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Applied and Environmental Microbiology, September 1999, p. 4264-4267, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Lactobacillus Isolates
from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA
Gene Intergenic Spacer Region Sequence Comparisons
G. W.
Tannock,1,*
A.
Tilsala-Timisjarvi,2
S.
Rodtong,3
J.
Ng,1
K.
Munro,1 and
T.
Alatossava2,4
Department of Microbiology, University of
Otago, Dunedin, New Zealand1; Department
of Biology, University of Oulu, Oulu,2 and
Biotechnology Laboratory, REDEC of Kajaani, University of Oulu,
Sotkamo,4 Finland; and School of
Microbiology, Suranaree University of Technology, Nakhon Ratchasima,
Thailand3
Received 16 February 1999/Accepted 1 July 1999
 |
ABSTRACT |
Lactobacillus isolates were identified by PCR
amplification and sequencing of the region between the 16S and 23S rRNA
genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of
97.5% or greater was considered to provide identification. To check
the reliability of the method, the V2-V3 region of the 16S rRNA gene
was amplified and sequenced in the case of isolates whose spacer region
sequences were less than 99% similar to that of a reference strain.
Confirmation of identity was obtained in all instances. Spacer region
sequencing provided rapid and accurate identification of
Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of
Lactobacillus casei and Lactobacillus
rhamnosus.
| |
TEXT |
The members of the genus
Lactobacillus are gram-positive organisms that belong to the
general category of lactic acid bacteria. They inhabit a wide variety
of habitats, including the gastrointestinal tracts of animals and
vegetation, and are used in the manufacture of fermented foods
(8). Interest in the lactobacilli has been stimulated in
recent years by the use of these bacteria in products that are claimed
to confer health benefits on the consumer (probiotics) (4).
The identification of Lactobacillus isolates by phenotypic
methods is difficult because it requires, in several cases,
determination of bacterial properties beyond those of the common
fermentation tests (for example, cell wall analysis and electrophoretic
mobility of lactate dehydrogenase) (8). In general, about 17 phenotypic tests are required to identify a Lactobacillus
isolate accurately to the species level (6). The derivation
of simple yet rapid identification methods is therefore required in
order to deal with the large numbers of Lactobacillus
isolates obtained during microbial ecological studies of ecosystems
such as the intestinal tract, silage, and food products.
Nucleotide base sequences of Lactobacillus 16S ribosomal DNA
(rDNA) provide an accurate basis for phylogenetic analysis and identification (2, 5, 17). The sequence obtained from an
isolate can be compared to those of Lactobacillus species
held in data banks. Although the species-specific sequences are
contained in the first half of the 16S rRNA gene (V1-V3 region),
identification is more accurate if the whole gene is sequenced
(13). This means that about 1.5 kb of DNA would have to be sequenced.
Studies by Tilsala-Timisjarvi and Alatossava (15), Berthier
and Ehrlich (3), Nour (11), and Nakagawa and
colleagues (10) have demonstrated that the DNA sequence
between the 16S and 23S genes of lactobacilli is hypervariable. This
intergenic spacer region is about 200 bases in length if tRNA genes are
absent (small spacer sequence) (3). The 16S-23S spacer
sequences of lactobacilli are sufficiently species specific for the
derivation of PCR primers that can be used to identify
Lactobacillus species (3, 10, 15).
Because a relatively large number of different species (at least 18 from monogastric animals) have been described as intestinal inhabitants
(9), identification of lactobacilli by PCR using sets of
specific primers is daunting logistically. We have therefore sequenced
the 16S-23S small spacer regions of Lactobacillus isolates and compared them to the sequences of type cultures and other valid
strains recorded in GenBank (National Center for Biotechnology Information, Bethesda, Md.). Our results show that this is a relatively simple and rapid method by which lactobacilli can be identified without
resorting to the use of species-specific PCR primers.
Twenty-eight intestinal isolates (from human feces, rodent
gastrointestinal samples, and porcine gastrointestinal contents), 10 isolates from probiotic yoghurts retailed in Dunedin supermarkets, and
2 silage isolates from Thailand were used in the study (Table 1). They were determined to belong to the
genus Lactobacillus by culture on Rogosa SL agar (Difco
Laboratories, Detroit, Mich.), Gram stain appearance, catalase test,
and determination of fermentation products by gas-liquid chromatography
(7). The 16S-23S intergenic spacer region from each isolate
was amplified by using primers (15) that annealed to
conserved regions of the 16S and 23S genes (primer 16-1A,
5'-GAATCGCTAGTAATCG-3', corresponding to nucleotides 1361 to
1380 of the 16S rRNA gene according to Lactobacillus casei numbering [18]; primer 23-1B,
5'-GGGTTCCCCCATTCGGA-3', corresponding to nucleotides 123 to
113 of the 23S rRNA of L. casei [10]). PCR
mixtures contained 5 µl of 10× polymerase buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 200 µM each deoxynucleoside triphosphate, 80 pM each primer, 2 µl of Lactobacillus
cell suspension (a few colonies emulsified in sterile MilliQ
[Millipore Corp.] water), and 2.6 U of Expand High Fidelity PCR
System (Boehringer Mannheim) DNA polymerase in a total volume of 50 µl. The PCR program began with a preincubation at 94°C for 2 min.
The amplification profile was 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. This was repeated for 30 cycles. The
program finished with a 5-min incubation at 72°C. PCR products were
electrophoresed in a 1% agarose gel and visualized by UV
transillumination after being stained in ethidium bromide solution (5 µg/ml). Lactobacillus species frequently contain both
small and large spacer regions, in which case the smallest product
(about 500 to 600 bp) was excised from the gel and extracted by using a
QIAEX kit (Qiagen, Hilden, Germany). The extract was used in a repeat
PCR amplification, and the resulting DNA was purified from primers and
unincorporated nucleotides by using a QIAquick kit (Qiagen).
Both polynucleotide strands of the purified DNA were
sequenced, using 16-1A and 23-1B as forward and reverse primers,
respectively. Sequencing was carried out either at the Centre for Gene
Research, University of Otago, by the dideoxy method of Sanger et al.
(12), using a PRISM BigDye Terminator Cycle Sequencing Ready
Reaction kit (Applied Biosystems Inc., Foster City, Calif.) in
combination with an Applied Biosystems model 377A automated sequencing
system, or manually by using a Circum Vent Thermal Cycle Dideoxy kit
(New England Biolabs, Beverly, Mass.). Analysis of nucleotide sequence
data was carried out by using the SeqEd program, version 1.0.3 (Applied
Biosystems Inc.). Further sequence editing and analysis were carried
out with either EditSeq version 3.98 (DNA Star Inc., Madison, Wis.) and
Megalign version 3.1.7 (DNA Star Inc.) or LKB DNASIS (version 7.0). The small intergenic spacer region sequences obtained by these methods were
compared to sequences from type culture and other
Lactobacillus strains held in GenBank (Table
2).
View this table:
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TABLE 1.
Lactobacillus isolates identified on the basis
of percent similarity to 16S-23S small spacer region sequences
in GenBank
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|
To test the reliability of the method, we extracted DNA from L. acidophilus ATCC 4356T on five separate occasions and
sequenced the amplified 16S-23S spacer region. The five sequences were
100.0% similar to each other, as well as to the sequence held in GenBank.
Comparisons of 16S-23S small spacer region sequences held in GenBank
showed that all were less than 97.5% similar except for L. salivarius subsp. salivarius and L. salivarius subsp. salicinius (98.6% similar), L. plantarum and L. pentosus (99.0% similar), and
L. curvatus and L. graminis (98.6% similar).
With these exceptions, in which small spacer region sequences can only
aid in grouping isolates, we arbitrarily chose a value for similarity
between sequences of 97.5% or greater to indicate species identity.
This cutoff value is supported by the observation that in cases where 16S rDNA sequence (whole gene) homologies are below about 97%, it is
unlikely that two organisms have more than 60 to 70% DNA similarity
and, hence, that they are related at the species level (13).
Comparisons of 16S-23S small spacer region sequences obtained from
Lactobacillus isolates with those held in GenBank enabled the identification of 35 of 40 isolates (Table 1). Intergenic spacer
region sequences from isolates GTH2, GTH8, GTH22, GTH24, and GTH26 did
not correspond to sequences in the data bank. Nine isolates
(GTH5, GTH6, GTH18, GTH28, GTH29, JN1, JN4, GTP5, and SR2) had
spacer sequences that were between 97.5 and 99.0% similar to sequences
in the database. To confirm that the cutoff similarity value used
(97.5%) was valid, we amplified and sequenced (one polynucleotide
strand only) the V2-V3 regions of the 16S rRNA genes of these isolates
and conducted a search of sequences deposited in the GenBank DNA
database by using the BLAST algorithm (1). Amplification of
the V2-V3 region was accomplished by using primers HDA1
(5'-ACTCCTACGGGAGGCAGCAGT-3') and HDA2
(5'-GTATTACCGCGGCTGCTGGCAC-3'), described by Turner et al.
(16), and the thermal cycler program described above. The
PCR product corresponded to positions 339 to 539 in the
Escherichia coli gene. BLAST searches confirmed the
identities (on the basis of highest score) obtained by spacer sequence
analysis of all nine isolates and identified isolates GTH8, GTH26,
GTH2, GTH22, and GTH24 as Weissella confusa (previously known as Lactobacillus confusus), for which the 16S-23S
spacer region of the type culture is not yet available (Table 1).
Additionally, we sequenced the V2-V3 regions of GTH15 and GTH30, which
had been identified as L. casei on the basis of spacer
region sequence. The highest scores for the V2-V3 sequence, for both
isolates, were obtained for members of the L. casei group
(L. casei, L. paracasei, and L. rhamnosus).
Our study shows that comparison of the percentages of similarity of
16S-23S spacer region sequences of lactobacilli provides a practical
method of strain identification. The small 16S-23S spacer sequences of
lactobacilli are about 200 bp in length. These relatively short
sequences can be easily sequenced on both polynucleotide strands and
provide reliable information for comparative work. The spacer sequence
identifications that showed less than 99.0% similarity to those of
reference strains were confirmed in all cases by sequencing the V2-V3
regions of their 16S rDNAs. Moreover, the spacer region method had the
advantage of distinguishing between L. rhamnosus and
L. casei strains, which cannot be accomplished by comparison
of 16S V2-V3 region sequences. These species are commonly used in the
production of probiotic products (14). As we have
demonstrated here, amplification of the spacer regions by PCR can
be carried out with suspensions of whole Lactobacillus cells, so colonies picked from agar plates can be used directly in
identification of an isolate. DNA of a quality suitable for sequencing
can be obtained within 48 h of culture of lactobacilli. For the
majority of our Lactobacillus isolates, a clear species identification could be made on the basis of percent similarity to GenBank sequences (97.5 to 100.0% similarity). Even when
16S-23S sequences do not differ greatly between species (e.g.,
the L. pentosus/L. plantarum group),
identification is at least aided by grouping of the isolate, as was the
case with the silage strains. The use of 16S-23S spacer sequences in
the identification of lactobacilli promises to be a valuable aid in
advancing our knowledge of the species composition of
Lactobacillus populations.
| |
ACKNOWLEDGMENTS |
S. Rodtong was supported by a Teaching and Research Observation
Fellowship from the Ministry of University Affairs, Thailand. Work
conducted in Finland was aided by the Technology Development Centre of
Finland, and A. Tilsala-Timisjarvi was the recipient of a grant from
the Finnish Cultural Foundation.
The support of the University of Otago Research Committee is
gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Otago, P.O. Box 56, Dunedin, New Zealand. Phone: 64-3-479-7713. Fax: 64-3-479-8540. E-mail:
gerald.tannock{at}stonebow.otago.ac.nz.
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Applied and Environmental Microbiology, September 1999, p. 4264-4267, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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