA from a known
number of C. proteoclasticum cells was coamplified with the internal
control to construct a standard curve. Rumen samples were collected
from eight dairy cows fed four diets in rotation: high nitrogen, high
nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen
supplemented with carbohydrate. DNA extracted from these and spiked
with internal control DNA was amplified with the C. proteoclasticum
primer pair. The relative intensities of the PCR products were used to
quantitate the numbers of C. proteoclasticum cell equivalents from the
rumen samples. The numbers ranged from 2.01 × 106 ml-1 to 3.12 × 107
ml-1. There was no significant effect on the numbers of C.
proteoclasticum detected in rumen samples among cows fed the four
diets. The utility of the competitive PCR approach for quantifying
ruminal bacterial populations in vivo and the occurrence of C.
proteoclasticum in forage-fed dairy cows are discussed.
New Zealand ruminants graze a fresh forage diet which is high in
protein and low in soluble carbohydrates (10). Up to 50% of the
protein available from this diet can be lost due to the rapid microbial
breakdown of plant protein (16). Bacteria are thought to be
responsible for the majority of this protein degradation in the rumen
(8). The proteolytic bacteria in forage-fed New Zealand cattle are
dominated by species of the genera Streptococcus, Eubacterium, and
Butyrivibrio (2), while a novel, highly proteolytic strain,
Clostridium proteoclasticum, has also been isolated. To determine the
significance of these particular proteolytic bacteria within the New
Zealand ruminant, we have set out to develop a sensitive and specific
method of quantitation of microbial populations directly from rumen
samples.
rRNA probes have been successfully used for the detection and
enumeration of bacteria within the rumen (7, 11, 17-20,
27). This technique expresses the abundance of a particular rRNA
sequence in relation to total RNA extracted from a sample. However, it
is relatively insensitive, only detecting down to 106 bacteria per ml
of rumen fluid (27). PCR has been used to detect bacteria in many
environments (5, 6), and its sensitivity has allowed the
detection of a single bacterium (28). Conventional PCR amplifies
target DNA exponentially, making it difficult to use the technique in a
quantitative manner. Competitive PCR (cPCR), however, has been used to
determine bacterial numbers in a range of environments (12, 14,
15). The addition of an internal DNA standard controls for the
variation among reactions, allowing reliable PCR quantitation. The
internal DNA standard contains the same primer binding sites as the
target, and the two DNAs compete for reaction reagents to produce PCR
products of different sizes, which can be separated in an agarose gel.
The log ratio of intensities of amplified target DNA to internal
control DNA is determined by the equation log (N[n1]/N[n2]) = log
(N[01]/N[02]) + n log (eff/eff) (29). If the efficiencies of
amplification (eff and eff) are equal, the ratio of amplified
products (N[n1]/N[n2]) is dependent on the log ratio of starting
products (N[01]/N[02]) (29). Even if the efficiencies of the two
reactions are not equal, the values still hold assuming that
eff/eff is constant and amplification is in the exponential
phase. With this technique, amounts of target DNA can be determined by
amplification with known amounts of internal control DNA. The resulting
log ratio of intensities of PCR products is compared to standard curves
derived from serial dilutions of known target DNA amplified with known
amounts of internal control DNA.
C. proteoclasticum is a gram-positive, straight to slightly curved rod
which was first isolated from a pasture-grazed cow in New Zealand
(4). Its most distinguishing feature is its extremely high
proteinase activity, and because of this feature we were interested in
quantifying this organism to estimate its contribution to rumen
proteolysis. Since its description as a new species, it has become
apparent that the 16S rRNA gene (rDNA) sequence of C. proteoclasticum
is very similar to those of Butyrivibrio fibrisolvens NCDO 2435, 2434,
2222, and 2398. However, the description of C. proteoclasticum as a new
species is justified since C. proteoclasticum and these Butyrivibrio
fibrisolvens strains are phylogenetically closely related to each
another (96.9 to 99.5% 16S rRNA sequence similarity) but are distantly
related to the Butyrivibrio fibrisolvens type strain NCDO 2221T (93.1
to 94.1% similarity). We have developed a cPCR technique which detects
C. proteoclasticum and these closely related B. fibrisolvens strains
directly from rumen samples. PCR primers were designed against a common
region of the C. proteoclasticum and B. fibrisolvens 16S rRNA genes.
These primers have been tested for specificity against DNA from 85
rumen bacterial strains, and the sensitivity of detection in a mixed
rumen bacterial background has been investigated. We have used this
cPCR approach to enumerate C. proteoclasticum cell equivalents in rumen
samples from dairy cows fed four different diets: high nitrogen, high
nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen
supplemented with carbohydrate.
MATERIALS AND METHODS
Bacterial strains and growth.
The bacterial strains used are listed in Table Table1.1. Bacteria
were grown on CC medium (13), except that the rumen fluid was not
preincubated to remove soluble carbohydrates and carbon sources were
replaced by either 1.0% (wt/vol) glucose or cellobiose. Clostridium
aminophilum, C. sticklandii, and Peptostreptococcus anaerobius were
grown in CC medium in which no carbon source was added and tryptone
(1.5% [wt/vol]; Difco, Detroit, Mich.) replaced trypticase.
Rumen samples.
Eight fistulated, lactating, Friesian dairy cows were fed four
different diets in rotation: high nitrogen, high nitrogen with
carbohydrate, low nitrogen, and low nitrogen with carbohydrate.
Ryegrass pasture, containing less than 5% clover, was top dressed with
20 kg of nitrogen (as urea) per ha for low-nitrogen diets and 90 kg of
nitrogen per ha for high-nitrogen diets. Urea was applied 21 to 28 days
before cutting, and the nitrogen was 2.11 and 2.82% of dry matter for
the low- and high-nitrogen diets, respectively. Carbohydrate was
supplied as a 50:50 mixture of dextrose and corn flour on an energy
basis and was drenched at 9:00 a.m., 11:00 a.m., 4:00 p.m., and 6:00
p.m., supplying 10% of the minimum energy intake. The cows were fed at
9:00 a.m. and 4:00 p.m. and were maintained on each diet for 12 days
before the samples were taken. Whole rumen samples of approximately 250
ml were collected before the last 4:00 p.m. feed, frozen immediately in
liquid nitrogen, and stored at -80°C until DNA extraction. Immediately
before DNA extraction, the samples were thawed in a 37°C water bath and
diluted with an equal weight of mineral salts (MS) buffer (3)
before homogenization in a Sorvall tissue homogenizer (Du Pont Co.,
Wilmington, Del.).
DNA extraction.
General techniques of DNA precipitation and phenol-chloroform
extractions were performed as described by Sambrook et al. (26).
For determination of primer specificity, bacterial cultures were grown
overnight and DNA was extracted by the enzymatic lysis procedure
described by Saito and Miura (25). To eliminate possible bias
introduced by enzymatic cell wall lysis and to maximize DNA extraction
efficiencies, physical disruption was used to extract DNA from rumen
samples and from C. proteoclasticum for sensitivity experiments and
standard curve construction. Unless otherwise stated, DNA was extracted
from triplicate samples. A 1-ml volume of homogenized rumen contents or
1010 C. proteoclasticum cells was added to 1.2 g of sterile
zirconia-silica beads (Biospec Products) followed by centrifugation at
12,000 × g for 10 min at room temperature. The pellet and beads were
rinsed twice in saline-EDTA solution (0.15 M NaCl, 0.1 M EDTA) before
final resuspension in 750 μl of saline-EDTA. Physical disruption was
then performed with a Mini-beadbeater (Biospec Products) at maximum
speed for two intervals of 2 min each, with a 1-min incubation on ice
between each treatment. Phenol-chloroform-isoamyl alcohol (25:24:1) was
added and mixed, and the mixture was centrifuged at 12,000 × g for 5
min at room temperature. The aqueous phase was removed, and the
interface was reextracted with TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH
8.0]). The combined aqueous phases were repeatedly extracted with the
phenol-chloroform-isoamyl alcohol until no protein remained at the
interface. A final chloroform-isoamyl alcohol extraction was performed
before the nucleic acids were precipitated with ethanol and centrifuged
at 10,000 × g for 20 min at 4°C. The air-dried pellet was resuspended
in TE buffer, RNase A (1 mg ml-1 [final concentration]) was added, and
the mixture was incubated at 37°C for 60 min. RNase A was removed by
phenol-chloroform-isoamyl alcohol extraction followed by ethanol
precipitation and centrifugation. The air-dried DNA pellet was
resuspended in TE buffer and was stored at -20°C, until required.
DNAzol reagent (Gibco BRL, Life Technologies, Auckland, New Zealand)
was used for chemical extraction of DNA. Cells were suspended in 1 ml
of DNAzol reagent before being subjected to bead beater treatment as
described above. The DNA was then precipitated as specified by the
manufacturer. The concentration and purity of the DNA were determined
spectrophotometrically by measuring the absorbances at 260 and 280 nm
(A[260/280]) with a Spectramax microplate spectrophotometer (Molecular
Dynamics).
To check that there was no amplification of plant material with the
primer pair, DNA was extracted from 20 g of pasture plant tissue. This
plant tissue was diluted with 4 volumes of MS buffer to ensure complete
homogenization. The DNA was extracted by the mechanical disruption
method described above.
PCR primers and amplification.
The primers used for the amplification of the 16S rRNA genes were as
follows: universal forward primer (S-*-Univ-0008-a-S-19; 5′ GAG TTT GAT
CCT GGC TCA G 3′), universal reverse primer (S-*-Univ-1528-a-A-17; 5′
AAG GAG GTG ATC CAG CC 3′), and the C. proteoclasticum primer
(S-S-Cprot-0832-a-A-21; 5′ CTG AAT GCC TAT GGC ACC CAA 3′). The
S-S-Cprot-0832-a-A-21 primer was screened for specificity with the
PROBE CHECK program of the Ribosomal Database Project (21) and the
BLAST (Basic Local Alignment Search Tool) facility at the National
Center for Biotechnology. PCR amplification of C. proteoclasticum DNA
produces an 830-bp product when amplified with the universal forward
primer and S-S-Cprot-0832-a-A-21. Because the S-S-Cprot-0832-a-A-21
primer also detects some closely related B. fibrisolvens strains in
rumen samples, in these instances cell numbers are expressed as C.
proteoclasticum cell equivalents.
The PCR mixtures contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM
MgCl, 0.2 mM each dATP, dCTP, dGTP and dTTP, 1 μM each primer, and
0.5 U of Taq DNA polymerase (Gibco BRL). The PCRs were performed in a
final volume of 20 μl sealed in a capillary tip, and thermocycling was
carried out in a model FTS-1 capillary thermal sequencer (Corbett
Research, Sydney, Australia). The PCR amplification conditions for
S-S-Cprot-0832-a-A-21 and the universal forward primers were as
follows: denaturation at 95°C for 3 min followed by 6 cycles of 95°C
for 30 s, 62°C for 15 s, and 72°C for 30 s and 25 cycles of 95°C for 15
s, 62°C for 5 s, and 72°C for 30 s, with a final cycle of 72°C for 3
min. Amplification with the universal forward and reverse primers
differed only in the annealing temperature, which was 55°C. PCR
products were separated by electrophoresis in agarose gels, stained
with ethidium bromide, and visualized by UV transillumination.
Construction of the internal control.
The 830-bp PCR product from C. proteoclasticum DNA amplified with the
universal forward and S-S-Cprot-0832-a-A-21 primers contains two AluI
sites (Fig. (Fig.1).1). To produce an AluI deletion, the 830-bp
fragment was digested with AluI as specified by the manufacturer
(Boehringer, Mannheim, Germany) and the restriction endonuclease was
removed with phenol treatment followed by ethanol precipitation. The
AluI fragments were ligated with T4 DNA ligase (Gibco BRL), 2 μl of the
ligation reaction was used in a PCR with the universal forward and
S-S-Cprot-0832-a-A-21 primers, and the products were separated by
agarose gel electrophoresis. One of the PCR products was a 480-bp
fragment expected from the deletion of the 350-bp internal AluI
fragment. The 480-bp fragment was purified from the gel and used as an
internal control for the cPCRs. The concentration of the internal
control was estimated from photographs of the gel to be approximately
100 ng ml-1. However, the DNA concentration was too low to obtain an
accurate A[260/280] reading, and so the internal control was expressed
as a dilution of the concentrated mixture.
FIG. 1
FIG. 1
Construction of the internal control. The 830-bp PCR product was
digested with AluI to give three fragments of 90, 350, and 390 bp. The
fragments were ligated and PCR amplified. The 480-bp product was
purified and used as the internal control.
Preparation of C. proteoclasticum cells for sensitivity testing.
C. proteoclasticum was grown in 100-ml overnight cultures, and a sample
was counted by phase-contrast microscopy with a WSI counting chamber
(Weber Scientific International Ltd., Middlesex, England). The cells
were pelleted by centrifugation and resuspended to a final
concentration of 1010 cells ml-1. DNA extractions were performed on
1010 cells; 10-fold serial dilutions of this DNA were amplified to
determine the detection limit.
Quantitation of PCR products.
PCR products were quantitated by photographing agarose gels with
Polaroid 665 film (Polaroid, St. Albans, England), which produces a
negative image of the photograph. The negative was scanned with a
GS-670 imaging densitometer (Bio-Rad, Hercules, Calif.) and analyzed
with Molecular Analyst software version 1.4 (Bio-Rad). To correct for
differences in the fluorescence of ethidium bromide-stained PCR
fragments of different sizes (23), the intensity of the internal
control was multiplied by the ratio 830/480.
RESULTS
Primer specificity and sensitivity.
Alignment of the C. proteoclasticum 16S rDNA sequence with other
sequences from the Ribosomal Database Project and closely related B.
fibrisolvens sequences retrieved from BLAST searches identified a
region at bp 832 to 851 (E. coli numbering) that was common to C.
proteoclasticum and B. fibrisolvens NCDO 2435, 2222, 2398, and 2434. A
primer, S-S-Cprot-0832-a-A-21, complementary to the sense strand of
this region facing the 5′ end of the 16S rRNA gene was designed. This
enabled it to be used with the universal forward primer in PCRs to
generate an 830-bp product. The primer pair was tested for specificity
against DNA from 85 bacterial strains, mostly of rumen origin (Table
(Table1).1). Only C. proteoclasticum genomic DNA amplified with
these two primers at an annealing temperature of 62°C (Table
(Table1);1); closely related strains (B. fibrisolvens-like C21b
and C122b) failed to amplify under these conditions. At an annealing
temperature of 60°C, some amplification occurred from unrelated
Enterococcus faecalis NCTC 775 and Streptococcus bovis RF-2. This
nonspecific amplification was eliminated once the annealing temperature
was raised to 62°C. The DNA from each of the rumen strains was also
tested with the universal forward and reverse primers at an annealing
temperature of 55°C. All the strains produced a PCR fragment of
approximately 1,500 bp, corresponding to the approximate size of the
16S rRNA gene, demonstrating that the DNA from each strain was
amplifiable.
The sensitivity of the S-S-Cprot-0832-a-A-21 primer in PCRs was
investigated by amplification of a serial dilution of DNA from known
numbers of C. proteoclasticum cells. The results show that 50 fg of DNA
(the equivalent of DNA from 25 C. proteoclasticum cells) was the lower
limit of detection when coamplified with a 2 × 106 dilution of the
internal control DNA (Fig. (Fig.2).2).
FIG. 2
FIG. 2
Detection limit of cPCR. DNA extracted from 1010 C. proteoclasticum
cells ml-1 was serially diluted and coamplified with a 2 × 106
dilution of the internal control to determine the detection limit of
the cPCR assay. The results are expressed (more ...)
DNA extraction.
To determine the most efficient method of recovering DNA from bacterial
samples, 1010 C. proteoclasticum cells were subjected to three methods
of DNA extraction: enzymatic cell lysis, chemical extraction, and
physical disruption. A[260/280] readings demonstrated that physical
disruption was the most efficient method of extraction, recovering 67
μg of DNA from 1010 cells. Enzymatic lysis and chemical extraction
recovered 43 and 1.01 μg of DNA per 1010 cells, respectively.
Internal-control amplification efficiency.
The relative amplification efficiencies of target and internal-control
DNAs can be determined from a plot of the ratio of log target intensity
to internal control intensity against the log concentration of internal
control DNA. Coamplification of DNA from 103 C. proteoclasticum cells
with dilutions of the internal control (Fig. (Fig.3)3) results in
a line with a slope of 0.94 and a regression of 0.999. This indicates
equivalent amplification efficiencies of the target and control DNAs.
The line intersects the x axis at -5.9, indicating that the optimal
dilution of internal control for detection of 103 C. proteoclasticum
cells is 1.26 × 106.
FIG. 3
FIG. 3
Internal-control amplification efficiency. Dilutions of the internal
control were coamplified with DNA from 103 C. proteoclasticum cells,
and the ratio of the intensities of internal control to the target DNA
was plotted against the dilution of the internal (more ...)
Standard curve.
Since cPCR is most accurate when the target and internal control are
coamplified in equimolar proportions, it was necessary to determine the
optimal internal-control concentrations to use with DNA extracted from
rumen samples. Dilutions of internal control were coamplified with DNA
from selected samples, and the optimal dilution of the internal control
was found to be 10-6. This concentration of internal control was used
to construct a standard curve by coamplification with C.
proteoclasticum DNA extracted from a known number of cells (Fig.
(Fig.4).4). The results show that DNA from 1 × 104 to 5 × 101
cells gave a linear response and could be used for quantitation of
samples within this range.
FIG. 4
FIG. 4
Standard curve construction. (a) DNA extracted from 1010 C.
proteoclasticum cells ml-1 was serially diluted and coamplified with a
106 dilution of the internal control. The results are expressed as C.
proteoclasticum cell equivalents based on (more ...)
Detection of C. proteoclasticum added to rumen fluid.
To test whether C. proteoclasticum could be detected by cPCR within a
background of nonspecific DNA from rumen fluid, samples of rumen
contents were spiked with known numbers of C. proteoclasticum cells.
DNA from each of the samples was coamplified under optimal cPCR
conditions, and the results show a linear relationship between the
number of cells added and the number of cells detected. The assay
slightly overestimated the number of C. proteoclasticum cells added
compared to the ideal (y = x), and this was particularly evident at the
higher concentrations of cells (Fig. (Fig.5).5). A population of
6.25 × 106 C. proteoclasticum cell equivalents ml-1 was detected in
the unspiked rumen samples.
FIG. 5
FIG. 5
In vivo detection of C. proteoclasticum. Rumen samples were spiked with
known numbers of C. proteoclasticum cells. DNA was extracted from these
mixtures and assayed for the presence of C. proteoclasticum. Solid
squares denote numbers of C. proteoclasticum (more ...)
Detection of C. proteoclasticum and closely related strains in vivo.
To examine the application of the cPCR approach to determining
bacterial numbers directly from animals, rumen samples were collected
from eight lactating dairy cows fed four different diets in rotation.
The number of C. proteoclasticum cell equivalents detected ranged from
2.01 × 106 to 3.12 × 107 ml-1 of rumen contents (Fig.
(Fig.6).6). Within individual animals there were significant
responses to diet, but overall there was no significant difference
between the diets.
FIG. 6
FIG. 6
Populations of C. proteoclasticum and closely related B. fibrisolvens
strains in dairy cows under four different feeding regimens. Numbers 1
through 8 represent cows numbered 709, 710, 727, 788, 1758, 8702, 9754,
and 9775, respectively. The results are (more ...)
DISCUSSION
C. proteoclasticum is a proteolytic bacterium that was isolated from
the rumen contents of a forage-fed cow (2). It has a predominately
serine-type proteinase activity (3) but also some cysteine- and
metallo-type proteinase activity. It seems most likely to be involved
with primary hydrolysis of feed protein, but the significance of C.
proteoclasticum to rumen microbial ecology has not yet been determined.
Since no suitable selective media are available for enumeration of C.
proteoclasticum, we set out to develop a method to quantify this
organism directly from rumen samples.
PCR is an extremely sensitive and specific method for the amplification
of DNA, and when it is used in conjunction with an internal DNA
control, the products of the amplification reactions can be quantified.
This technique, cPCR, was originally developed for quantitation of
human immunodeficiency virus type 1 3B long terminal repeat DNA
(29). Its use has since been extended to bacterial quantitation in
the environment (12, 14, 15). In the present study, a cPCR
method was developed to quantify C. proteoclasticum and closely related
strains from rumen samples. PCR primers, based on both conserved and
hypervariable regions of the 16S rRNA gene, were used to amplify DNA in
the presence of an internal control which allowed quantitation of the
PCR products. The primer pair used circumscribes C. proteoclasticum and
four closely related B. fibrisolvens strains and did not amplify DNA
from any other bacterium tested. The specificity of the assay stems
from the selection of 16S rDNA as the target for amplification and the
primer design. The semiconserved nature of rRNA genes allows the use of
regions of hypervariable sequence as targets for group-, genus-,
species-, and strain-specific amplification (22). The primer was
designed to match, as closely as possible, the length, G+C content, and
T[m] of the universal forward primer so that the forward primer
“anchored” the PCR at the 5′ end of the 16S rRNA gene. This approach
limits the range of suitable specific primers which can be designed
within a particular region but avoids the need to design two specific
primers for a given organism. Using the 16S rRNA gene as the
amplification target also allows primers to be checked against the
existing entries in DNA sequence databases, thereby reducing the effort
needed to test primer specificity.
A critical factor in ensuring the accuracy of cPCR is to demonstrate
that coamplifications of the target and internal control are equivalent
(24). This can be done by plotting the log ratio of target to
internal-control DNA intensities against the log dilution of
internal-control DNA. Coamplification of C. proteoclasticum DNA with
dilutions of internal control gave a straight line with a slope of
-0.94, indicating that the amplification efficiencies of the two DNAs
are essentially equal. The small deviation from equivalence may be due
to a slightly more efficient amplification of the smaller internal
control (480 bp) than of the target (830 bp). This effect was taken
into account by using standard curves to relate the log ratio of target
to internal control to the log of cell numbers. These standard curves
also account for the rRNA gene copy number, which can vary anywhere
from 2 to 10 copies per genome (1, 9). Each standard curve,
generated by coamplification of a single dilution of internal control
with DNA from different numbers of C. proteoclasticum cells, gives a
substantial working range of cell numbers of approximately 103- to
104-fold. Indeed, in the application of the assay to rumen samples,
only a single standard curve was required to encompass the entire range
of C. proteoclasticum numbers encountered in differently fed dairy
cows.
In a similar manner, dilutions of internal control can be used to
determine the absolute sensitivity of the assay. Theoretically, the
detection of one copy of the target sequence is possible (28).
However, in cPCR, the target and internal control compete for
amplification reagents, reducing the sensitivity of detection. In our
assay, DNA from the equivalent of 25 C. proteoclasticum cells could be
detected when coamplified with a 2 × 106 dilution of the internal
control. However, it was found that a 100-fold dilution was necessary
to overcome an inhibitory effect in DNA extracted from rumen samples.
Thus, in practice, the sensitivity of the assay is limited to 2.5 ×
103 cells. The exact nature of the inhibitory factor is not known, but
high A[260/280] readings from these DNA samples indicate that it is not
proteinaceous and that it may be a water-soluble polysaccharide or
polyphenolic compound similar in nature to the humic acids described by
Leser (14) as interfering with the PCRs. The level of detection
compares favorably with that in similar studies of bacterial
populations from other environments. Leser (14) detected DNA from
40 Pseudomonas cells in cPCR assays of samples from a marine
environment. Radiolabelled oligonucleotides used to probe rRNA from
rumen bacteria (7, 11, 17-20, 27) are less
sensitive, being able to detect 0.01% of the total rumen population, or
approximately 106 cells ml-1 (27). It should be noted that the
cPCR technique does not discriminate between living and dead cells and
therefore is likely to overestimate viable populations. This is in
contrast with oligonucleotide probing, where microbial abundance is
expressed in terms of a proportion of total rRNA. Since the rRNA
content in cells changes according to the growth phase, this technique
provides an approximation of relative cell numbers (20), which
reflects their contribution to total metabolic activity (27).
However, the estimation of total rRNA abundance depends on universal
probes, and recent evidence suggests that domain-specific variations in
dissociation temperatures of universal probes could lead to significant
biases in quantifying microbial populations from environmental samples
(30). Estimates of both absolute cell numbers and the percent
contribution to total rRNA may be the best approach to gain an accurate
assessment of the importance of microbial populations in environmental
samples.
After the development of the cPCR assay in vitro, it was necessary to
test its ability to detect C. proteoclasticum cells in the complex
mixture of plant and microbial DNA present in rumen fluid. Mechanical
disruption was chosen for DNA extraction from rumen contents since the
efficiency of extraction was greatest with bead beating followed by
phenol-chloroform extractions. Also, this method allowed entire rumen
contents to be sampled, thereby enabling the quantitation of
populations adherent to plant tissue. Previous methods have sampled
only filtered rumen contents (7, 17, 27). The addition
of C. proteoclasticum cells to rumen fluid followed by cPCR
quantitation demonstrated the usefulness of the technique in vivo.
There was good correlation between the number of cells added and those
detected by the assay. A slight overestimation of absolute numbers of
cells compared to the ideal (y = x) is probably due to the resident
population of C. proteoclasticum and closely related B. fibrisolvens
strains in the rumen fluid used.
When the assay was applied to rumen samples collected from animals fed
diets differing in nitrogen and carbohydrate content, the C.
proteoclasticum cell equivalents detected ranged from 2.01 × 106 cells
per ml in the carbohydrate-supplemented, high-nitrogen diet to 3.12 ×
107 cells per ml in the high-nitrogen diet. These numbers represent
the population of C. proteoclasticum and closely related B.
fibrisolvens strains present in the samples and are consistent with the
original isolation of C. proteoclasticum from a 108 dilution of rumen
contents (4). These results indicate that this group of organisms
is common among forage-fed ruminants in New Zealand. Within individual
animals, the diet had some effects on C. proteoclasticum cell
equivalents, but overall, between the animals there were no significant
differences (Fig. (Fig.6).6). The relatively small range of cell
numbers detected indicates that the population is stable and
unresponsive to changes in dietary content. This is a little surprising
since one might expect the proteolytic population of C. proteoclasticum
to be influenced by nitrogen supply. However, the population density of
C. proteoclasticum does not necessarily reflect its overall
contribution to proteolytic activity, which may well vary significantly
under the conditions tested. Previously, it was shown that C.
proteoclasticum has a high chymotrypsin-like proteinase activity
(3), and, based on its specific activity for the artificial
chymotrypsin substrate N-succinyl
alanine-alanine-proline-phenylalanine-p-nitroanilide and the
populations detected in this study, it may be responsible for up to 20%
of this type of activity in forage-fed dairy cows. More detailed
investigations of specific C. proteoclasticum proteinase activities are
required before the contribution of this organism to ruminal protein
breakdown can be estimated more precisely.
We have found cPCR to be an easy, accurate, and reliable method of
bacterial quantitation. Collection of rumen samples, DNA extraction,
preliminary PCR amplification with internal control dilutions, and cPCR
followed by scanning densitometry can be accomplished within 10 h.
Testing primers for specificity and constructing internal DNA controls
are the most time-consuming steps in developing the method, but once
these have been carried out, they need not be repeated. The precision
of the technique is good, with the coefficient of variation between
replicate PCRs of the same sample averaging 2.5% and that between
samples from the same animal averaging 7.5%. The technique combines the
specificity of 16S rDNA-targeted oligonucleotides with the sensitivity
of PCR in a format which allows quantitation of bacterial populations
from a complex microbial ecosystem. The technique is currently being
extended to other protein-degrading genera and will eventually allow us
to examine how proteolytic bacterial populations are influenced by
changes in the rumen ecosystem.
TABLE 1
Bacterial strains used in this study
Species Strain Source or reference Reaction with:
__________________________________________________________________
Universal primersa Specific primersb
Butyrivibrio fibrisolvens C130a, C211, C219 2 + -
Butyrivibrio fibrisolvens CF3, H17c M. P. Bryant, University of
Illinois + -
Butyrivibrio fibrisolvens WV1 Laboratory strain + -
Butyrivibrio fibrisolvens OR509 R. Teather, Centre for Food and Animal
Research, Agriculture and Agri-food, Canada + -
Butyrivibrio fibrisolvens like C122a, C21b 2 + -
Clostridium aminophilum F J. B. Russell, Cornell University + -
Clostridium clostridioforme ATCC 25537, ATCC 29084 ATCCc + -
Clostridium proteoclasticum ATCC 51982 2 + +
Clostridium sticklandii SR J. B. Russell, Cornell University + -
Enterococcus faecalis NCTC 775 S. Flint, New Zealand Dairy Research
Institute + -
Enterococcus faecalis 68a Laboratory strain + -
Eubacterium cellulosolvens 5494 M. P. Bryant + -
Eubacterium limosum ATCC 8486 M. P. Bryant + -
Eubacterium ruminantium M. P. Bryant + -
Eubacterium sp. C11, C12b, C13b, C14b#2, C118b, C119b, C120a, C124b,
C125b, C260 2 + -
Fibrobacter succinogenes AC3 M. P. Bryant + -
Lachnospira multiparus ATCC 19307 M. P. Bryant + -
Megasphaera elsdenii T81 M. P. Bryant + -
Peptostreptococcus anaerobius C J. B. Russell + -
Peptostreptococcus productus SF-50 M. P. Bryant + -
Prevotella ruminicola C21a 2 + -
Prevotella ruminicola subsp. brevis 118b, B4 M. P. Bryant + -
Prevotella ruminicola subsp. ruminicola 23 M. P. Bryant + -
Ruminococcus albus 7, 8 M. P. Bryant + -
Ruminococcus flavefaciens ATCC 19208, FD1 M. P. Bryant + -
Selenomonas ruminantium subsp. lactilytica GA31 M. P. Bryant + -
Selenomonas ruminantium subsp. ruminantium ATCC 12561, ATCC 27209 M. P.
Bryant + -
Streptococcus bovis JB1 M. P. Bryant + -
Streptococcus bovis NCFB 2476 A. G. Williams, Hannah Research Institute
+ -
Streptococcus bovis 3-31, 3-36, 3-39, 5-21, 7-2, 7-25, 7-26 J. B.
Russell + -
Streptococcus bovis A12, A14, A120, A166, A191b, B11, B32a, B314, B315,
B327, B337, B342, B346, B348, B350, B360, B372, B382, B385, B395, B396,
B398, C14b#1, C17, C123b, C271 2 + -
Streptococcus bovis RF-1, RF-2, UDY-KF Laboratory strains + -
Succinomonas amylolytica ATCC 19206 M. P. Bryant + -
Succinovibrio dextrinosolvens ATCC 27209 M. P. Bryant + -
aPCR amplification with universal forward and universal reverse
primers.
bPCR amplification with universal forward primer and
S-S-Cprot-0832-a-A-21.
cATCC, American Type Culture Collection, Rockville, Md.
ACKNOWLEDGMENTS
This work was funded by Public Good Science Funding from the Foundation
for Research in Science and Technology and a Lottery Science Research
Grant from the New Zealand Lotteries Grant Board.
We thank Vicky Carruthers of the Dairying Research Corporation for the
rumen samples and Raymond Bennett and Doug Hopcroft of HortResearch for
the development of photographs. Constructive criticisms of the
manuscript by Barry Scott and K. N. Joblin are also gratefully
acknowledged.