Nucleotide base sequence-targeted methods of analysis provide considerable advantages over culture-based approaches for the analysis of complex bacterial communities. Both cultivable and noncultivable populations can be detected, and international cooperative studies can be initiated because freshly collected samples can be frozen and sent to analytical laboratories in other locations. The provision of laboratory conditions for the culture of fastidious anaerobic bacteria is unnecessary. Moreover, nucleotide base sequences provide tangible evidence of the identity of a bacterial isolate compared to the sometimes subjective interpretation of phenotypic observations. PCR coupled with DGGE provides a valuable tool in microbial ecological studies because the composition of the bacterial community can be screened in numerous samples with a minimal expenditure of resources and time (34
). Real-time quantitative PCR for the enumeration of specific groups of bacteria in complex communities holds much promise because, relative to culture-based techniques, it removes bias related to selective culture media. Doubtless other biases are introduced, because PCRs are known to amplify DNA sequences from mixed populations with different efficiencies (22
). Real-time PCR assays can be performed rapidly (about 1 h 50 min per run).
Nucleotide base sequence-based analytical methods in microbial ecology target mainly the V regions of 16 rRNA genes. It is common for multiple copies of the rRNA operon to be present on the bacterial chromosome (6
), which might affect the accuracy of real-time quantitative PCR (multiple 16S target sites). Additionally, there is heterogeneity with regard to 16S rRNA sequences even within a bacterial cell that might affect the binding of a probe to target DNA (21
). We reasoned, therefore, that an alternative target sequence might be preferable for the enumeration, detection, and identification of bifidobacteria. The transaldolase gene was worthy of consideration because it is common to bifidobacteria, was present as a single copy on the genome of B. infantis
ATCC 15702, and contained conserved sequences that were suitable targets for PCR primers and a real-time quantitative PCR probe. A BLAST search of GenBank showed that the primers would not anneal with the sequences of transaldolase genes of other bacterial genera, and only sequences resembling those obtained from bifidobacterial cultures were detected in DGGE gels in which PCR amplicons from fecal DNA had been tested.
We tested the ability of PCR-DGGE to identify bifidobacterial species isolated from human feces. Our “gold standards” for comparison were identification using species-specific PCR primers (18
) and 16S rDNA sequence. We observed that the majority of type cultures of bifidobacterial species reported to occur in human feces could be differentiated by transaldolase-specific PCR-DGGE. Intraspecies heterogeneity in the transaldolase target sequence resulted in the recognition of two subtypes of B. longum
, two subtypes of B. pseudocatenulatum
, and five subtypes of B. adolescentis
among the fecal isolates. Screening the identity of bifidobacterial isolates was simpler with PCR-DGGE than with species-specific primers because only a single PCR with Tal primers was required. Thus, PCR-DGGE targeting the transaldolase gene will prove to be a useful method for bifidobacterial identification in microbial ecological studies. It has the advantage over 16S rDNA-based differentiation of species (18
) that B. catenulatum
and B. pseudocatenulatum
can be differentiated from each other. From our observations, B. pseudocatenulatum
is relatively common in human feces (Table ). Moreover, we were able to recognize subgroups within B. longum
, B. adolescentis
, and B. pseudocatenulatum
. This may be useful information in tracking the fate of particular cultures of bifidobacteria in future probiotic and prebiotic studies. Transaldolase-specific PCR-DGGE did not differentiate B. catenulatum
from B. angulatum
, however, although these species can be separated on the basis of 16S rDNA sequences (26
PCR-DGGE detection of bifidobacterial species in feces was not as sensitive as species-specific PCR. We speculate that only bifidobacterial species whose cells were the most numerous were detected in feces by PCR targeting the transaldolase gene and DGGE. For comparison, we detected species in some of the fecal samples by using primers targeting the 16S rRNA gene and DGGE (26
), and although intensely staining DNA fragments matching the migration of the B. adolescentis
markers were detected in the DGGE gel, other faintly staining fragments did not match the migration distances of fragments generated from type cultures (observations not shown). Thus, PCR-DGGE can be used to detect bifidobacterial populations in fecal samples, but some of the constituent species detected by species-specific PCR will be missed.
Real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture from adult feces. Culture-based enumeration was a satisfactory gold standard because we have demonstrated previously that results using our culture method are the same as those obtained by fluorescent in situ hybridization (31
). Results obtained from the enumeration of bifidobacteria in infant feces by using transaldolase and Taqman did not correlate well with culture results. This was probably because under Taqman conditions, the transaldolase target region did not amplify well from B. bifidum
. This was the species present in the infant samples. B. bifidum
has been detected in the feces of adult humans occasionally (20
). If the cells of this species were numerous in an adult sample, PCR targeting the transaldolase gene would give an inaccurate result. Quantitative PCR targeting the 16S rDNA provided results that correlated adequately with culture results from both adult and infant feces.
We conclude that PCR-DGGE targeting the transaldolase gene provides a useful method for the identification of bifidobacterial isolates from human feces. Although culture of the bacteria is necessary, DGGE of a single PCR amplicon per isolate can provide an identification. It can probably also be used to screen fecal samples for the presence of predominant bifidobacterial species, but this requires confirmation by a study in which individual bifidobacterial species in the fecal samples would be enumerated. Both transaldolase- and 16S rDNA-targeted quantitative PCR methods can be used to enumerate bifidobacteria in feces obtained from adult humans. These methodologies promise to enhance the scope and accuracy of microbial ecological studies of the gut microflora.