Lactobacillus rhamnosus strain GG (ATCC 53103) is one of the most widely studied and commercialized probiotic strains, and thus strain-specific identification for the strain is highly valuable. In this study, two published PCR-based identification methods for strain GG, a transposase gene-targeting system and a phage-related gene-targeting system, were evaluated. The former produced amplicons from eight of the 41 strains tested and the phage-related system from five of the tested strains, including the strain GG. Fingerprinting analysis indicated that the strains LMG 18025, LMG 18030, and LMG 18038, which had an amplicon by the former system but none by the latter, were genetically distinguishable from L. rhamnosus GG at strain level. Strains LMG 23320, LMG 23325, LMG 23534, and LMG 25859 showed profiles very similar to that of the strain GG, suggesting that these strains might be identical to GG or derivative strains of it. The results here indicated that the phage-related gene-targeting system is a good tool for accurate identification of L. rhamnosus GG. This system would be able to detect both the original L. rhamnosus GG and its derivative strains.
Lactobacillus rhamnosus strain GG (=ATCC 53103) is one of the most widely studied and commercialized probiotic strains. Several functional and health-associated characteristics of the strain have been demonstrated, including enhancement of the mucus adhesion of other beneficial microorganisms (Ouwehand et al., 2000) and competitive exclusion of human pathogens (Lee et al., 2003). In addition, in vivo intervention studies have suggested that administration of L. rhamnosus GG has an impact on atopic eczema in infants (Kalliomäki et al., 2001, 2003), healthcare-associated diarrhea, including rotavirus gastroenteritis in hospitalized children (Szajewska et al., 2011), the frequency and severity of abdominal pain with irritable bowel syndrome in children (Francavilla et al., 2010), and upper respiratory tract infections in children attending day-care centers (Hojsak et al., 2010). In view of the importance of the organism for both research and industrial applications, a strain-specific identification system would be the most valuable means of verifying the quality and presence of the strain both in food products and in human intestinal samples in follow-up and future intervention studies. Strain-specific identification was originally carried out by specific culture properties and then by DNA fingerprinting or enzyme-linked immunosorbent assay (ELISA) monoclonal antibodies (Yuki et al., 1999; Yeung et al., 2004; Coudeyras et al., 2008). However, these methods are time-consuming and need experienced experts to perform. PCR-based methods for strain-specific identification using strain-specific primers are now regarded as a fast and accurate identification method, and such methods have been developed for several industrially valuable probiotic strains (Maruo et al., 2006; Sisto et al., 2009; Fujimoto et al., 2011). Two primer sets have hitherto been reported as L. rhamnosus GG-specific primer sets (Brandt & Alatossava, 2003; Ahlroos & Tynkkynen, 2009). However, few studies have used the strain-specific primer sets, and the qualities of the sets remain to be characterized. In this study, the two published L. rhamnosus GG-specific primer sets were evaluated by focusing on strain specificities of the sets for future use.
Materials and methods
Bacterial strains, culture conditions, and DNA extraction
All strains used in this study are shown in Table 1. L. rhamnosus GG (=ATCC 53103) was obtained from the American Type Culture Collection and used as positive control. L. rhamnosus DSM 20021T was from the German Collection of Microorganisms and Cell Cultures and used as negative control. A number of dairy isolates and human clinical isolates originating from different countries and identified as L. rhamnosus were obtained from the Belgian Coordinated Collection of Microorganisms/LMG. The strains were cultured in MRS broth at 37 °C for 20 h. Bacterial DNA was extracted from 1 mL of the cultured cells as previously described (Endo et al., 2007).
Table 1. L. rhamnosus strains used in this study and their PCR reactions with two LGG strain-specific primer sets
Two different L. rhamnosus GG strain-specific PCR systems were used in this study, and all PCR primers used are shown in supporting information Table S1. The first PCR system targets a putative transposase gene in L. rhamnosus GG as described by Ahlroos & Tynkkynen (2009). Preparation of the reaction mixture and amplification of DNA were conducted as described by Ahlroos & Tynkkynen (2009). The second PCR system targets a phage-related gene in L. rhamnosus GG as described by Brandt & Alatossava (2003). Preparation of the reaction mixture and amplification of DNA were according to a method previously described (Brandt & Alatossava, 2003). The amplification products were subjected to gel electrophoresis in 1.0% agarose, followed by ethidium bromide staining.
Fingerprinting and data analysis
Rep-PCR, RAPD, and ERIC PCR fingerprinting were carried out for strain differentiation in L. rhamnosus strains. (GTG)5 primer and a primer set REP1R-I and REP2-I were used for rep-PCR (Table S1). Preparation of the reaction mixture and amplification of DNA were according to the method described by Gevers et al. (2001). For RAPD fingerprinting, six different primers (C0540, 1251, OPA-03, D, E, and F) were used (Table S1). Preparation of the reaction mixture and amplification of DNA were performed as described elsewhere (Endo & Okada, 2006). PCR primers ERIC-1 and ERIC-2 were used for the ERIC PCR (Table S1). Preparation of the reaction mixture and amplification of DNA were by the method of Ventura et al. (2003). The amplification products were subjected to gel electrophoresis in 1.0% agarose, followed by ethidium bromide staining. Digitalized electrophoresis images were imported into the bionumerics software ver. 6.6 (Applied Maths, Belgium) for normalization and band detection. Band search and band matching using a band tolerance of 1% were performed as implemented in the BioNumerics. All fingerprinting data were combined to make a composite data set using the BioNumerics. The dendrogram was constructed from the composite data using Dice coefficients with the unweighted pair-group method using arithmetic averages (UPGMA) clustering method.
Lactobacillus rhamnosus GG strain-specific PCR
The L. rhamnosus GG strain-specific PCR system targeting the putative transposase gene described by Ahlroos & Tynkkynen (2009) produced an approximately 760 bp of amplicon from eight of the tested 41 strains of L. rhamnosus, including strain GG (Table 1). Sequence analysis indicated that the eight strains, including L. rhamnosus GG, shared completely identical sequences of the putative transposase gene among the strains (accession numbers AB685214-AB685217 and AB743581-AB743583).
The second L. rhamnosus GG strain-specific PCR system targeting a phage-related gene described by Brandt & Alatossava (2003) produced an approximately 480 bp of amplicon from five of the 41 strains tested (Table 1). The five amplified strains were included in the eight detected by the specific PCR system targeting the putative transposase gene. Strains LMG 18025, LMG 18030, and LMG 18038, originating from zabady and domiatti cheese, Egyptian fermented milk products, produced an amplicon by the first system but not by the second (Table 1).
Fingerprinting and numerical analysis
Rep-PCR, RAPD, and ERIC PCR fingerprinting were carried out to identify L. rhamnosus strains at strain level. The eight strains which produced an expected size of amplicon by the L. rhamnosus GG strain-specific PCR system targeting the putative transposase gene (Table 1) were used in this study. Strain DSM 20021T was included as reference. Rep-PCR with the REP1R-I/REP2-I primer set clearly indicated that strains LMG 18025, LMG 18030, LMG 18038, and DSM 20021 are genotypically distinct from L. rhamnosus GG at strain level (Fig. 1a). Strains LMG 23320 and LMG 23325 originating from human blood in Finland, LMG 23534 originating from human feces in Finland, and a dairy starter strain LMG 25859 produced profiles quite similar to L. rhamnosus GG (Fig. 1a). Rep-PCR with the (GTG)5 primer produced a number of bands in the tested strains, but the banding patterns were similar among the strains (Fig. 1b).
RAPD fingerprinting using six different primers also demonstrated that strains LMG 18025, LMG 18030, LMG 18038, and DSM 20021T are distinguishable from strain GG (Fig. 2). Strains LMG 23320, LMG 23325, LMG 23534, and LMG 25859 produced profiles very similar to that of strain GG, and any differences were hardly visible (Fig. 2). These tendencies were also observed in ERIC PCR (Fig. 3).
All fingerprinting data were imported into BioNumerics software ver. 6.6 and numerically analyzed. Clustering analysis of the fingerprinting data produced two clusters in the strains tested (Fig. 4). The first cluster contained three strains originating from Egyptian fermented milk products (Egyptian fermented milk cluster). They shared high similarities among the strains but showed < 60% similarities against strain GG. The second cluster contained strain GG, LMG 23520, LMG 23525, LMG 23534, and LMG 25859 (LGG and derivative strains cluster). These strains shared over 90% similarities among the strains. DSM 20021T was located distantly from other tested strains (Fig. 4).
PCR-based strain-specific identification using strain-specific primers has been reported for several probiotics (Maruo et al., 2006; Sisto et al., 2009). This technique is a valuable tool for identifying probiotics in commercialized products and monitoring the population of probiotics in human specimens in intervention studies. The specificity of the primers used is a key to accuracy in this technique.
The present findings clearly indicated that the L. rhamnosus GG strain-specific PCR system targeting the putative transposase gene produces an amplicon from human clinical isolates and dairy isolates (Table 1). This result is contradictory to the findings of Ahlroos & Tynkkynen (2009). The difference may be attributable to the small number of L. rhamnosus strains, only six strains of L. rhamnosus including the strain GG, used for evaluation of specificity by these authors. Egyptian dairy isolates, strains LMG 18025, LMG 18030, and LMG 18038, were clearly distinguished from L. rhamnosus GG by fingerprinting (Figs. 1, 2, and 3) but produced an amplicon by the PCR (Table 1). These strains belonged to the same cluster elicited by the numerical analysis (Fig. 4), but showed only weak similarities to LGG, meaning that the primer pair involves a risk of false detection of non-LGG strains. Interestingly, all these strains originated from Egyptian dairy products, suggesting that the transposase gene might be transferred horizontally between strains in Egyptian fermented food. These strains had no amplicons by the specific PCR system targeting the phage-related gene (Table 1). Among the set of strains tested, none produced amplicons in the PCR system targeting the phage-related gene when the strains had no amplicons in the system targeting the transposase gene. These results suggest that the detection system targeting the phage-related gene described by Brandt & Alatossava (2003) is more specific than that targeting the transposase gene described by Ahlroos & Tynkkynen (2009). Phage-related genes have been used to design strain-specific primers in related taxa (Fujimoto et al., 2011).
Strains LMG 23320, LMG 23325, LMG 23534, and LMG 25859 produced an expected size of amplicon in both systems (Table 1). These strains produced profiles very similar to strain GG by fingerprinting analyses (Figs. 1, 2, and 3) and showed marked similarities to strain GG based on numerical analysis (Fig. 4). This might imply that they are identical to LGG or derivative strains of it. Interestingly, three of the four strains (LMG 23320, 23325, and 23534) originated from human clinical samples in Finland, and DSM 25859 is a dairy starter isolated from human feces. Finland has a long history in providing L. rhamnosus GG for several food matrices. It would, thus, not be surprising if L. rhamnosus GG colonizes and produces derivative strains in the human body, and this may apply to other probiotic strains in Western countries and Japan, as probiotic products are popular and widely consumed in these countries. The implication here is that isolation of probiotic candidates from human samples in these countries might involve a risk of reisolation of potentially protected probiotic strains.
In conclusion, for strain-specific identification of L. rhamnosus GG, the specific PCR system targeting the phage-related gene described by Brandt & Alatossava (2003) is the best tool, and this system can detect L. rhamnosus GG and its derivative strains. L. rhamnosus GG is one of the most intensively researched and also commercialized probiotic strains and has been used for numerous intervention studies (Kalliomäki et al., 2001; Rautava et al., 2009). The PCR-based L. rhamnosus GG-specific identification system targeting the phage-related gene will be a valuable tool in monitoring the population of L. rhamnosus GG in probiotic products and in human specimens, where the accuracy and specificity of the identification is of the utmost importance. The results of this study suggest that the next step might be to combine this method with real-time qPCR and propidium monoazide to identify viable cells of L. rhamnosus GG in complex microbiota compositions, as has been suggested for other probiotic strains (Fujimoto et al., 2011).