Nucleic acidbased techniques to type bifidobacteria 1751 Ribotyping

Ribotyping has been used to estimate polymorphism within the 16S or 23S rRNA genes in bifidobacteria. During ribotyping, DNA is extracted and cleaved with a frequently-cutting restriction enzyme. The fragments are electrophoresed and then allowed to hybridize to a labeled rRNA gene probe. The resulting pattern is then used to differentiate bifidobacteria. A modification to this basic technique involves embedding the cells in low-melting point agarose prior to lysis and DNA extraction (McCartney and Tannock, 1995).

Ribotyping is more effective in characterizing bacteria that contain multiple copies of the rRNA genes, like bifidobacteria (Bourget et al., 1993, Yoon et al., 1999, Satokari et al., 2001a, Schell et al., 2002), rather than a single copy. Ribotyping demonstrates high reproducibility among laboratories, is applicable to all bacteria, and does not require knowledge of the genomic sequence. However, this technique is labor- and time-intensive. Automated ribotypers are available, along with a library of patterns of reference strains, which allow for ease of processing and identification of a large number of samples. When comparing a pattern from an isolate to a library, it is important to consider the number and diversity of reference strains representing the genera or species of interest.

Sakata et al. (2006) compared species delineations to species identification from an automated ribotyping device. Clusters of the EcoRI ribotype patterns did not correlate well with Bifidobacterium species, suggesting that although ribotyping may be useful in discriminating among bifidobacteria strains, the technique was not suitable for species identification. The discriminatory power of ribotyping may vary with the restriction enzyme selected and the specificity of the probe. More than one restriction enzyme may be needed to differentiate closely related strains (McCartney and Tannock, 1995, McCartney et al., 1996, Kimura et al., 1997). While 16S rRNA probes are most commonly used, bifidobacteria have been differentiated using 23S rRNA probes as well (Mangin et al., 1994, 1995, 1996, 1999). Ribotyping has been used to track a specific bifidobacteria strain after human consumption (Mangin et al., 1994) and, in combination with PFGE, has been used to assess the variability of bifidobacteria populations in humans (McCartney et al., 1996, Kimura et al., 1997, Matto et al., 2004). Ribotyping has also been used with RAPD-PCR and other molecular characterization methods to conclude that B. infantis and B. suis should be included as biotypes in the species B. longum (Sakata et al., 2002).

17.5.2 RFLP

Restriction fragment length polymorphism (RFLP) is one means of assessing the variability in genes without sequence determination. A segment of DNA is amplified by PCR, subsequently digested with a restriction enzyme, and then electrophoresed through an agarose gel, yielding a unique pattern of fragments. The banding pattern varies with the primers and the amplicon size, the actual variation in the amplified sequence, the restriction enzyme used, and the number of copies of the sequence in the chromosome (as may be the case with multiple 16S rRNA operons). 16S rDNA-RFLP, or amplified rDNA restriction analysis (ARDRA), was used to uniquely identify a probiotic strain from among the indigenous microbiota of human subjects using the restriction enzyme HaelH (Kullen et al., 1997a). With this technique, it was possible to monitor the appearance and disappearance of the ingested strain in fecal samples. Depending on the variability of the amplified sequences, it may be necessary to screen a number of restriction enzymes or to include patterns from more than one digest to characterize strains. If the sequence of the amplified gene is known, in silico digests may be performed using computer software to aid in restriction enzyme selection. Two enzymes, Alul and TaqI, were required to differentiate between bifidobacterial species of human and animal sources with ARDRA (Delcenserie et al., 2004). Only 12 of 16 bifidobacterial groups and/or species could be differentiated by ARDRA with Sau3AI (Ventura et al., 2001a). By adding BamH1 patterns, it was possible to differentiate 14 of the species - except for B. animalis/lactis and B. longum/suis. However, because of recent taxonomical changes unifying each of these groups, this method does provide accurate discrimination at least to the species level. Similar results were obtained when Roy and Sirois used three restriction enzymes (BamH1, Sau3AI, and Taq I) to differentiate the species of B. infantis, B. longum, and B. animalis with ARDRA, but could not differentiate the type strain of B. lactis from B. animalis (Roy and Sirois, 2000).

Venema and Maathius (2003) were able to classify bifidobacterial species common to the human GI tract using six restriction enzymes. This allowed for identification of 14 species, including B. longum and B. suis, as well as B. animalis and B. lactis. However, this increase in discriminatory power -to the subspecies level - makes the assay cumbersome for large numbers of isolates and results in increased assay costs because of the need for large amounts of restriction enzymes.

One advantage of ARDRA is that, as a PCR-based method, it may be fairly rapid (if few restriction enzymes are used), allowing many samples to be processed in a single day. However, because of the high 16S rRNA sequence similarity among bifidobacteria, the discriminatory power is lower than other typing methods. Thus, strains of different species may display similar ARDRA patterns.

To discriminate among bifidobacteria, other DNA sequences have been targeted to identify regions with more variation. Ventura and zink (2003) compared the tuf and recA gene sequences of B. lactis and B. animalis strains, each theoretically digested with two restriction enzymes. Analysis of the 16S-23S rRNA ITS region revealed greater sequence variability than either the 16S rDNA or tuf and recA sequences, suggesting the ITS region may be able to distinguish between these two closely related groups of strains. Using Sau3AI, it was possible to differentiate between the ITS-RFLP patterns of B. animalis and B. lactis strains. All sequence analysis and RFLP patterns revealed high homogeneity among the B. lactis strains and strong similarity between the two groups, ultimately leading the authors to suggest B. animalis and B. lactis be separated at the subspecies level.

17.5.3 REP-PCR

With the introduction of PCR came the development of a new group of bacterial typing methods. The basis of these methods is the amplification of polymorphic DNA through the selection of primers whose annealing sites are variable in number and location around the chromosome. The advantages of these methods, as with traditional PCR, include they are rapid, require equipment and reagents which are typical of most research labs, and are widely applicable. They also share the disadvantages associated with traditional PCR, including differential or incomplete amplification of strains (for a review of PCR biases, see von Wintzingerode et al. (1997)).

REP-PCR (repetitive extragenic palindromic-PCR) targets highly conserved, non-coding, repetitive chromosomal elements which are 38-bp sequences containing a stem-loop structure with a 5-bp variable loop. REP-PCR was introduced to differentiate among strains of bacteria ((Versalovic et al., 1991); review (Versalovic et al., 1994)). Similarly, ERIC-PCR is based on ERIC (enterobacterial repetitive intergenic consensus) sequences, conserved 126-bp elements. Other conserved repetitive sequences targeted by PCR include BOX elements, which were among the first repetitive elements identified in Gram-positive microorganisms, and interspersed polytrinucleotides, including (GTG)5. When the PCR primer annealing sites are on opposite DNA strands and are within a few thousand bases of each other, amplification occurs. The resulting amplicons vary in size and are separated by electrophoresis to create a unique banding pattern.

Although ERIC sequences were originally identified in the Enterobacteriaceae, they have also been identified among other prokaryotes. Shuhaimi et al. (2001) first identified ERIC elements in bifidobacteria and other Gram-positive probiotic microorganisms. It was possible to differentiate among five species of bifidobacteria, and between B. longum and B. infantis, and among strains of B. pseudocatenulatum. With the same ERIC-PCR primers, Ventura and Zink (2002) were also able to differentiate B. lactis and B. animalis. Ventura et al. (2003b) were also able to apply ERIC-PCR to the differentiation of 26 species of bifidobacteria, including strains from culture collections and fecal samples, and were able to use ERIC-PCR patterns to identify bifidobacteria isolated from food products.

BOX-PCR was used to differentiate reference strains of bifidobacteria (Gomez Zavaglia et al., 2000). These patterns were then compared to those obtained from isolates obtained from fermented milk products to identify species, which correlated well with those obtained by whole cell protein SDS-PAGE. Using BOX-PCR for species identification, the majority of 58 food and dietary supplements evaluated were found to contain B. animalis ssp. lactis (Masco et al., 2005).

Masco et al. (2003) evaluated 35 strains of seven species of dairy-related bifidobacteria (B. adolescentis, B. animalis, B. breve, B. bifidum, B. infantis, B. lactis, and B. longum) with primers for ERIC, BOX, (GTG)5, and REP sequences. BOX-PCR was selected for further study because it was the most discriminatory, yielding more than 20 bands and exhibiting the greatest inter-strain variation, and was able to differentiate among species, subspecies, and strains. It was possible to separate the groups of B. longum/infantis/suis and B. animalis/lactis. There was good correlation between identification with BOX-PCR and identification with species-specific primers. Also, multiple strains of each species were clustered together, except for strains of B. asteroides and B. pseudolongum ssp. pseudolongum.

The sensitivity and reproducibility of REP-PCR (REP, ERIC, BOX, etc.) depends on a number of factors, including the method of preparing the template DNA. While crude cell lysates may be used, and in fact, some research has demonstrated reproducibility is independent of template preparation, accurately quantified and purified genomic DNA was recommended for optimum reproducibility by Versalovic et al. (1994). Primer length and mole% GC content are also factors that contribute to pattern reproducibility.

AP-PCR (arbitrarily primed-PCR) and RAPD-PCR (randomly amplified polymorphic DNA-PCR) are similar in concept to the amplification of DNA sequences interspersed around the chromosome, except that AP- and RAPD-PCR employ a single primer with a completely arbitrary sequence and PCR conditions of reduced stringency (Welsh and McClelland, 1990, Williams et al., 1990). The lower stringency conditions allow the primers to anneal at multiple locations despite imperfect matches to the template DNA. As with REP-PCR, annealing sites on opposite strands that are close enough together result in the amplification of the region in between. Amplicons of various lengths then represent a banding pattern characteristic of the strain. There is no strict consensus delineation of the terms AP- and RAPD-PCR and these terms are often used interchangeably, although a system of nomenclature has been proposed (Vaneechoutte, 1996). Both methods usually involve single-primer reactions, although AP-PCR typically refers to methods that use primers of greater lengths (perhaps 18-25 bases) while RAPD-PCR involves much shorter primers (~6-10 bases) (Towner and Grundmann, 2001). According to the original method description, AP-PCR also employed two cycles of low stringency (annealing temperature 40°C for 5 minutes) followed by ten cycles of higher stringency (annealing temperature 60°C for 1 minute) with primers of 20 or 30 bases in length (Welsh and McClelland, 1990). RAPD-PCR employs reduced stringency during all amplification cycles (Williams et al., 1990).

Because the amplification conditions of AP- and RAPD-PCR employ lower annealing temperatures than typical PCR, these methods are very sensitive to slight alteration in procedure and suffer from a lack of reproducibility. Because of this sensitivity to variations in reaction conditions, it is important to include reference strains as positive controls for comparisons.

Towner and Grundman (2001) emphasized the need to adhere to a standardized protocol, beginning with preparation or isolation of the DNA for amplification through the conditions of staining and destaining the gel, which is especially important when comparing patterns among strains on different gels. An overview of the factors which influence pattern reproducibility includes template DNA, primer design, primer:template DNA ratio, reaction conditions or reagent sources, transposable elements and plasmids.

AP-PCR was used to differentiate between the indigenous microbiota of four subjects fed yogurt containing a strain of B. bifidum (Chen et al., 1999). Two primers were included in the study, one of which was a primer specific for ERIC elements and the other an arbitrary primer 20 bases in length. The first four cycles of PCR included a low stringency annealing temperature (37°C).

RAPD-PCR was used to assess the variability of bifidobacteria isolated from rats fed kidney beans (Fanedl et al., 1998). The RAPD patterns of the 15 isolates were compared to those of the type strains of 20 species of bifidobacteria. The unknown isolates did not form a coherent cluster, nor did they cluster with any of the type strains. These results indicated a large degree of genetic variability among the isolates, and therefore species identification was not possible. Eighty primers were initially screened and seven were selected that yielded between one and ten fragments. A low (36°C) annealing temperature was used and an extension time of two minutes was selected to produce bands no larger than 2 kb, with most bands being smaller than 750 bp. These screening criteria may have resulted in the selection of primers that were more useful for strain differentiation rather than species identification. However, the discriminatory power of this technique is easily modified. Varying the sequence or length of the PCR primers and/or the stringency of the annealing conditions might have produced banding patterns that would have been useful for species identification.

Five single-primer RAPD-PCR reactions, selected from among one hundred primers on the basis of discriminatory power, were able to differentiate among culture collection strains and commercial isolates (Vincent et al., 1998). It was possible to separate strains of B. adolescentis, B. bifidum, and B. breve into groups. It was also possible to group strains into clusters of B. infantis/longum and B. animalis/lactis and then to divide each into their respective subclusters.

RAPD-PCR has been employed to characterize the genetic variability among strains of B. longum, B. infantis, and B. suis using three 10-base long primers and a low (30°C) annealing temperature (Sakata et al., 2002). RAPD-PCR was more discriminatory than ribotyping, was able to differentiate among these species, and also indicated high levels of similarity among the three species. RAPD-PCR was also used with other molecular methods to demonstrate intraspecies genetic heterogeneity of bifidobacteria isolated from human fecal samples (Matto et al., 2004).

A variation of AP-PCR, termed TAP-PCR (triplicate arbitrarily primed-

PCR) was developed for strain differentiation and identification (Cusick and O'Sullivan, 2000). TAP-PCR capitalized on the sensitivity of AP-PCR to varied reaction conditions by creating a master mix of template DNA and the PCR amplification mixture containing a single 18-base primer specific for a conserved region of 16S rRNA. The mixture was then split into three aliquots, each of which employed a different annealing temperature (38, 40, or 42°C). The resulting amplicons were electrophoresed in adjacent wells. Bands appearing in at least two of the three lanes were considered less sensitive to varied reaction conditions and are included in the pattern analysis. With this technique it was possible to differentiate among species and strains. The added advantage of this technique was increased reproducibility compared to typical AP-PCR. However, a large collection of bifidobacteria strains, representing a variety of species, should be characterized with TAP-PCR to better assess the specificity and applicability of this method.

17.5.5 PFGE

PFGE (pulsed-field gel electrophoresis) is a widely-used and highly discriminatory molecular typing method, based on the comparison of restriction-digested total cellular DNA fragment patterns (Basim and Basim, 2001). In PFGE, cells are embedded in agarose plugs and treated with enzymes and detergents to lyse the bacteria. The chromosomal DNA is then digested with rare-cutting restriction endonucleases recognizing 6- or 8-bp sequences. Slices of the agarose plugs are inserted into a gel and electrophoresis is performed while the electric field changes direction and the pulse times continually increase. The resulting banding pattern is related to polymorphisms around the chromosome. The choice of restriction endonuclease depends largely on the G+C content of the strains to be characterized and enzymes should be selected that yield an optimum number of bands for pattern differentiation and for maximum discriminatory power. Comparing PFGE patterns obtained with multiple single-enzyme digests can increase the discriminatory power of the method, but the additional reagents are costly. Using PFGE, DNA molecules as large as 12 Mb can be separated. This technology has been used to physically map chromosomes, to estimate chromosome size, to aid in the precise selection of cloning fragments, to estimate double-strand breaks in DNA, to fingerprint bacteria, and to estimate relatedness among bacterial strains.

PFGE has been applied to strains of Bifidobacterium by various researchers to examine strain relatedness. Bourget et al. (1993) used PFGE to estimate chromosome size and compared the restriction patterns of five strains of B. breve obtained from culture collections. Digestion with Xbal, Dral or Spel yielded unique restriction patterns for each strain, except for ATCC 15698 and CIP 6466 which produced identical patterns when digested with DraI and XbaI. This observation of strain-specific patterns first suggested the feasibility of PFGE in distinguishing and differentiating closely related strains of bifidobacteria.

McCartney et al., and subsequently Kimura et al., used PFGE and ribotyping to assess differences in populations of lactic acid bacteria and bifidobacteria within and between fecal samples taken from human subjects over time (McCartney et al., 1996, Kimura et al., 1997). Isolates displaying different PFGE patterns were considered unique strains and therefore could be differentiated at the strain level. Over a one-year period, McCartney et al. identified a subject that displayed a 'simple' gut microbiota containing five strains of bifidobacteria and lactobacilli, while another subject displayed a 'complex' microbiota consisting of more than 30 strains.

Expanding on McCartney's work, Kimura et al. identified individuals as having either simple (less than four strains of bifidobacteria) or complex gut microbiota (up to ten strains). Surprisingly, isolates of bifidobacteria from fecal samples from each of two different subjects were identified as being the same strain. The conclusions drawn by these researchers about microbiota composition, complexity, and stability in these subjects depended upon the assumption that strains with identical PFGE and ribotyping patterns were identical strains. While it is true that isolates with different PFGE profiles and ribotypes may reasonably be considered different strains, the reverse is not necessarily true. For example, Crittenden et al. did not observe strain-specific PFGE patterns among four distinct strains of B. lactis. The strains displayed the same XbaI and SpeI patterns, which suggested a lack of heterogeneity among these isolates. However, despite having similar PFGE profiles, phenotypic differences related to amylase activity were observed (Crittenden et al., 2001). Other researchers have similarly used PFGE to differentiate closely related strains of bifidobacteria from various sources (Bonaparte and Reuter, 1997, Grand et al., 2003, Roy et al., 1996, Simpson et al., 2003, Ventura and Zink, 2002).

To compare the discriminatory power of some nucleic acid-based methods used for the differentiation of bifidobacteria, Matto et al. characterized isolates by ribotyping, RAPD-PCR, and PFGE (Matto et al., 2004). An equal number of ribotypes and PFGE types were observed, greater than the number of RAPD types. However, six of the isolates included in the study were not analyzed by PFGE, probably because of insufficient cell lysis. These strains were, however, characterized by ribotyping and RAPD. The discriminatory power of PFGE, as revealed by the relative number of pattern types compared to ribotyping and RAPD-PCR, may have increased if the strains not lysed by PFGE were discarded from the comparison of the methods, or if a different restriction enzyme was included in the survey.

In general, PFGE is a more discriminatory method than RAPD-PCR, which is more discriminatory than ribotyping or other common typing methods (O'Sullivan and Kullen, 1998, Busch and Nitschko, 1999). In applying PFGE to bifidobacteria, this technique 'is perhaps the most discriminatory technique at the strain level, even if it has no value in taxonomic identification' (Biavati and Mattarelli, 2001). In fact, for the differentiation of probiotic strains, PFGE is considered the 'gold standard' (FAO/WHO, 2002).

PFGE was applied by Simpson et al. (2003) in their analysis of 160 strains of bifidobacteria isolated from pigs. They were able to divide the isolates into 15 distinct profiles, comprising seven major pattern types. Other analyses, including RAPD-PCR, cell morphology, whole-cell protein electrophoresis, 16S rRNA sequence analysis and DNA hybridization, were used to evaluate the groupings established by PFGE. Results from these other methods supported the PFGE analysis and led Simpson et al. to conclude the seven PFGE types represented four distinct species, including two previously unreported species that shared less than 92% similarity in their 16S rDNA sequences to known Bifidobacterium species.

Roy et al. (1996) used PFGE with Xbal or Spel to differentiate strains of B. animalis, B. bifidum, B. breve, B. infantis, and B. longum obtained from culture collections and commercial suppliers. It was possible to differentiate strains belonging to B. infantis and B. longum, two species traditionally difficult to separate using more traditional methods. It was also possible to identify unique patterns for many of the other strains evaluated and to identify closely related strains. Within a species, some of the culture collection strains did not exhibit unique patterns, and the PFGE patterns were not useful in definitively identifying species; however, many of the PFGE profiles were strain-specific. They also identified similar profiles between some of the culture collection and commercial strains and identified similar profiles among commercial strains, suggesting PFGE could be used as a typing method to differentiate bifidobacteria at the strain level or to identify the original source of commercial starters. Their work, and later work by Bonaparte and Reuter (1997), revealed identical PFGE patterns between B. animalis ATCC 27536 and eleven and four commercial strains of B. animalis, respectively.

PFGE is one of the most discriminatory methods and is very reproducible. However, PFGE is culture-dependent, requires a substantial investment of equipment, and is intensive in terms of time, labor, and reagents. Most published PFGE protocols used with bifidobacteria require five to seven days to complete. Recently, our lab developed a rapid method, requiring only 24 hours between obtaining a broth culture and obtaining a digital image of a gel (Briczinski and Roberts, 2006). This method was specifically developed for bifidobacteria and was assessed with 34 strains of bifidobacteria, representing seven species commonly employed in the dairy industry. Banding patterns and intensities were similar to those obtained with a longer, traditional method. With the logistical constraints of any PFGE method, we have found that no more than 30 strains may easily be prepared per day. While our rapid method does allow for a faster result to be obtained and is ideal for a research setting, the PFGE method is still quite labor-intensive and is not likely suited for routine analysis in a commercial setting or for high sample throughput.

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