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Laboratory of Microbiology, Wageningen University, CT Wageningen, The Netherlands
* To whom correspondence should be addressed. E-mail: willem.devos{at}wur.nl.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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Overview of molecular tools
The tools that have been developed for identifying microbes and analyzing their activity can be divided into those based on nucleic acids and other macromolecules and approaches directed at analyzing the activity of complete cells. The nucleic acid–based tools are more frequently used because of the high throughput potential provided by using PCR amplification or ex situ or in situ hybridization with DNA, RNA, or even peptide nucleic acid probes. Notably, these include 16S rDNA sequences that can be used to place diagnostics into a phylogenetic framework and can be linked to databases providing up to 100,000 sequences (1). These 16S rDNA–based methodologies are robust and superior to traditional methods based on phenotypic approaches, which are often unreliable and lack the resolving power to analyze the microbial composition and activity of bacterial populations. In addition, a panoply of approaches that are based on DNA sequences other than rDNAs have been applied frequently to probiotic bacteria. These have been shown to be particularly useful for strain identification, as detailed below.
Recently, approaches based on complete or partial genomes have been introduced in the food industry. These include DNA arrays that can be used in comparative genomics or genome-wide expression profiling (2). These omics approaches have now become feasible for probiotic bacteria since the recent realization of the complete genome sequences of human isolates of Bifidobacterium longum (3) and Lactobacillus plantarum (4). Other functional approaches may be based on the properties of other macromolecules such as proteins. Notably, the link with the complete or partial genomes provides the basis for the further development of proteomics and other omics-related approaches to detect, identify, and analyze the functionality of LAB and bifidobacteria (5).
In addition to analysis of individual macromolecules or their collective set in a LAB strain, whole cells can also be targeted. This offers the possibility of analyzing the physiological properties of intact cells in situ using fluorescently labeled probes or substrates in combination with high-throughput approaches such as flow cytometry. These systems are notably useful to provide information on the viability and stresses in lactic cultures, as described below.
16S Ribosomal RNA probing: genus to species identification
Over the last decade, hybridizations with ribosomal RNA (rRNA)-targeted probes have provided a unique insight into the structure and spatiotemporal dynamics of complex microbial communities (6). Nucleic acid probes can be designed to specifically target taxonomic groups at different levels of specificity (from species to domain) by virtue of variable evolutionary conservation of the rRNA molecules. Appropriate software environments such as the ARB package, a software environment for sequence data (http://www.arb-home.de/) and availability of large databases (http://rdp.cme.msu.edu/html/), or the online resource for oligonucleotide probes probeBase (http://www.microbial-ecology.de/probebase/index.html) offer powerful platforms for a rapid probe design and in silico specificity profiling. Oligonucleotide probes that are complementary to regions of 16S or 23S rRNA have been successfully used for the identification of LAB, and hence, they offer the potential to be used as reliable and rapid diagnostic tools.
An overview of the currently available and validated 16S rRNA targeted oligonucleotide probes for the identification of LAB that belong to the low-GC-content gram-positive bacteria is provided with a distinction between Lactobacillus spp. (Table 1) and Lactococcus, Leuconostoc, Pediococcus, and Enterococcus spp. (Table 2). These probes have different degrees of hierarchy ranging from group and genus to species and subspecies level. Although most target the species or subspecies level, the Lab-158 and Lab-722 probes detect nearly all species of the genera Lactobacillus, Enterococcus, Pediococcus, Weissela, Vagococcus, Leuconostoc, and Oenococcus (7,8).
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Genotypic typing: strain identification
Several molecular typing techniques have been developed during the past decade for the identification and classification of bacteria at or near the strain level. The most powerful of these are genetic-based molecular methods known as DNA fingerprinting techniques, e.g., pulsed-field gel electrophoresis (PFGE) of rare-cutting restriction fragments, ribotyping, randomly amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP), which have been applied extensively for the infraspecific identification and genotyping of LAB and bifidobacteria isolated from fermented food products as well as from the human gastrointestinal tract (12). Basically, these methods rely on the detection of DNA polymorphisms between species or strains and differ in their dynamic range of taxonomic discriminatory power, reproducibility, ease of interpretation, and standardization. Genetic fingerprinting techniques that are currently being used for typing dairy LAB and especially probiotic cultures are described below.
Pulse field gel electrophoresis
Restriction fragment length polymorphism (RFLP) analysis of bacterial DNA involves the digestion of genomic DNA with rare-cutting restriction enzymes to yield a few relatively large fragments. The restriction fragments are then size-fractionated using PFGE that allows separation of large genomic fragments. The generated DNA fingerprint obtained depends on the specificity of the restriction enzyme used and the sequence of the bacterial genome and is therefore characteristic of a particular species or strain of bacteria. This fingerprint represents the complete genome and thus can detect specific changes (DNA deletion, insertions, or rearrangements) within a particular strain over time. Its high discriminatory power has been reported for the differentiation between strains of important probiotic bacteria, such as Bifidobacterium longum and B. animalis (13), Lactobacillus casei and Lb. rhamnosus (14), Lb. acidophilus complex (15), Lb. helveticus (16), and Lb. johnsonii (17). More recently, a new approach combining RFLP with DNA fragment sizing by flow cytometry has been reported for bacterial strain identification (18). DNA fragment sizing by flow cytometry was found to be faster and more sensitive than PFGE, and this technique is also amenable to automation.
Ribotyping
Ribotyping is a variation of the conventional RFLP analysis. It combines Southern hybridization of the DNA fingerprints, generated from the electrophoretic analysis of genomic DNA digests, with rDNA-targeted probing. The probes used in ribotyping vary from partial sequences of the rDNA genes or the intergenic spacer regions to the whole rDNA operon (19). Ribotyping has been used to characterize strains of Lactobacillus and Bifidobacterium from commercial products as well as from human fecal samples (20,21). However, ribotyping provides high discriminatory power at the species and subspecies level rather than on the strain level. PFGE was shown to be more discriminatory in typing closely related Lb. casei and Lb. rhamnosus as well as Lb. johnsonii strains than either ribotyping or RAPD analysis (14,17).
Randomly amplified polymorphic DNA
Arbitrary amplification, also known as RAPD, has been widely reported as a rapid, sensitive, and inexpensive method for genetic typing of different strains of LAB and bifidobacteria. This PCR-based technique makes use of arbitrary primers that are able to bind under low stringency to a number of partially or perfectly complementary sequences of unknown location in the genome of an organism. If binding sites occur in a spacing and orientation that allow amplification of DNA fragments, fingerprint patterns are generated that are specific to each strain (19). RAPD profiling has been applied to distinguish between strains of Bifidobacterium (22) and between strains of the Lb. acidophilus group and related strains (14,15,23–25). Several factors have been reported to influence the reproducibility and discriminatory power of the RAPD fingerprints, i.e., annealing temperature, DNA template purity and concentration, and primer combinations. The use of 5 single-primer reactions under optimized conditions improved the resolution and accuracy of the RAPD method for the characterization of dairy-related bifidobacteria including B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, and B. longum (22).
Amplified restriction length polymorphism
AFLP combines the power of RFLP with the flexibility of PCR-based methods by ligating primer-recognition sequences (adaptors) to the digested DNA. Total genomic DNA is digested using 2 restriction enzymes, 1 with an average cutting frequency and a second with higher cutting frequency. Double-stranded nucleotide adapters are usually ligated to the DNA fragments serving as primer binding sites for PCR amplification. The use of PCR primers complementary to the adapter and the restriction site sequence yields strain-specific amplification patterns (26). At present, AFLP has mostly been employed in clinical studies, but its successful application for strain typing of the Lb. acidophilus group and Lb. johnsonii isolates has recently been reported (17,23).
Other PCR approaches
PCR-based approaches other than RAPD and AFLP have been used for molecular typing, such as amplified ribosomal DNA restriction analysis (ARDRA) (27–29), repetitive extragenic palindromic PCR (Rep-PCR) (30), and triplicate arbitrary primed PCR (TAP-PCR) (31), and have been shown to offer a high discriminatory power for the identification and differentiation of LAB.
Although these genotypic fingerprinting methods have been successfully applied to the identification and taxonomic classification of a number LAB and bifidobacteria, the outcome can be highly variable between laboratories. Furthermore, a basic limitation in genotypic typing procedures is that the organism to be typed must be isolated because DNA from other sources disturbs the DNA fingerprints. Considering the cost/time-effectiveness and ambiguities that are still inherent to some of these techniques, 16S rRNA sequence–based methods (PCR amplification or nucleic acid probing) offer a viable option for the rapid and reliable identification of LAB and probiotic strains in mixed populations. Yet, DNA fingerprinting is a very powerful tool for the intraspecific classification of LAB and bifidobacteria provided that its methods are used in combination with other approaches. Subsequently, these methods may offer a useful means for the quality assurance of LAB starters and probiotic strains used in food products by monitoring their genetic stability and integrity over time.
Characterization of microbial communities and detection of lactic acid bacteria
The separation of PCR-amplified segments of 16S rRNA genes different in sequence by denaturing gradient gel electrophoresis (DGGE) offers a unique and comprehensive tool for the characterization of bacterial communities. With DGGE, double-stranded DNA is denatured in a linearly increasing denaturing gradient of urea and formamide at elevated temperatures. As a result a mixture of amplified PCR products will form a banding pattern after staining that reflects the different melting behavior of the various sequences. The DGGE-generated patterns make it possible to monitor shifts in the structure of microbial communities over time and/or following different treatments. Subsequent identification of specific bacterial groups or species present in the sample can be achieved either by cloning and sequencing of the excised bands or by hybridization of the profile using phylogenetic probes (32). Since its application to study the intestinal microbiota, PCR-DGGE fingerprinting has provided a sound knowledge of the succession and temporal changes of this complex microbial ecosystem (33). The predominant microbiota was shown to be remarkably stable over time and very complex in adults, less complex and more unstable in children, and unstable and developing in infants (34,35). With use of group-specific primers, the sensitivity of the method for detecting intestinal bifidobacteria and lactobacilli has been considerably enhanced (36,37). The application of these group-specific primers allowed the detection of novel species of lactobacilli in the human intestine, allowing for the development of novel probiotic cultures (36). These group-specific primers were also instrumental in monitoring the effect of the administration of a prebiotic (galactooligosaccharide) and/or probiotic (B. lactis Bb-12) on the composition of indigenous bifidobacterial species and in tracking the probiotic strain itself (38). The results showed that the simultaneous administration of the prebiotic and probiotic (synbiotic approach) did not improve the colonization of the probiotic strain in the gut. PCR-DGGE was successfully used to monitor the development of the microbial community and specifically the LAB population during the production and ripening of artisanal Sicilian cheese from milk to the ripened cheese (39). DGGE was also used to differentiate among the predominant species in a commercial mix of probiotic cultures (40). Thus, PCR-DGGE can offer an alternative tool for rapid detection and identification of LAB in food products as well as in the gastrointestinal tract.
One major limitation of DGGE fingerprinting is its low sensitivity in detecting rare members of the community (<1%). However, with group- or species-specific primers, the sensitivity of detecting less-frequent bacteria has been significantly improved. Furthermore, the detection limit of PCR-DGGE for the major intestinal bacterial groups is 
105 cells/mL fecal sample depending on the DNA extraction method used (41). Additionally, the detection of heteroduplex formation of heterogeneous rRNA operons, which can lead to an overestimation of the bacterial diversity, has been reported. Simple modifications to current PCR amplification protocols, however, were shown to be an effective approach to minimize such artifacts (42).
More high-throughput methods for determining the composition of LAB including probiotics may be achieved with DNA microarrays (43). Identification can be accomplished with designed diagnostic arrays within a matter of hours without prior cultivation and knowledge. Detection-type arrays generally contain oligonucleotides targeting a set of sequences, usually the 16S rRNA gene. The probes have to be designed so that they will hybridize with similar efficiencies to a target group of sequences. Essentially, the oligonucleotides are designed by in silico prediction using sequences from the databases and printed or arrayed onto slides. The target DNA or rRNA of the bacteria from the food or sample is purified and prepared to incorporate a fluorescent label, fragmented, and hybridized to the arrays. Although generally the 16S RNA is targeted, especially for a very complex microbial ecosystem such as that in the human gastrointestinal tract, highly specialized arrays may target specific microbes in fermented foods such as cheese and vegetables using other genes that are relatively conserved.
Analyzing the viability of lactic acid bacteria
It is crucial that the viability of the LAB strain and stability of the desirable characteristics be maintained during processing, storage, and delivery of the final product (44–46). In particular, for a microorganism to be potentially selected as a probiotic, it should be metabolically active toward the identified target in vivo. Therefore, it should survive during transit through the acidic conditions of the stomach and resist degradation by hydrolytic enzymes and bile salts in the small intestine. Although the plate count approach is often employed as the gold standard method to measure bacterial viability, it actually only indicates how many of the cells can replicate under the conditions provided for growth. The ability to reproduce might be repressed or blocked in a certain cell type, or reproduction might be limited to a certain set of conditions. Furthermore, cell populations that have been exposed to stress (e.g., oxidative, heat, freezing, osmotic stress, or starvation) can enter an unculturable state in which they still can maintain activity. Alternatively, fluorescent techniques in combination with flow cytometry (FCM) offer a powerful tool to analyze a cell population at the single-cell level because they can be used to measure different physical and biochemical parameters simultaneously and hence offer substantial information on the dynamics and physiological heterogeneity of a bacterial population (47). Ben Amor et al. used a set of fluorescent probes including carboxyfluorescein diacetate, oxonol, and propidium iodide to monitor the esterase activity, membrane potential, and membrane permeability of bile salt–stressed bifidobacteria (48). These physiological parameters serve as good indicators to assess viable, injured, and dead cells as illustrated for B. adolescentis (Fig. 1). Cell sorting confirmed that a fraction of the injured cells of B. adolescentis adopted a latent state in which they could not reproduce but could be induced to a physiologically active state after recovery. In addition, FCM has been used to detect and enumerate a number of lactobacilli and other LAB in milk and commercial probiotic products after chemical or enzymatic clearing of milk and staining of bacteria with fluorescent probes (49–51). The FCM assay was rapid (<1 h) and very sensitive (<104 bacteria/mL milk). Undoubtedly, FCM technique will provide a novel tool for the assessment of viability and stability of various lactic acid bacteria and probiotic-containing products.
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FOOTNOTES |
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2 Author disclosure: no relationships to disclose.
3 Part of this work was supported by the EU Quality of Life projects Microbe Diagnostics (QLK1-2000-00108), EU and Microfunction (QLK1-2001-00135) and Progid (QLK1-2000-00563).
4 Abbreviations used: AFLP, amplified fragment length polymorphism; DGGE, denaturing gradient gel electrophoresis; FCM, flow cytometry; FISH, fluorescent in situ hybridization; LAB, lactic acid bacteria; PFGE, pulsed-field gel electrophoresis; RAPD, randomly amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; rDNA, ribosomal DNA; rRNA, ribosomal RNA.
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