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Applied and Environmental Microbiology, July 2003, p. 4296-4301, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.4296-4301.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification and Tracing of Bifidobacterium Species by Use of Enterobacterial Repetitive Intergenic Consensus Sequences

Marco Ventura, Valerie Meylan, and Ralf Zink*

Nestlé Research Center, 1000 Lausanne 26, Switzerland

Received 2 December 2002/ Accepted 2 April 2003


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ABSTRACT
 
Eighty-nine Bifidobacterium strains from 26 species were identified and classified to the species level with an enterobacterial repetitive intergenic consensus (ERIC)-PCR approach. We demonstrated that ERIC-PCR is useful for a phylogenetic and taxonomical analysis but as well as for a species composition analysis of mixed bifidobacterial cultures isolated from dairy products and other environments.


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INTRODUCTION
 
The human colonic flora is a highly complex ecological environment, consisting of at least 400 to 500 different species (11). The genus Bifidobacterium constitutes a significant proportion of the normal colonic flora (6, 11, 16). Bifidobacterium species can be detected in various microecological environments, such as intestines, dairy products, dental caries, and sewage.

An accurate molecular identification at species and strain level is mandatory for any intestinal microbiota composition and clinical examination involved in monitoring bacteria during their passage through the gastrointestinal tract. Many studies have been carried out to design genus- or species-specific PCR primers for bifidobacteria (8-10). Another way of utilizing rRNA sequences in bifidobacterial ecology is to amplify rRNA gene fragments and separate the obtained PCR products in a sequence-specific manner in, e.g., a temperature gradient gel electrophoresis or denaturing gradient gel electrophoresis (13). Recently, it was demonstrated that amplified ribosomal DNA restriction analysis had powerful potential in the discrimination of various bifidobacteria to the species level (19). Enterobacterial repetitive intergenic consensus (ERIC)-PCR involves the use of oligonucleotides targeting short repetitive sequences dispersed throughout various bacterial genomes (21). Their location in bacterial genomes allows a discrimination at the genus, species, and strain levels based on their electrophoretic pattern of amplification products (1-5).

The aim of our study was the development of a rapid, reproducible, and easy-to-handle molecular tool to enable highly specific detection and identification of bifidobacterial species within a mix of other bifidobacteria or in pure culture concentrates. This ERIC-PCR approach generated species-specific patterns for all investigated species of Bifidobacterium. ERIC-based polymorphism was further applied to enumerate bifidobacterial species in various food systems, like yoghurts and infant formula, or in microecological environments appearing to contain bifidobacteria.


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Species-specific ERIC-PCR patterns.
 
Bacterial strains used in this study were either from culture collections or were isolated from human or animal fecal samples (Table 1) and were grown as described previously (19). PCR primers ERIC-1 (5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC-2 (5'-AAGTAAGTGACTGGGGTGAGCG-3') were applied (21). The 25-µl reaction mixture contained 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl2, 200 µM each deoxynucleoside triphosphate (Gibco BRL, Paisley, United Kingdom), 1 µM each primer, 2.5 U of Taq DNA polymerase (Gibco BRL), and 25 ng of template DNA (extracted as described in reference 22). Amplifications were done in a Perkin-Elmer Cetus-9700 thermal cycler with the following temperature profiles: 1 cycle at 94°C for 3 min; 35 cycles at 94°C for 30 s, at 48°C for 30 s, and at 72°C for 4 min; and 1 cycle at 72°C for 6 min. Aliquots of each amplification reaction (15 µl each) were electrophoresed in 2% (wt/vol) agarose gels at a voltage of 7 V/cm. Gels were ethidium bromide stained (0.5 µg/ml) and were photographed under 260-nm UV light.


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  		TABLE 1. Bifidobacterial type strains and additional strains applied to evaluate the specificity of the ERIC-PCR

We investigated 26 Bifidobacterium species and achieved amplicons showing different ERIC patterns for each bifidobacterial species (Fig. 1). The specificity of these ERIC patterns was confirmed by investigating DNA from various Bifidobacterium strains belonging to different bifidobacterial species (Table 1) as well as DNA isolated from nonbifidobacterial species (17). However, closely related species have highly similar ERIC-PCR patterns (see Bifidobacterium longum-B. suis, B. catenulatum-B. pseudocatenulatum, and B. animalis-B. lactis). PCR amplification of different bifidobacterial strains from the same species with the primers ERIC-1 and ERIC-2 revealed species-specific amplicons, with a few strain-specific bands visible for each ERIC-PCR pattern (Fig. 1). The reliability of these ERIC-PCR banding patterns was evaluated by using whole cells (19, 20) or extracted pure chromosomal DNA (22) as a direct DNA template.



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  		FIG. 1. ERIC-PCR patterns of reference strains of different Bifidobacterium species. Lane 1, B. lactis DSM 10140; lane 2, B. animalis ATCC 25527; lane 3, B. pseudocatenulatum DSM 20438; lane 4, B. catenulatum ATCC 27539; lane 5, B. subtile DSM 20096; lane 6, B. thermophilum DSM 20212; lane 7, B. minumum DSM 20102; lane 8, B. coryneforme DSM 20216; lane 9, B. angulatum DSM 20098; lane 10, B. choerinum ATCC 27686; lane 11, B. gallinarum DSM 20670; lane 12, B. boum DSM 20432; lane 13, B. mericycum DSM 6492; lane 14, B. suis ATCC 27533; lane 15, B. pullorum DSM 20433; lane 16, B. pseudolongum DSM 20099; lane 17, B. infantis ATCC 15697; lane 18, B. bifidum ATCC 29521; lane 19, B. breve ATCC 15700; lane 20, B. cuniculi ATCC 27916; lane 21, B. dentium ATCC 27534; lane 22, B. asteroides DSM 20089; lane 23, B. ruminantium DSM 6489; lane 24, B. magnum ATCC 27540; lane 25, B. adolescentis ATCC 15703; lane 26, negative control (complete PCR mixture without DNA); lane 27, B. longum ATCC 15707; lane M, 1-kb DNA ladder (Gibco BRL). Strain variation in the ERIC pattern of B. breve species is indicated by the arrows.


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Identification of isolated Bifidobacterium strains.
 
ERIC-PCR resulted in a clear identification to the species level for all 89 different Bifidobacterium strains, mainly isolated from human feces. Their ERIC-PCR fingerprints were compared with those retrieved from reference strains (data not shown). The taxonomical allocation of 53 isolates was identified as 24 strains of B. breve, 8 strains of B. lactis, 13 strains of B. longum, 2 strains of B. infantis, 2 strains of B. pseudocatenulatum, and 4 strains of B. bifidum, respectively. ERIC-PCR profiles of B. breve strains show a greater variability in their banding pattern than found in other bifidobacterial species, but notably its depicted species specificity relied on highly amplified amplicons, underlined by minor strain-specific variations (indicated by the arrows in Fig. 1). This ERIC-PCR identification assay was compared with classical identification tools (e.g., carbohydrate fermentation profiling), species-specific primers (9, 10), species-specific amplified ribosomal DNA restriction analysis, and multiplex PCR (17, 19). A taxonomical positioning of some bifidobacterial isolates strictly by their carbohydrate fermentation profiling remained rather doubtful or even unacceptable. By use of this method, only a few strains belonging actually to B. infantis, B. longum, and B. breve were initially misidentified as B. bifidum and two strains belonging to B. longum were initially wrongly attributed to B. infantis.


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ERIC-PCR fingerprinting of simple mixed-strain communities.
 
Equal volumes of all purified bacteria were combined to create a mixed culture. DNA was subsequently extracted (22) and was applied for the PCR. The overall applied DNA concentration for each tested species was defined by mixing different amounts of pure chromosomal DNA. All PCRs using DNA extracted from mixed bifidobacterial cultures were highly reproducible. ERIC-PCR fingerprinting of mixed bifidobacterial strains was additive for their individual species fingerprints and contained signature amplicons that specifically represented the molecular patterns of B. breve, B. bifidum, B. lactis, and B. longum (Fig. 2).



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  		FIG. 2. ERIC-PCR patterns of four Bifidobacterium species and their mixture (mixed strains). Shown are ERIC-PCR patterns of four individual Bifidobacterium strains from the four species (B. bifidum, B. breve, B. longum, and B. lactis) and a combination of these very same applied strains in a mixed approach created by using various template DNA (a) and different quantities of the template DNAs in an ERIC-PCR assay of the two species B. breve and B. longum (b). (a) Lane 1, B. bifidum ATCC 15696; lane 2, B. breve ATCC 15701; lane 3, B. longum ATCC 15708; lane 4, B. lactis DSM 10140; lane 5, B. bifidum and B. breve species; lane 6, B. longum and B. breve species; lane 7, B. longum and B. bifidum species; lane 8, B. bifidum and B. lactis species; lane 9, B. breve and B. lactis species; lane 10, B. longum and B. lactis species; lane M, 1-kb DNA ladder (Gibco BRL). (b) Lane 11, ERIC-PCR with 70 ng of DNA as PCR template for both species; lane 12, 70 ng of B. breve and 7 ng of B. longum as DNA template; lane 13, 70 ng of B. breve and 0.7 ng of B. longum as DNA template; lane 14, 7 ng of B. breve and 70 ng of B. longum as DNA template; lane 15, 0.7 ng of B. breve and 70 ng of B. longum as DNA template; and lane M, 1-kb DNA ladder (Gibco BRL).


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Specificity of the amplification method.
 
The sensitivity of the ERIC-PCR was evaluated by using whole cells (19, 20) or extracted pure chromosomal DNA (22). Different amounts of chromosomal DNA from different species were mixed in various ERIC-PCRs. Species-specific PCR-fragments were still visible with 3.7 pg of DNA template even in a 10-fold or 100-fold mix with 37 or 370 ng of DNA derived from other bifidobacterial species (Fig. 3a). The sensitivity of these ERIC-PCR amplifications was evaluated by direct application of whole cells and not only with purified bacterial DNA (22). Bifidobacterial cells were collected from late-log-phase cultures. After a rapid cell lysis by bead beating, these lysed bacterial cells were serially diluted and aliquots were assayed for their determinable sensitivity (20). We showed clearly that with 35 PCR cycles a bifidobacterial amount of 103 CFU/ml is still easily detectable and depicted ERIC-PCR amplicons for all investigated bifidobacterial species, whereas, at a dilution corresponding to 100 bacterial cells, only very few ERIC-PCR amplicons could be discernible (Fig. 3a). Some bands generated by ERIC-PCR were stable overall with 102 CFU/ml, while others were disrupted by a 10-fold change in their initial template concentration. Therefore, the sensitivity of the applied ERIC primers seems to be highly dependent on the method of analysis (e.g., cells, cell lysates, or extracted pure chromosomal DNA).



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  		FIG. 3. Quantitative ERIC-PCR results with regard to the detection limit of ERIC-PCR primers that used whole bacterial cells from culture concentrates and subsequent 10-fold dilutions containing 106 to 10 CFU/ml (a). ERIC-PCR patterns obtained from different food samples with declared content of bifidobacteria (b). (a) Lanes 1 to 6, direct detection of B. lactis; lane 7 to lane 12, direct detection of B. bifidum ATCC 35914; lanes 13 to 18, direct detection of B. breve ATCC 15701; and lanes 19 to 24, direct detection of B. longum ATCC 15708. (b) Lane 1, sample 15 containing only B. bifidum; lane 2, sample 16 containing only B. breve; lane 3, sample 13 containing only B. longum; lane 4, sample 1 containing only B. lactis; lane 5, sample 6 containing B. longum and B. bifidum; lane 6, sample 7 containing B. longum and B. bifidum; lane 7, sample 8 containing B. longum and B. bifidum; and lane M, 1-kb DNA ladder (Gibco BRL).


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Suitability of ERIC-PCR for a quantification of various bifidobacterial species in various food systems.
 
To evaluate the ability of ERIC-PCR to identify bifidobacterial species in different food preparations (e.g., yoghurt and infant formula) commercially available products with bifidobacteria were analyzed. The DNA preparation was performed by a slight modification of the method of Romero et al. (12). One gram of sample was dissolved in 2 ml of water and was kept at -20°C for 16 h. Frozen milk was thawed at room temperature, and 500 µl of sample was mixed with 100 µl of Tris-EDTA buffer (1 mM EDTA and 10 mM Tris-HCl [pH 8]). Sodium dodecyl sulfate (final concentration, 3.4%) was added and was incubated at 80°C for 10 min. Proteinase K digestion (500 µg/ml) was performed for 2 h at 50°C. All purification steps were performed in accordance with the method described by Romero et al. (12). The species composition (Fig. 3b) was determined by a comparison of the ERIC-PCR patterns of pure and mixed cultures (Table 2).


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  		TABLE 2. Direct ERIC-PCR analysis of commercially available product samples with declared content of bifidobacteria

The ERIC-PCR results reported here identified it as a powerful molecular identification tool for bifidobacterial species and support the potential ubiquitous nature of these sequences in all bacteria (21). The full genome sequence of B. longum NCC 2705 was recently completed (15). Computer searches of ERIC sequences in both genomes identified a large number of sequences that showed a very high and consistent degree of identity or similarity to these ERIC sequences (21). For all bifidobacterial species that exhibit a close phylogenetic proximity (e.g., B. catenulatum-B. pseudocatenulatum, B. adolescentis-B. ruminantium, and B. suis-B. longum), it may be difficult to design specific primer sets based on rRNA genes. The application of such an ERIC-PCR for bifidobacterial identification and detection therefore offers considerable potential as a rapid method and combines the necessary criteria, i.e., specificity and sensitivity. ERIC-PCR data and data from classical molecular tools for identification to the species level (ribosomal DNA sequencing and species-specific oligonucleotide probes) should be incorporated in a modern polyphasic approach for bifidobacterial taxonomy. Our results suggest that ERIC-PCR has specific advantages compared to other molecular tools for species identification (e.g., ribosomal DNA species-specific primer). In fact, ERIC-PCR targets the complete genome and not just one gene's single region. This might be more advantageous because the interpretation and power of rRNA-based data (e.g., the use of a single gene or operon) in molecular taxonomy appear sometimes to be questionable (23). In addition, the application of ERIC-PCR for our species-specific identification allows simultaneous handling and comparison of many isolates, in contrast to a required repeated PCR amplification with species-specific primers.

ERIC-PCR has already supported the differentiation of two closely bifidobacterial species (18). In fact, B. lactis DSM 10140 and the type strain of B. animalis ATCC 25527 showed clear and distinct ERIC profiles and generated species-specific DNA markers easily suitable for tracing each of these species.

We have now extended the application of ERIC-PCR for the genotyping of pure bacterial strains as well as for molecular fingerprinting of mixed bifidobacterial cultures or communities of hitherto low species complexity. Many available food products, supplements, or pharmaceutical preparations claim to contain bifidobacterial strains. The predominantly used species are B. lactis, B. animalis, B. longum, B. breve, and B. bifidum (7, 14). Our results confirmed that all analyzed commercially available products revealed at least one of those species. However, we demonstrated that most of the isolated strains from dairy products actually belong to B. lactis. All ERIC-PCR results were in complete agreement with recently published results for already existing Bifidobacterium species-specific primers targeting the 16S and the 16S-23S spacer region (9, 10, 20). ERIC-PCR in detecting various bifidobacterial species in commercial products claiming only bifidobacteria can be a very useful tool for a rapid identification of the underlying Bifidobacterium species, since it does not require any bacterial cultivation step and can yield results with very small numbers of cells. So far, ERIC-PCR approach is evaluated for directly tracing bifidobacteria in dairy products or in infant formulae containing only bifidobacteria and not for any other microorganisms without any purification steps (e.g., mixture with Lactobacillus and Streptococcus). By employing fluorescence-labeled primers and polyacrylamide gels to obtain a better separation of ERIC-PCR amplicons or by using automated gene scanners, it seems possible to significantly increase the overall detection sensitivity and data scanning. With such an approach, it is possible to evaluate the fecal and intestinal microbiota by extending this bacterial analysis to other relevant taxa in gastrointestinal or microecological environments (e.g., Lactobacillus, Clostridium, Bacteroides, and Enterococcus).


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FOOTNOTES
 
* 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. Back


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Applied and Environmental Microbiology, July 2003, p. 4296-4301, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.4296-4301.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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