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Applied and Environmental Microbiology, June 2001, p. 2760-2765, Vol. 67, No. 6
Nestlé Research Center,
Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland
Received 12 December 2000/Accepted 20 March 2001
The species Bifidobacterium lactis, with its main
representative strain Bb12 (DSM 10140), is a yoghurt
isolate used as a probiotic strain and is commercially applied in
different types of yoghurts and infant formulas. In order to ensure the
genetic identity and safety of this bacterial isolate, species- and
strain-specific molecular tools for genetic fingerprinting must be
available to identify isolated bifidobacteria or lactic acid bacteria
from, e.g., various clinical environments of relevance in medical
microbiology. Two opposing rRNA gene-targeted primers have been
developed for specific detection of this microorganism by PCR. The
specificity of this approach was evaluated and verified with DNA
samples isolated from single and mixed cultures of bifidobacteria and
lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus
species). Furthermore, we performed a Multiplex-PCR using
oligonucleotide primers targeting a specific region of the 16S rRNA
gene for the genus Bifidobacterium and a conserved
eubacterial 16S rDNA sequence. The specificity and sensitivity
of this detection with a pure culture of B. lactis were,
respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.
The genera
Bifidobacterium and Lactobacillus constitute a
significant proportion of the probiotic cultures used in the food industry (15, 24, 25). The utilization of strains
belonging to Bifidobacterium animalis, Bifidobacterium
longum, Bifidobacterium bifidum, and
Bifidobacterium infantis as probiotic starter cultures is
due to their important role played in the large intestine, namely,
control of the pH value and reduction of growth of many potential
pathogens and putrefactive bacteria (4, 8, 19). An
accurate species and strain identification is mandatory in clinical
studies involved in monitoring the probiotic value of cells during
passage through the human gastrointestinal tract or potentially any
public health-related monitoring. Furthermore, such monitoring of
Bifidobacterium cells directly from yoghurt, cheese, infant
formula, and other supposedly Bifidobacterium-containing products is an important quality control tool of this probiotic strain
in culture.
An identification of bifidobacteria at the genus and species level is
possible by use of several probes targeting different genes, e.g., 16S
rRNA (11, 16, 17), 23S rRNA, and recA
(13). Sequence comparison of 16S and 23S rRNAs has
demonstrated its suitability as a genetic tool for identification of
many bacterial species (6, 28). The advantages of using
these genes as targets in hybridization experiments or in amplification
reactions depend highly on the fact that they exist in large copy
numbers within one single bacterial cell, exceeding by far the number
of chromosomally encoded genes.
Several studies have been carried out on Bifidobacterium
ribosomal DNA (rDNA) sequences in order to develop species-specific primers. Furthermore, Kaufmann et al. (11) described PCR
oligonucleotide primers based on specific 16S rRNA sequences for
the identification of isolates belonging to the genus
Bifidobacterium. Recently, interest has been focused on the
generation of 16S rRNA gene probes, allowing the exclusive
detection of bifidobacteria in fecal samples through an in situ
hybridization approach (14). Another way to utilize rDNA
sequences in Bifidobacterium identification is to generate
rDNA by a PCR approach and then separate the achievable amplicons in a
sequence-specific manner in a temperature gradient gel electrophoresis
or denaturing gradient gel electrophoresis (21, 27).
Specific Multiplex-PCR assays based on the gene amplification of, e.g.,
parts of the 16S rRNA or surface layers by two pairs of primers in
a single reaction were already demonstrated to be useful for a
species-specific identification in the genus Lactobacillus
(20, 29).
In the present study, a new set of PCR primers for a specific species
identification within a mixture of bifidobacteria or as a pure culture
of B. lactis was developed. This probiotic species is used
in various infant formulas and yoghurts and is technologically favored
by its high tolerance of increased oxygen levels and its unusual acid
tolerance (18). The aim of our work was the development of
a rapid and reliable method for the highly specific detection, identification, and safety assurance (due to genetic identity) of
B. lactis isolates (by means of PCR amplification)
frequently detectable in infant feces and from different commercially
available products. This was undertaken by a Multiplex-PCR approach
using simultaneously strain- and species-specific (for B. lactis), genus-specific (for bifidobacteria), and
eubacterium-specific conserved oligonucleotide primers.
Bacterial strains and growth conditions.
All strains used in
the present study are listed in Table 1.
Strains were grown anaerobically in MRS broth (Difco, Detroit, Mich.)
generally supplemented with 0.5 g of cysteine hydrochloride per
liter and incubated for 16 h at 37°C under anaerobic conditions (Gas-pack, AnaeroGen; Oxoid, Basingstoke, United Kingdom).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2760-2765.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Specific Identification and Targeted Characterization of
Bifidobacterium lactis from Different Environmental
Isolates by a Combined Multiplex-PCR Approach
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial type strains (29 bifidobacteria and 9 lactobacilli) and nine additional Bifidobacterium and
Lactobacillus isolates utilized to evaluate the specificity
of the Multiplex-PCR
Preparation of template DNA.
DNA was extracted following a
new rapid method of cell lysis: 2 ml of an overnight culture was
collected by centrifugation at 12,000 × g (10,000 rpm)
for 10 min at 4°C, and the pellet was washed twice with 2 ml of TE
buffer (10 mM Tris [pH 8], 10 mM EDTA). One gram of glass beads (106 µm; Sigma, St. Louis, Mo.) was added to the bacterial cell
suspension. Maximal occurring cell lysis was performed with the
Mini-Beadbeater (Biospec product) for 3 min at maximum speed.
Subsequently, the suspension was centrifuged at 12,000 × g (10,000 rpm) for 2 min at 4°C, and 5 µl of the
supernatant was applied directly into the PCR. Chromosomal DNA was
further purified by phenol-chloroform extraction (26) only
for those experiments designed to detect the limit of sensitivity of
the applied PCR amplifications. Chromosomal DNA was quantified
on the basis of the absorbance (
) at 260 and 280 nm
(26).
Development of specific strain primers.
All available
bifidobacterial 16S rDNA and 16S-23S rDNA spacer region sequences were
retrieved from two databases, EMBL and GenBank, and aligned by using
the Clustal program. Two potentially strain- and species-specific
primers for B. lactis could be identified. These primers
were synthesized (MWG Oligo Synthesis) and used with the other listed
primers in this study as outlined in Fig. 1 and summarized in Table
2.
|
|
PCR amplification. Amplification reactions were performed in a total volume of 50 µl of a solution containing 1.5 mM MgCl2; 10 mM Tris HCl, pH 8.3; 50 mM KCl; each deoxynucleoside triphosphate at 200 µM; 10 pmol of P0 primer and Lm3 primer; 5 pmol of 338F primer; 50 pmol of each of the two B. lactis-specific primers, Bflact2 and Bflact5; 5 µl of the respective template DNA (from the supernatants after rapid DNA extraction, which equals about 25 ng of DNA); and 2.5 U of Taq polymerase (Gibco BRL). Amplifications were performed with a DNA thermocycler (Cetus 9700; Perkin Elmer, Norwalk, Conn.) with the following temperature profiles: 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 30 s, 58°C for 1 min, and 72°C for 4 min; and finally 1 cycle of 72°C for 7 min.
Gel electrophoresis.
Aliquots of each amplification reaction
(15 µl each) were analyzed by 1.5% (wt/vol) agarose gel
electrophoresis in Tris-acetate-EDTA buffer (26) at a
constant voltage of 7 V cm
1 and visualized with
ethidium bromide (0.5 µg/ml) and photographed under UV light
(wavelength, 260 nm).
Detection of B. lactis in food systems. Different commercially available dairy products and infant formula containing bifidobacteria and lactobacilli were investigated for the presence of the species B. lactis. In summary, 1 g of product was dissolved in 1 ml of sterilized water. An equivalent amount of glass beads (Sigma) was added to the suspension, and the mixture was treated with the Mini-Beadbeater (Biospec product) for 3 min at maximum speed in order to achieve a maximal cell lysis. After cell lysis, the suspension was centrifuged 12.000 × g (10,000 rpm) for 2 min at 4°C to remove the majority of cell fractions and debris. The DNA in the supernatant was purified by using DNAzol (Gibco BRL) according to the supplier's instructions. About 25 ng of the chromosomal DNA was loaded into the PCR amplification under the conditions described above in order to apply this Multiplex-PCR approach directly on various final, commercially available products.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Selection of a specific primer pair for PCR. The analysis of the 16S rDNA sequence of the B. lactis type strain (DSM 10140) indicated that the strain is very closely related (98.6% similarity) to B. animalis ATCC 25527 (18). Based on the comparison of both nucleotide sequences, one PCR primer pair (Bflact2 and Bflact5) was designed for the specific detection of only B. lactis. The oligonucleotide Bflact2 targets a region of the 16S rRNA gene, whereas the Bflact5 primer was designed to target the 16S-23S rRNA spacer region. The specificity of this primer pair could be demonstrated in a convincing manner with all DNA samples coming from strains (type strains and various isolates) summarized in Table 1.
Species-specific and genus-specific detection with the
Multiplex-PCR.
The application of the oligonucleotide pair
Bflact2-Bflact5 resulted in a PCR amplicon of 680 bp only
with DNA derived from B. lactis DSM 10140, whereas
absolutely no PCR product could be detected with those primers for any
other Bifidobacterium and Lactobacillus isolates,
including 38 type strains (Fig. 2).
|
Sensitivity of amplification.
Generally, a PCR amplification
allows a very sensitive detection of specific DNA sequences. The
sensitivity of amplifications was evaluated by using whole cells or
extracted pure chromosomal DNA. Lysed bacterial cells were serially
diluted, and aliquots were assayed for any occurring and detectable
sensitivity with Bflact2 and Bflact5. After 25 PCR cycles, this set of
primers yielded, with a quantitative PCR approach, a product of the
expected size with a culture dilution of as few as 100 bacterial cells per ml. Sensitivity can be improved further by the application of a
nested-PCR approach (Fig. 3). In such
a nested-PCR approach, 2 µl of the reaction mixture was transferred
after the first 25 PCR cycles to a second (not yet inoculated) reaction
mixture and amplified for an additional 25 PCR cycles. In such a way,
we have been able to reduce the detection limit to 1 to 10 bacterial
cells per ml. In order to establish the sensitivity of the method
described here, different amounts of chromosomal DNA from B. lactis DSM 10140 were used in various PCRs. Visible amplicons were
still obtained from the amplification of 3.4 pg of DNA template, even in a mixture with 60 or 200 ng of DNA derived from other
bifidobacterial species (data not shown).
|
Efficacy of primers for the application of B.
lactis from various food systems.
To evaluate the ability
of Bflact2 and Bflact5 primers to monitor B. lactis in
various food systems such as yoghurts and infant formula, 18 commercially available products claiming to contain B. lactis were analyzed. In 10 samples we were able to detect B. lactis, whereas a further 7 samples resulted in PCR
amplicons specific for only the genus Bifidobacterium and
only one sample contained bacterial species not belonging to the genus
Bifidobacterium at all (Table
3). All Multiplex-PCR results for the
various products analyzed for the presence of B. lactis
were in agreement with the labeling of bacterial strains as stated by
the producers. Furthermore, our Multiplex-PCR was also performed with
an additional 56 bifidobacterial strains previously isolated from
infant feces and identified with species-specific primers (16,
17). B. lactis-specific primers resulted in a
PCR product of the expected size with DNA of only two additional
isolates, which are currently under investigation for their taxonomic
affiliation (NCC 311 and NCC 363).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In modern bacterial taxonomy, analysis of 16S and 23S rDNA sequences is considered to be an overall powerful tool to investigate phylogenetic relationships. These sequences are used to design species-specific and genus-specific oligonucleotide probes for a rapid identification of, e.g., lactic acid bacteria and bifidobacteria.
Several reports have already described the use of PCR-specific primers targeting rDNA sequences to identify various Bifidobacterium species. Cai et al. (3) reported that the relative taxonomic position of B. lactis is still under discussion and that B. lactis could be considered a junior subjective synonym for B. animalis. A decision on this issue by the International Committee on Systematic Bacteriology is still outstanding (9). However, our study demonstrated clear differences in rDNA sequences between B. lactis DSM 10140 and the type strain of B. animalis, ATCC 25527, overcoming the so far rather difficult selective and reliable species distinction between these two species. Only a few nucleotide mismatches were found when the rDNA sequences of these two Bifidobacterium species were compared. To design primers that would specifically identify B. lactis species, we had to screen at least four regions in the 16S-23S interspacer region showing the highest variability within these two closely related species. Subsequently, for each species-specific PCR amplification each of the putative species-specific primers was paired with the primer Bflact2, originating from the 16S rRNA gene. The four primers designed were subjected to further specificity testing using DNA from all strains listed in Table 1. Only the primer pair Bflact2-Bflact5 allowed a very specific amplification for the species B. lactis and not for any other Bifidobacterium or Lactobacillus species (Fig. 2). No rapid discrimination tool for these closely related species is available so far (18).
Unfortunately, at present the species B. lactis comprises basically only one strain, the type strain DSM 10140, isolated from a dairy product (yoghurt), as described by Meile et al. (18). Prasad et al. (22) and Kok et al. (12) described two other supposed B. lactis isolates, potentially members of the same species, confirming their allocation to the species B. lactis after sequencing of their 16S-23S interspacer region and comparison to the deposited B. lactis DSM 10140 sequence. Complete nucleotide sequence analysis of 16S rRNA and 16S-23S spacer region genes provides the most accurate basis for an identification of B. lactis; however, such a complete gene sequencing cannot currently be considered to be a reasonable approach for any routine identification of multiple isolates from clinical and environmental studies or from a product or contamination tracing. While the utilization of B. lactis species-specific primers described here could allow the rapid identification of further isolates, they should be also considered as a valuable tool for use in identifying new strains belonging to these rather limited bifidobacterial species.
The investigation of infant fecal samples for B. lactis by using this very specific set of primers allowed us to identify two additional strains as being potential members of the species B. lactis, but the strains expressed clearly different and distinguishable randomly amplified polymorphic DNA patterns (data not shown). Further investigation will be necessary to redefine with various oligonucleotides an overall strain allocation or relocation from and to the species B. animalis. As described by Roy et al. (23), B. animalis ATCC 27536 has unique pulsed-field gel electrophoresis patterns, therefore being more similar to the strain B. lactis DSM 10140 than to the type strain of its own species, B. animalis. This raises questions with regard to the taxonomic borderlines between these two species (B. lactis and B. animalis) specifically and among other bifidobacterial species in general (7). DNA isolated from B. animalis ATCC 27536 yielded an amplicon of 680 bp by use of the B. lactis-specific primers. Further analysis for this specific strain seems to be required to finally confirm its taxonomic assignment (23).
The application of this Multiplex-PCR in the analysis of commercially available products can be a very useful tool for any rapid monitoring of the species B. lactis (product claims, bacterial counts, strain and species identification) and might simultaneously give indications of other occurring bifidobacterial cells. This Multiplex-PCR can be considered, in terms of reduction of tedious labor time, easy application, reliable and repeatable PCR results and intrinsic controls, and low costs, a uniquely powerful tool for studying any bifidobacterial ecology (e.g., that of the gastrointestinal tract) and furthermore the fecal environment of newborns, babies, toddlers, and adults fed with products containing members of the genus Bifidobacterium in general and B. lactis specifically. Unfortunately, such a PCR amplification that uses chromosomal DNA as a target cannot distinguish between viable and nonviable bacterial cells (10). Therefore, in the determination of the shelf life of a product containing B. lactis with regard to bacterial stability and viability, it remains essential that all detectable and viable bacterial cells are additionally plate counted. Due to the extremely short half-life of the majority of bacterial mRNAs, it seems to be possible and maybe more appropriate to select mRNA as a PCR-based target to detect bacteria by PCR amplification. In particular, total RNA extraction followed by a reverse transcriptase PCR using primers Bflact2 and Bflact5 will represent a reliable tool for detection of only the viable fraction of B. lactis cells present in various food systems.
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FOOTNOTES |
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* Corresponding author. Mailing address: Nestlé Research Center, Route du Jourat 57, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland. Phone: 41-21-785-8901. Fax: 41-21-785-8925. E-mail: ralf.zink{at}rdls.nestle.com.
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Discussion
References
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Applied and Environmental Microbiology, June 2001, p. 2760-2765, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2760-2765.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Ventura, M., Canchaya, C., van Sinderen, D., Fitzgerald, G. F., Zink, R.
(2004). Bifidobacterium lactis DSM 10140: Identification of the atp (atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny. Appl. Environ. Microbiol.
70: 3110-3121
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Bonjoch, X., Balleste, E., Blanch, A. R.
(2004). Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution. Appl. Environ. Microbiol.
70: 3171-3175
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Ventura, M., Zink, R.
(2003). Comparative Sequence Analysis of the tuf and recA Genes and Restriction Fragment Length Polymorphism of the Internal Transcribed Spacer Region Sequences Supply Additional Tools for Discriminating Bifidobacterium lactis from Bifidobacterium animalis. Appl. Environ. Microbiol.
69: 7517-7522
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Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R., Zink, R.
(2003). Analysis, Characterization, and Loci of the tuf Genes in Lactobacillus and Bifidobacterium Species and Their Direct Application for Species Identification. Appl. Environ. Microbiol.
69: 6908-6922
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Ventura, M., Meylan, V., Zink, R.
(2003). Identification and Tracing of Bifidobacterium Species by Use of Enterobacterial Repetitive Intergenic Consensus Sequences. Appl. Environ. Microbiol.
69: 4296-4301
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Nebra, Y., Bonjoch, X., Blanch, A. R.
(2003). Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution. Appl. Environ. Microbiol.
69: 2651-2656
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Ventura, M., Zink, R.
(2002). Rapid Identification, Differentiation, and Proposed New Taxonomic Classification of Bifidobacterium lactis. Appl. Environ. Microbiol.
68: 6429-6434
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