Lactobacilli is the largest and most heterogeneous group among lactic producing bacteria. It is composed of several species commonly used as starter cultures and probiotics. Due to their economical interest, the precise identification of species in this group often requires molecular identification [8]. The taxonomy of lactobacilli became clearer after the genome sequencing technologies appearance and L plantarum WCFS1 genome was the first to be sequenced [13]. 16S rRNA sequences were widely used for the first diagnostics and classification of bacterial species because extensive databases of sequences, primer sets, and enzymes for analysis of 16S rRNA length polymorphism are well established [14, 15]. However, some species within lactobacilli groups share similar 16S rRNA genes (more than 99%) and are undistinguishable on basis of their 16S rRNA phylogeny [16, 17]. The use of RFLP of 16S rRNA genes resulted in efficient discrimination of lactobacilli except for some species in L casei-, L plantarum- groups and Lactobacillus genus for which limitations were encountered specially to separate L casei from L paracasei and L plantarum from L paraplantarum [17,18,19,20]. Likewise, there were some difficulties in distinguishing L zeae from L rhamonosus and L casei or Lb gasseri from Lb johnsonii on basis of 16S rRNA phylogenetic [11, 14, 21]. For all these authors the correct choice of restriction endonucleases was suspected. Therefore, other molecular approaches like SDS-PAGE protein profiles [22], PFGE fingerprinting [18], protein-encoding genes as hsp60 marker [14, 23], and dnaK marker [16, 17] have been added to the RFLP analysis for better discrimination of lactobacilli closed species. Approaches based on analysis of recA gene, partial Tuf gene, mal gene, pepC gene, pepN gene, htrA gene, and rpoB gene were also used in cases of species sharing more than 99% 16S rRNA sequences similarities [21, 24].
As pointed out in Figs. 1, 2, 3, and 4 different RFLP patterns were obtained by the selected restriction endonucleases making it possible to distinguish clearly between L casei, L paracasei, L zeae, and L rhamnosus; between L plantarum, L paraplantarum, and L pentosus; between L gasseri and L johnsonii; and between L curvatus, L sakei, and L graminis.
L casei and L paracasei were usually confused each one to the other because of the closed relationship between many strains of L paracasei species and the L casei type strain ATCC 393 [20]. Results illustrated in Fig. 1 showed that L casei and L paracasei could be discriminated effectively on the basis of their RFLP patterns by using AlwI, BpuEI, BsgI, BsrDI, BstYI, EarI, MluCI, and NsPI among the restriction endonucleases tested in silico. It is interesting to note that real digestion by restriction enzymes resulted usually in similar fragment sizes to those in the in silico experiments [25]. However, the use of the inadequate enzymes limited some authors to distinguish L rhamnosus and L paracasei from the L casei type strain on basis of small-fragments (PCR product of approximately 295 bp) by Not1 restriction enzyme patterns and neither by using large-fragment PFGE [18] or on basis of PCR amplification and digestion products (fragments of 1500 bp in size) using AluI and MspI restriction enzymes [20]. There has been also a controversy about the classification of Lb zeae which was usually classified as a subspecies of Lb casei or Lb rhamnosus [21]. These species were differentiated from its closest neighbour only when considering other markers like Dnak-PCR RFLP/apoI [16], Tuf-PCR RFLP/HaeIII [21], or 16S-23S rDNA ITS-PCR RFLP/MseI [26]. Our results indicate that the partial 16SrRNA RFLP using both BsrDI and MluCI key enzymes are valuable method to differentiate Lb zeae although 16S rRNA gene is significantly less polymorphic than other genes because similarities are significantly higher in 16S rRNA sequences (from 98.9 to 99.9%).
Also, the discrimination between L plantarum, L pentosus, and L paraplantarum species produced ambiguous outcomes because molecular analysis of 16S rRNA polymorphism by some endonucleases was not sufficient enough to reveal significant differences [16, 18, 20]. Huang and Lee [16] noted also that species in L plantarum group were indistinguishable using HaeIII, MspI, and AluI for dnaK amplicons digestion. These two authors pointed that the crucial element in RFLP techniques is the good selection of the restriction enzymes [16]. In addition, hsp60 RFLP patterns obtainable by using both endonucleases AluI and TacI were insufficient to distinguish between L plantarum and L pentosus [23]. In our study, we showed that MucI, NspI, and TspDTI selected as key enzymes produced three different restriction profiles and distinguished these three related species.
On the other hand, comparison of AflI and NspI restriction enzyme patterns showed good species distinction between these following species of L curvatus, L sakei and L graminis. Similar to our finding (data not shown) previous in vitro restriction analysis using Hind III endonuclease discriminates L sakei from L curvatus but no data were reported for L graminis species [27]. In the present study, only restriction enzyme showing at least one sequence digestion are selected, therefore Hind III endonuclease could not be considered as key enzyme because it showed digestion in both L curvatus and L graminis (data not shown).
In the case of L gasseri and L johnsonii belonging to Lactobacillus genus, 16S rRNA gene sequence analysis is not able to reveal significant differences between these two species and their differentiation leads usually to ambiguous results using several powerful approaches like MALDI-TOF MS [26], for this reason, various molecular tools have been combined for the precise differentiation of L johnsonii from L gasseri [14, 28, 29].
In the present study, we showed that the partial 16S rRNA RFLP generated by the key enzymes DraI, MseI, and TaqI could rapidly differentiate between L gasseri and L johnsonii although their highest sequences homologies [11]. A previous study showed that these two closed species could also differentiate each one from the other on basis of ITS 16S-23S rDNA RFLP/TaqI but not with ITS RFLP/MseI [26].
