PCR-DGGE clearly has potential in the analysis of gastrointestinal communities. Using the defined system consisting of our unique lactobacillus-free mice, we were able to detect the addition of Lactobacillus species to the gastric microflora of the animals. The sensitivity of the method was limited to detection of different Lactobacillus species, rather than strains, as expected of a method based on 16S rRNA gene sequences. It will be interesting to apply this method to more complex communities, such as those inhabiting large-bowel ecosystems.
PCR-DGGE also proved a practical addition to available identification methods for lactobacilli of gastrointestinal origin. Multiple fragments of different sizes were sometimes present in PCR products from pure cultures (see Fig. , lanes 8 and 11, for examples). There was, however, always one major fragment (most dense in the DGGE gel) which indicated the identity of the isolate. The minor (less dense fragments) were probably PCR artifacts resulting from the highly folded (loops and stems) nature of the V2-V3 region (Turner et al., New Zealand Microbiol. Soc. Ann. Meet.). Lactobacillus ruminis, L. reuteri, L. fermentum, Lactobacillus vitulinus, and Lactobacillus agilis could be identified directly on the basis of the migration of their V2-V3 sequences. DNA fragments from Lactobacillus plantarum and Lactobacillus delbrueckii subsp. bulgaricus had characteristic migration behaviors, but we have not tested representative strains of all of the members of the L. plantarum and L. delbrueckii taxa.
DGGE results provided a useful initial screen for the remaining gastrointestinal species because it narrowed the possible identities of the isolates. These grouped isolates could then be identified by application of the species-specific PCR primers. This meant that an isolate needed to be tested only with two or three primer pairs to obtain a specific identification. We do not yet have species-specific primers for all of the gastrointestinal species of lactobacilli, but this would be a desirable goal for future research.
The species-specific PCR primers based on the 16S-23S rRNA intergenic spacer regions were particularly valuable in the identification of the L. casei
group isolates. They permitted discrimination to be made between L. casei/L. paracasei
, L. rhamnosus
, and L. zeae
. These species cannot be differentiated by DGGE or BLAST comparisons of V2-V3 sequences. The information that we have obtained using these species-specific primers may be helpful in unraveling the somewhat confused situation regarding the taxonomy of the L. casei
group. Dellaglio and colleagues (2
), on the basis of DNA-DNA homology studies, requested in 1991 that strain ATCC 334 replace ATCC 393 as the neotype strain of Lactobacillus casei
. They rejected the species name L. paracasei
. In 1996, Dicks et al. (3
) proposed that strain ATCC 393 be reclassified as L. zeae
. Subsequently, Mori and colleagues (9
) proposed that the L. casei
group be reclassified to include three species: L. zeae
containing strains ATCC 15820T
(DSM 20178) and ATCC 393, a species containing L. paracasei
and ATCC 334, and L. rhamnosus
. Our PCR primer pair 7, based on the 16S-23S spacer region sequences of ATCC 393 (which we have considered on the basis of the work of Dicks et al. [3
] to be L. zeae
), produced a PCR product with DNA from both ATCC 393 and ATCC 15820 (DSM 20178), but not with DNA from ATCC 334, L. paracasei
ATCC 25302, or L. rhamnosus
strains. PCR primer pair 6, based on the intergenic spacer region of ATCC 334, produced products only from L. casei
and L. paracasei
cultures. Primer pair 8, based on the intergenic spacer region of L. rhamnosus
), produced PCR products only from L. rhamnosus
cultures. The application of our primers to a collection of strains belonging to L. casei
may therefore assist in future taxonomic considerations of this group. Additionally, it may be possible to differentiate between the members of this group by DGGE if primers that targeted another region of the 16S rRNA gene are used. A potentially useful region is located between nucleotides 73 and 111 (L. casei
numbering), where variation in sequences has been observed among the members of the L. casei
Identifications obtained by a BLAST search of the GenBank database with V2-V3 region sequences correlated with those obtained by DGGE and species-specific PCR primers. We are therefore confident that the approaches to the detection and identification of Lactobacillus species that we have described in this report will contribute to future studies of the composition of the intestinal microflora and to a better understanding of Lactobacillus taxonomy.