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Applied and Environmental Microbiology, June 2001, p. 2578-2585, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2578-2585.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Lactobacillus, Pediococcus, Leuconostoc,
and Weissella Species in Human Feces by Using Group-Specific
PCR Primers and Denaturing Gradient Gel Electrophoresis
Jens
Walter,1
Christian
Hertel,1,*
Gerald
W.
Tannock,2
Claudia M.
Lis,1
Karen
Munro,2 and
Walter P.
Hammes1
Institute of Food Technology, University of
Hohenheim, Stuttgart, Germany,1 and
Department of Microbiology, University of Otago, Dunedin, New
Zealand2
Received 1 December 2000/Accepted 20 March 2001
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ABSTRACT |
Denaturing gradient gel electrophoresis (DGGE) of DNA fragments
generated by PCR with 16S ribosomal DNA-targeted group-specific primers
was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and
Weissella in human feces. Analysis of fecal samples of four
subjects revealed individual profiles of DNA fragments originating not
only from species that have been described as intestinal inhabitants
but also from characteristically food-associated bacteria such as
Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc
mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the
fecal samples by plating on Rogosa agar. On the other hand, all of the
LAB species cultured from feces were detected in the DGGE profile. We
also detected changes in the types of LAB present in human feces during
consumption of a milk product containing the probiotic strain
Lactobacillus rhamnosus DR20. The analysis of fecal samples
from two subjects taken before, during, and after administration of the
probiotic revealed that L. rhamnosus was detectable by
PCR-DGGE during the test period in the feces of both subjects, whereas
it was detectable by culture in only one of the subjects.
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INTRODUCTION |
The distal human intestinal tract is
colonized by numerous bacteria that form a complex community rich in
bacterial species (22). This community is referred to as
the intestinal microflora. Most current knowledge of the intestinal
microflora has been obtained by using bacteriological culture
techniques and microscopy. Bacteriological culture has been shown to
have deficiencies in microbial ecological studies, particularly due to
the presence of bacterial cells that are not detectable by culture
methods (7). The application of recently developed
culture-independent molecular techniques permits more detailed
investigations of the intestinal microflora (16, 23). Most
of the molecular techniques for the detection, identification, and
classification of bacteria are based on the nucleotide sequence of 16S
rRNA. Denaturing gradient gel electrophoresis (DGGE) and
temperature gradient gel electrophoresis (TGGE) of 16S ribosomal
DNA (rDNA) amplicons have been demonstrated to be suitable tools for
the analysis of microbial communities because they permit the detection
of species and changes in community structure quickly and economically
(14). Total bacterial DNA from the habitat of interest is
extracted and a region with a hypervariable nucleotide base sequence of
the 16S rRNA gene is amplified by PCR. The resulting mixture of 16S
rDNA fragments is subjected to a denaturing gradient, established in a
polyacrylamide gel with urea and formamide or increasing temperature,
in order to separate the fragments and generate a "genetic
fingerprint" of the community. The usefulness of PCR (universal
primers) and DGGE and TGGE in the analysis of the biodiversity of
gastrointestinal microfloras has already been clearly demonstrated
(20, 24, 29).
Lactobacillus species are commonly present in human fecal
samples (5). Some Lactobacillus species have
also received considerable attention with respect to their putative
healthful properties when ingested as probiotics (17, 25).
The detection of particular bacterial species in human feces using
single PCRs with species-specific primers is logistically daunting
(9). PCR-DGGE has the potential to provide a more
practical approach because lactobacilli can be detected and identified
(or at least grouped) by comparison of the migration distance of their
PCR amplicons in DGGE gels with those of reference strains. This
approach has already been applied for the detection and identification
of lactobacilli in the stomach contents of mice (26). In
this ecosystem, the microbial community has a simple composition with
large numbers of Lactobacillus cells compared to the
microbial community human feces. In human feces, lactobacilli generally
make up less than 1% of the community (19). The fecal
Lactobacillus population cannot be detected by DGGE analysis
of DNA fragments obtained from a PCR amplification with universal
primers because this method detects the 90 to 99% most numerous
species in the community (14, 29). There is thus a need
for the derivation of specific primers to enable the monitoring of
bacteria present as minority members of the microflora. The use of
group-specific primers for actinomycetes (6) and cyanobacteria (15) in combination with DGGE has been
reported for soil and lichens, respectively. Recently, PCR-DGGE with
primers specific for the Lactobacillus reuteri phylogenetic
group was applied to detect these lactobacilli in the feces of pigs
(20).
In this paper, we describe the derivation and use of PCR primers
specific for lactic acid bacteria (LAB) of the genera
Lactobacillus, Pediococcus, Weissella, and
Leuconostoc in conjunction with DGGE. This technique was
used to detect these LAB in human feces. The bacterial species detected
by this method were identified by sequencing of DNA excised from the
gels or by comparison of the migration distances of the DNA amplicons
with those of reference strains. We also compared the identity of LAB
cultured from fecal samples with the results obtained by PCR-DGGE.
Finally, changes in the LAB population during consumption of a product
containing the probiotic strain Lactobacillus rhamnosus DR20
were investigated.
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MATERIALS AND METHODS |
Primer design.
The PROBE-DESIGN tool of the software package
ARB
(http://www.biol.chemie.tu-muenchen.de/pub/ARB/documentation/arb.ps)
was used to derive 16S rDNA-targeted primers specific for
lactobacilli. The potential target sequences for the deduced primers
were compared with all sequences in the ARB database using the
PROBE-MATCH tool. A 40-bp GC clamp
(5'-CGCCCGGGGCGCGCCCCGGGCGGCCCGGGGGCACCGGGGG-3') was
attached to the reverse primer (Lac2) to obtain PCR fragments suitable
for DGGE analysis (28). The melting behavior of the PCR
fragments, as revealed by the software of the Poland server (http://www.biophys.uni-duesseldorf.de/POLAND/poland.html), served as a basis for primer optimization.
Bacterial strains and growth conditions.
The following
bacteria were used in this study: Lactobacillus acidophilus
DSM 20079T, L. brevis DSM 20054T,
L. casei DSM 20011T, L. crispatus DSM
20584T, L. curvatus subsp. curvatus
DSM 20019T, L. delbrueckii subsp.
bulgaricus DSM 20081T, L. gasseri DSM
20243T, L. johnsonii DSM 10533T,
L. plantarum DSM 20174T, L. paracasei
subsp. paracasei LTH 2579, L. paracasei subsp. paracasei DSM 5622T, L. reuteri DSM
20016T, L. rhamnosus DSM 20021T,
L. ruminis DSM 20403T, L. sakei DSM
20017T, L. salivarius subsp.
salicinius DSM 20554T, L. sharpeae
DSM 20505T, Bacteroides distasonis DSM
20701T, Bifidobacterium dentium ATCC 27678, Clostridium perfringens ATCC 13124, Enterococcus
faecium DSM 20477T, Escherichia coli Shure
(Stratagene), Eubacterium limosum DSM 20543T,
Staphylococcus epidermidis DSM 20044T,
Veillonella parvula DSM 2008T, and
Weissella viridescens DSM 20410T.
Lactobacilli and W. viridescens were grown anaerobically in
MRS medium (Difco) at 37°C. Enterococci and staphylococci were grown
aerobically in M53 medium (Deutsche Sammlung von Mikroorganismen und
Zellkulturen) at 37°C, and E. coli was grown under the
same conditions in Luria-Bertani medium. All other bacteria were
cultured anaerobically in brain heart infusion broth (Difco) at 37°C.
Fecal samples.
Samples from two studies were investigated.
In the first study, fecal samples were obtained from four healthy
subjects (A, B, C, D; two female, two male) aged 28 to 37 years.
Freshly collected samples were diluted 10-fold in phosphate buffer
(0.05 M [pH 7.0]) and stored at 
82°C in 1-ml aliquots for later
DNA extraction. To compare DGGE results with those of bacteriological
culture, fresh fecal samples of subjects A, C, and D were homogenized
and diluted 10-fold in prereduced diluent (containing 8.5 g of
NaCl, 1.0 g of peptone, and 0.1 g of cysteine per liter [pH
7.0]) in an anaerobic chamber. Viable cell counts of lactobacilli were determined by plating on Rogosa SL agar (Difco) after incubation for 2 days. Eleven colonies were picked randomly from an agar plate
containing 30 to 300 colonies. These subcultured isolates represented
the predominant strains comprising the population selected on Rogosa
agar (10). Finally, fecal samples of two subjects (C, D)
were collected once a month for 6 months. The second study used fecal
samples of two subjects that had been collected previously during a
probiotic trial with L. rhamnosus DR20 (24).
DNA extraction.
Chromosomal DNA was isolated from an
overnight culture of L. paracasei LTH 2579 as described
previously (18). Total DNA of pure cultures was extracted
as described previously (26). For extraction of the total
DNA from fecal samples (1 ml), the frozen samples were thawed on ice
and purified as described by Wang et al. (27) but, after
washing of the cells, the pellet was resuspended in 100 µl of lysis
buffer (6.7% sucrose, 50 mM Tris HCl [pH 8.0], 10 mM EDTA, 20 mg of
lysozyme per ml, 1,000 U of mutanolysin per ml, 100 µg of RNaseA per
ml). After incubation for 1 h at 37°C, 6 µl of sodium dodecyl
sulfate (20%) and 5 µl of proteinase K solution (15 mg/ml) were
added and the mixture was further incubated for ca. 15 min at 60°C
until the cells lysed. After cooling on ice, 400 µl of Tris HCl (pH
8.0) was added and the mixture was extracted once with
phenol-chloroform-isoamyl alcohol (25:24:1) and twice with chloroform.
After ethanol precipitation the DNA was dissolved in 100 µl of Tris
HCl (pH 8.0).
PCR amplification.
Amplification was carried out using a
GeneAmp 2400 Thermocycler (Perkin-Elmer) and the specific primers Lac1
and Lac2GC (Fig. 1). The reaction mixture
(50 µl) contained 25-pmol amounts of each primer, 0.2 mM
concentrations of each deoxyribonucleotide triphosphate, reaction
buffer, 20 mM tetramethylammonium chloride, 25 µg of bovine serum
albumin, 2.5 U of rTaq polymerase (Amersham Pharmacia
Biotech), and 1 µl of DNA solution. The amplification program was
94°C for 2 min; 35 cycles of 94°C for 30 s, 61°C for 1 min,
and 68°C for 1 min; and finally 68°C for 7 min. To determine the
lower limit of PCR detection, a solution of chromosomal DNA (ca. 10 ng/µl) of L. paracasei LTH 2579 was serially diluted and subjected to PCR.
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FIG. 1.
Primers for the specific amplification of 16S rDNA
sequences of species of the Lactobacillus, Pediococcus,
Leuconostoc, and Weissella group and alignment of the
primer binding regions within the 16S rDNA sequences of related and
nonrelated intestinal bacteria.
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DGGE and excision of DNA fragments.
DGGE was performed as
described previously (26), with the following
modifications: the gel contained a 32.5 to 40% gradient of urea and
formamide increasing in the direction of electrophoresis. Excision and
purification of DNA fragments from DGGE gels were performed as
described by Ben Omar and Ampe (2).
Bias determination in PCR-DGGE.
To determine whether DNA
extraction and template annealing in the PCR amplification introduced
bias to the results, cells of L. acidophilus DSM
20079T, L. paracasei subsp. paracasei
DSM 5622T, L. reuteri DSM 20016T,
L. salivarius subsp. salicinius DSM
20554T, and L. sharpae DSM 20505T
were mixed to obtain final counts for each species of 5 × 107 and 5 × 108 cells/ml. DNA was
extracted from a 1-ml aliquot of the mixtures, and PCR-DGGE was carried
out as described for the fecal samples.
Sequencing and sequence analysis.
DNA sequences of PCR
fragments obtained from pure cultures or from purified DGGE bands were
determined by the dideoxy chain termination method using the AutoRead
sequencing kit (Amersham Pharmacia Biotech), the LI-COR system (MWG
Biotech), and the IRD 800 labeled primer Lac2Seq
(5'-ATTTCACCGCTACACATG-3'). To determine the closest
relatives of the partial 16S rDNA sequences, a search of the GenBank
DNA database was conducted by using the BLAST algorithm (1). A similarity of >99% to 16S rDNA sequences of type
strains was used as the criterion for identification.
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RESULTS |
Construction and evaluation of primers.
PCR primers Lac1 and
Lac2 (Fig. 1) were derived for the amplification of 340 bp of the 16S
rRNA gene (16S rDNA) of lactobacilli. In silico primer specificity
analysis showed that the primers would not bind exclusively to the 16S
rDNA of lactobacilli but would also anneal to that of
Pediococcus spp., Leuconostoc spp., and
Weissella spp. Alignment of amplified 16S rDNA sequences
theoretically permitted the differentiation of Lactobacillus
species with the exception of the L. casei group (L. casei, L. paracasei, L. rhamnosus, and L. zeae). For
DGGE analysis, a GC clamp of 40 bases was linked to primer Lac2,
resulting in primer Lac2GC. The PCR-amplified 16S rDNA fragments of
various lactobacilli containing this GC clamp exhibited a melting
behavior suitable for DGGE (data not shown).
The specificity of primers Lac1 and Lac2GC was evaluated using DNA
extracted from all of the bacterial strains listed in Materials
and
Methods. A PCR product was only obtained when using DNA templates
from
the
Lactobacillus species and from
W. viridescens
DSM 20410
T. DGGE of the PCR amplicons from representative
lactobacilli showed
that most of the
Lactobacillus species
could be differentiated
according to the migration distances of their
respective 16S rDNA
fragments. Examples are depicted in Fig.
2 (lanes 1 to 5). The
amplicons from
members of the
L. casei group showed similar migration
distances. The sensitivity of the PCR system was determined using
chromosomal DNA of
L. paracasei subsp.
paracasei
LTH 2579. A minimum
of 100 fg of DNA was necessary to obtain a PCR
product.
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FIG. 2.
PCR-DGGE analysis of 16S rDNA fragments generated by PCR
with the specific primers Lac1 and Lac2GC and DNA extracted from
Lactobacillus species or mixed populations. Lanes: 1, L. sharpeae DSM 20505T; 2, L. acidophilus DSM 20079T; 3, L. salivarius
subsp. salicinius DSM 20554T; 4, L. paracasei subsp. paracasei DSM 5622T; 5, L. reuteri DSM 20016T; M1 and M2, mixture of the
Lactobacillus species containing 5 × 107
and 5 × 108 cells of each species per ml,
respectively.
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Detection of lactobacilli in fecal samples using DGGE.
An
initial experiment with mixtures of Lactobacillus species
prepared in vitro did not demonstrate difficulties with the detection of the various species. As shown in Fig. 2 (lanes M1 and M2), all five
species were detected in the Lactobacillus population. Based
on the intensity of the signals, a slight decrease was observed for the
species L. acidophilus and L. salivarius subsp.
salicinius in the mixture when compared to the other species
in the mixed culture.
The usefulness of the Lac1 and Lac2GC primers in conjunction with DGGE
in studying
Lactobacillus populations in fecal samples
of
human subjects was tested. DGGE analysis of the PCR-amplified
16S rDNA
fragments generated from the feces of four subjects (A
to D) revealed
different profiles in the case of each individual
(Fig.
3). Sequencing of excised and purified
DNA from gels showed
that the fragments originated from species of the
genera
Lactobacillus, Pediococcus, Leuconostoc, and
Weissella. This result was consistent
with the prediction of
the computer-based primer specificity analysis.
Interestingly, DGGE
revealed the presence of characteristically
food-associated bacteria in
the feces (e.g.,
L. sakei and
L. curvatus).
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FIG. 3.
DGGE analysis of PCR-amplified 16S rDNA fragments
obtained with primer pair Lac1 and Lac2GC and DNA isolated from human
feces. The fragments were excised and sequenced. Based on sequence
comparisons, the fragments were allotted to the following species: 1A
to 1C, Lactobacillus sakei; 2A to 2C, L. curvatus; 3, L. acidophilus; 4, L. crispatus; 5, W. confusa; 6, P. pentosaceus;
7A and 7B, Leuconostoc mesenteroides; 8, L. fructivorans; 9A and 9B, L. casei group. Bands not
resulting in sequences upon purification and sequencing are indicated
by arrows.
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Fecal samples from subjects C and D were collected monthly over a
period of 6 months and the samples were subjected to DGGE
analysis. As
shown in Fig.
4, in the case of both
subjects, fluctuations
in the 16S rDNA fragment profiles were observed.
Some fragments
could be detected for a longer period (e.g., for both
subjects
fragments 1 and 2, and for subject C fragment 3) than others.
Interestingly, these frequently detected fragments originated
from
Lactobacillus species typically found to be associated with
foods and often used as starter organisms.
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FIG. 4.
DGGE of PCR products obtained with primer pair Lac1 and
Lac2 GC of fecal samples from subjects C and D taken each month over a
period of 6 months. Sequence characterization of the excised fragments
indicated the presence of 1A to 1D, L. sakei; 2A to 2D,
L. curvatus; 3A, L. delbrueckii; and 4A, L. plantarum.
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Comparison of results of DGGE analyses with those of
bacteriological culture.
The fecal samples of subjects A, C, and D
(Fig. 3) were additionally used for bacteriological culture on Rogosa
agar. Although a DGGE profile could be obtained for subject A (Fig. 3,
lane A), bacteria were not cultured from the fecal sample. For subjects C and D, the CFU/g values were 1.4 × 107 and 1.2 × 106, respectively. To obtain insight into the
composition of the dominant Lactobacillus species of
subjects C and D, 11 colonies were randomly picked and subcultured. DNA
was extracted from the bacterial isolates and subjected to PCR
amplification. DGGE analysis of these amplicons revealed different
profiles for isolates from subjects C and D. These results are shown in
Fig. 5, together with that of the
corresponding fecal sample. The bacterial isolates were identified by
sequencing as belonging to the species L. acidophilus, L. crispatus, L. plantarum, Weissella confusa, and Pediococcus pentosaceus and to the L. casei group.
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FIG. 5.
Comparison of PCR-DGGE and bacteriological culture to
monitor the species composition of the Lactobacillus
population in human feces. DGGE analyses were performed with
PCR-amplified 16S rDNA fragments which were obtained with the primer
pair Lac1 and Lac2GC and DNA extracted from fecal samples (lane 1) or
the corresponding Lactobacillus isolates (subject C, lanes 2 to 5; subject D, lanes 2 to 4) cultured on Rogosa medium. The DNA
fragments were allotted by sequence analysis to the following species.
Isolates from subject C were as follows: lane 2, L. plantarum; lane 3, L. acidophilus; lane 4, L. crispatus; lane 5, L. casei group. DGGE profile of
subject C revealed the following: fragment 1, L. sakei;
fragment 2, L. curvatus; fragment 3, L. acidophilus; fragment 4, L. crispatus; fragment 5, Leuconostoc mesenteroides. Isolates from subject D were
identified as follows: lane 2, W. confusa; lane 3, Pediococcus acidilactici; lane 4, L. casei group.
DGGE profile of subject D identified the following: fragment 1, L. curvatus; fragment 2, W. confusa; fragment 3, Lactobacillus fructivorans; fragment 4, L. casei
group. Fragments that did not result in sequences upon purification and
sequencing are indicated by arrows.
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Table
1 summarizes the comparisons of
culture and PCR-DGGE results. All of the cultured species could be
detected by PCR-DGGE.
In three cases, faintly staining fragments
present in the DGGE
profiles of the fecal samples matched those
obtained from the
bacterial isolates (Fig.
5), but excision and
sequencing were
unsuccessful. DNA from several species (
L. sakei, L. curvatus,
and
L. fructivorans, Leuconostoc
mesenteroides, and
Pediococcus pentosaceus) was
detected by PCR-DGGE in the fecal samples of
all of the subjects, but
these species were not detected among
the colonies cultured on Rogosa
medium. These species are characteristically
involved in food
fermentations. They were present at least on
the same order of
magnitude as the cultured lactobacilli, as indicated
by the intensity
of staining of the DGGE fragments (e.g.,
L. mesenteroides in
subject C [Fig.
5, subject C, lane 1, fragment 5] and
L. curvatus in subject D [Fig.
5, subject D, lane 1, fragment 1]).
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TABLE 1.
Bacterial counts of fecal samples from subjects A, C, and
D and species detected by PCR-DGGE and by culture
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Investigation of fecal samples from probiotic trial.
Fecal
samples collected from two human subjects (subjects 2 and 4) who had
consumed a milk product containing the probiotic strain L. rhamnosus DR20 (24) were investigated. The extracted DNAs of type strains of those species (L. rhamnosus, L. ruminis, and L. acidophilus) which had been cultured
from these fecal samples on Rogosa agar during the probiotic trial were
used as identification standards in DGGE gels. As summarized in Table
2 and shown in Fig.
6, PCR-DGGE using the group-specific
primers Lac1 and Lac2GC detected the presence of L. rhamnosus only in the fecal samples of both subjects collected
during the test period. As shown during the probiotic trial
(24), L. rhamnosus was below detectable limits
in the feces of subject 2 when using bacteriological culture, but was
detectable using L. rhamnosus-specific PCR primers (Table 2). L. ruminis was cultured from all of the fecal samples
collected from subject 2 and was also detected by PCR-DGGE. The feces
of subject 4 harbored some species that were only detected by PCR-DGGE (Fig. 6, fragments 1 to 4). These species (L. sakei, L. delbrueckii, L. curvatus, and Leuconostoc
mesenteroides) are considered to be food-associated bacteria.
L. acidophilus was identified in the DGGE gel by reference
to the migration distance of the fragment from the type strain (Fig. 6,
R3 and sample 8). Detection of this species in the samples was in
agreement with the culture result. PCR amplicons were not obtained from
fecal samples 1 to 3 in the control period and sample 7 in the posttest
period of subject 4. L. crispatus and L. acidophilus were, however, detected at levels of 3.2 × 105 and 1.6 × 105 CFU/g in samples 1 and
7, respectively (Table 2).
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TABLE 2.
Bacterial counts of fecal samples from a probiotic trial
and species detected by PCR-DGGE and by culture
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FIG. 6.
DGGE analysis of 16S rDNA fragments obtained from the
fecal samples of subjects 2 and 4 before, during, and after consumption
of a probiotic product. Subject 2: R1, L. ruminis DSM
20403T; R2, L. rhamnosus DSM 20021T;
1 to 8, fecal samples taken during the control (1 to 3),
test (4 and 5), and posttest period (6 to 8).
Subject 4: R2, L. rhamnosus DSM 20021T; R3,
L. acidophilus DSM 20079T; 4 to 8, fecal samples
taken during the test (4 and 5) and posttest period
(6 and 8). Samples from the control period (1 to
3) and sample 7 of the posttest period of subject 4 did not give
PCR products. DNA fragments which did not match fragments of the
reference strains were allotted upon sequencing to the following
species: 1, L. sakei; 2, L. delbrueckii; 3, L. curvatus; and 4, Leuconostoc mesenteroides.
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DISCUSSION |
The presence of lactobacilli in fecal or intestinal samples has
traditionally been demonstrated by bacteriological culture. This work
has revealed that a relatively limited number of
Lactobacillus species are regularly detectable in human
feces (12). The PCR-DGGE system that we have described
permits the detection in feces of not only Lactobacillus
species considered to be intestinal inhabitants but also of LAB
commonly associated with food and often used as starter organisms
(21). At least half of the LAB that we detected in feces
belonged to this category. The staining intensity of the DNA fragments
in the gels indicated that the food-associated bacteria were present in
the feces in similar numbers to the typical intestinal LAB. The
food-associated species in the fecal samples were not cultivable on
Rogosa agar. Either target DNA in dead cells, DNA released from cells
that had lysed in the intestinal tract, or DNA from living cells in a
noncultivable state was detected. Culture media other than Rogosa agar
might support the growth of these latter cells, but we have not yet
investigated this possibility. We believe that the detection of
noncultivable LAB sequences in human feces was of interest because it
made us wonder about the impact of these ingested bacteria on the
consumer. It would be interesting to know, for example, in which part
of the digestive tract the LAB change from cultivable to noncultivable
and whether even in the noncultivable state they may have an impact on
the immune system as they transit the gut in the digesta (4,
11). The irregular detection in human feces of
noncultivable LAB originating in food strengthens the concept that only
some of the Lactobacillus species detected in human studies
are truly autochthonous to the intestinal ecosystem; even some of the
cultivable species may be transient bacteria consumed in food
(24).
LAB are difficult to detect in human feces when using universal PCR
primers because these bacteria constitute only a minor part of the
microflora. In the probiotic trial using L. rhamnosus DR20,
the strain was not detected in human fecal samples using PCR-DGGE and
universal primers HDA1GC and HDA2, although it was cultured from the
feces of the subjects and represented the predominant Lactobacillus strain during the test period
(24). The use of the Lac1 and Lac2GC primers lowered the
detection limit so that L. rhamnosus could be detected in
the fecal samples by PCR-DGGE. In general, Lactobacillus
species present in numbers of >106 CFU/g of feces (wet
weight) could be detected even in the presence of DNA from the
predominant members of the fecal microflora (about 1011
cells/g) in the PCR.
PCR with primers Lac1 and Lac2GC amplified a 340-bp fragment of the V3
region of the 16S rRNA gene. This sequence permitted most of the
Lactobacillus species to be differentiated. Members of the
L. casei group have sequence variation in the V1 region between nucleotides 73 and 111 of the 16S rRNA gene (13)
but none in the V3 region. Thus, they could not be differentiated in
our system. Sequencing all of the fragments in a DGGE profile is
laborious, and inaccurate identifications may occur because of the poor
quality of some sequences deposited in databases. We found that it is
easier to identify the bacteria by comparison of the PCR amplicon
migration distances in DGGE gels with those of reference strains. It is
noteworthy that the species comprising the L. acidophilus
group cannot easily be identified by classical phenotypic tests
(8) but can be easily differentiated by PCR-DGGE using the
Lac1 and Lac2GC primers.
Our study has led to the derivation of PCR primers that amplify
bacteria belonging to the Lactobacillus, Pediococcus,
Leuconostoc, and Weisella group of LAB. The primers
have been demonstrated to be useful in the analysis, at the species
level, of these populations in human feces. This can be achieved simply
and economically by a single PCR followed by DGGE. We think that the
primers Lac1 and Lac2GC and PCR-DGGE have potential use in the
following types of studies. Firstly, they could be used in studies to
monitor the LAB content of human subjects in probiotic or other trials aimed at manipulating the composition of the intestinal microflora. Secondly, they could be used in large-scale microbial ecological investigations of the intestinal tract of humans and other animals in
order to define the characteristic Lactobacillus microflora of particular hosts. Thirdly, they could be used to follow the bacterial successions that occur during the acquisition of the intestinal microflora. Finally, the results of our experiments suggest
that there is potential for the derivation of group-specific PCR
primers for the many phylogenetic groups of bacteria that comprise the
intestinal microflora. The use of group-specific primers could provide
information essential to an understanding of the dynamics, at the level
of bacterial species, of the intestinal ecosystem.
| |
ACKNOWLEDGMENTS |
We thank M. Kranz for excellent technical assistance during
sequencing. The participation of the subjects in this study is gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Food Technology, University of Hohenheim, Garbenstr. 28, D-70599
Stuttgart, Germany. Phone: 49 711 459 4255. Fax: 49 711 459 4199. E-mail: hertel{at}uni-hohenheim.de.
| |
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Applied and Environmental Microbiology, June 2001, p. 2578-2585, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2578-2585.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
