Wageningen Academic P u b l i s h e r s
Beneficial Microbes, 2014; 5(4): 471-481
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Lactobacillus species identification by amplified ribosomal 16S-23S rRNA restriction fragment length polymorphism analysis S.H.C. Sandes1#, L.B. Alvim1#, B.C. Silva1, D.F. Zanirati1, L.R.C. Jung1, J.R. Nicoli2, E. Neumann2 and A.C. Nunes1* 1Departamento
de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Campus Pampulha, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil; 2Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Campus Pampulha, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil; [email protected]; #These authors contibuted equally to this work Received: 18 December 2013 / Accepted: 2 April 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies. Keywords: Lactobacillus, species-level identification, ARDRA, 16S-23S rRNA intergenic region
1. Introduction Lactic acid bacteria (LAB) are often used for food production, preservation, and the generation of desirable organoleptic properties of certain foods (Florou-Paneri et al., 2013; Singh et al., 2012). Many strains have probiotic properties, promoting homeostasis of host intestinal microbiota, assisting in the digestion and assimilation of nutrients (Rabot et al., 2010), protecting against colonisation by pathogens (Gareau et al., 2010), and helping in the treatment of irritable bowel syndrome (Aragon et al., 2010). Among the main LAB genera, Lactobacillus, Lactococcus, Enterococcus and Leuconostoc are frequently isolated and characterised in terms of their physiological and probiotic properties with regard to consumption by animals (Maldonado et al., 2012; Ripamonti et al., 2011; Silva et al., 2013) and humans (Li
et al., 2010; Thirabunyanon and Hongwittayakorn, 2013). Species-level identification is a fundamental prerequisite of such characterisation (FAO/WHO, 2002), but is hindered by the absence of adequate selective media, which allows the co-isolation of different genera. The Lactobacillus genus is extremely diverse, with 190 known species (Euzéby, 2013) that have been identified and characterised by phenotypic and/or genotypic methods. Identification of Lactobacillus by phenotypic methods is difficult because it can require up to 17 phenotypic tests to identify a Lactobacillus isolate accurately to the species level (Tannock et al. 1999). Furthermore, misidentification of probiotic cultures employed in commercial products is frequently seen. For example, species of Lactobacillus gasseri and Lactobacillus johnsonii reported as Lactobacillus
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2013.0092471
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acidophilus and Lactobacillus casei strains described as Lactobacillus paracasei. Therefore, molecular approaches are regularly used for identification in prospective studies that seek to select new LAB with probiotic properties. Such approaches are not only more accurate but are also less laborious (Moreira et al., 2005). Molecular methods commonly applied include analysis of conserved gene sequences, such as 16S rRNA and rpo (Naser et al., 2007; Shevtsov et al., 2011); PCR amplification using repetitive sequences, such as GTG5 PCR, enterobacterial repetitive intergenic consensus sequence-based PCR, and BOX PCR (Gevers et al., 2001); and amplified ribosomal DNA restriction analysis (ARDRA) (Chenoll et al., 2003; Markiewicz et al., 2010). The use of ARDRA of the 16S-23S internal transcribed spacers (ITS) to identify LAB to the species level is one of the areas of focus of our working group. Microorganisms belonging to Lactobacillus typically exhibit three-length polymorphisms in the region (long, medium, and short spacers) due to the presence or absence of tRNA-encoding sequences (Moreira et al., 2005). Moreira et al. (2005) conducted in silico and in situ analyses of restriction sites in the short spacers of several Lactobacillus isolates, generating a simplified panel that allowed the identification of most of the evaluated microorganisms. However, the evaluation of only a single spacer is not sufficiently informative for the accurate distinction of species belonging to very diverse genera such as Lactobacillus. Therefore, the aim of this study was to describe restriction patterns for all three ITS types (long, medium, and short) in different Lactobacillus strains for application in species-level identification.
2. Materials and methods Bacterial strains, growth conditions, and DNA extraction The LAB strains used in this study and their isolation sources are listed in Table 1. All strains had their taxonomy affiliation confirmed by 16S rRNA gene sequencing as described further. The bacteria were stored at -80 °C in De Man Rogosa Sharpe broth (MRS) (Difco Laboratories Inc., Detroit, MI, USA) or M17 broth (Difco Laboratories Inc.) with 30% glycerol. Before experimental use, cultures were grown for 18 h in accordance with the specific metabolism of each strain, in anaerobic (85% N2, 10% H2 and 5% CO2) or in aerobic conditions, at 28 °C or 37 °C, and in MRS or M17 broth. They were then subcultured twice under the same conditions. Chromosomal DNAs were isolated from LAB cultures in 10 ml MRS or M17 broth. The entire cultures were centrifuged at 8,000×g for 10 min, the bacterial pellets washed with 1 ml of deionised water, suspended in 1 ml of 5 M LiCl, and incubated for 1 h under constant shaking. Cells were washed once more with 1 ml of deionised water and each pellet was resuspended in 1 ml of protoplasting buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 25 mM sucrose) supplemented with lysozyme (10 mg/ml), before incubating for 1 h at 37 °C. The samples were centrifuged once again and the genomic DNA isolated using a NucleoSpin Tissue XS kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s recommendations. DNA yield was quantified by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) at 260 nm, and DNA purity by 260/230 nm and 260/280 nm ratios. DNA
Table 1. Strains and sources of the bacterial species used in this study. Lactobacillus species Culture collection sources L. acidophilus L. alimentarius L. antri L. brevis L. farciminis L. fructivorans L. frumenti L. gastricus L. hilgardii L. kalixensis L. kefiranofaciens L. kefiri L. panis L. paralimentarius L. paraplantarum
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Strain1
Source (origin)
ATCC 4356T ATCC 29643T DSM 16041T ATCC 14869T ATCC 29644T ATCC 8288T DSM 13145T DSM 16045T ATCC 8290T DSM 16043 T ATCC 43761T ATCC 35411T DSM 6035T DSM 13238T ATCC 700211T
human marinated fish product stomach mucosa, human faeces, human sausage spoiled salad dressing rye-bran sourdough stomach mucosa, human wine stomach mucosa, human kefir grains kefir grains sourdough sourdough beer contaminant
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Table 1. Continued. Lactobacillus species
Strain1
Source (origin)
L. pentosus L. plantarum L. pontis L. rhamnosus L. rossiae L. sakei L. sanfranciscensis Human sources L. coleohominis L. crispatus L. gasseri L. iners L. jensenii L. reuteri L. ruminis L. vaginalis L. vaginalis L. delbrueckii Kefir sources L. casei L. diolivorans L. mali L. parafarraginis L. perolens L. satsumensis Animal and industrial sources L. agilis L. animalis L. camelliae L. curvatus L. fermentum L. johnsonii L. plantarum L. reuteri L. salivarius L. vini Other lactic acid bacteria Culture collection sources Pediococcus pentosaceus Streptococcus pyogenes Kefir sources Lactococcus lactis Leuconostoc mesenteroides Oenococcus oeni Human sources Propionibacterium acnes Animal sources Weissella confusa Enterococcus casseliflavus
ATCC 8041T ATCC 14917 T ATCC 51518T DSM 8745 ATCC BAA-822T ATCC 15521T ATCC 27651T
corn silage pickled cabbage rye sourdough endocarditis, human wheat sourdough moto, starter of sake San Francisco sourdough
97 567 452 512 1021 912 032 25C 923 UFV-H2B20
vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human newborn faeces, human
15U2 1Z 21U2 12P 11P2 2P3
sugar water kefir sugar water kefir sugar water kefir sugar water kefir sugar water kefir milk kefir
1AC41 06 29(L3) 16 V3B-08 B1F-34 1ANG4 B6F-02 B2F-38 TR7.5.7
caecum, chicken faeces, canine bioethanol contaminant mouth, maned wolves mouth, bovine faeces, bovine mouth, porcine faeces, bovine faeces, bovine bioethanol contaminant
ATCC 33314T ATCC 12344T
sake mash throat, human
2U 10U1 4R2
milk kefir milk kefir sugar water kefir
P6
prostate, human
01 14
faeces, equine faeces, equine
1 ATCC
= American Type Culture Collection; DSM = German collection of microorganisms and cell cultures; T = type strain.
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integrity was verified by 0.8% agarose gel electrophoresis as described further.
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PCR amplification of 16S-23S rRNA ITS regions and restriction enzyme digestion The present analysis was based on the ARDRA of Moreira et al. (2005), making use of the three-length polymorphism observed for the 16S-23S rRNA ITS in Lactobacillus species. The amplification was performed using 10 ρmols of 16-1A (5’ GAA TCG CTA GTA ATC G 3’), corresponding to nucleotides 1361 to 1380 of the 16S rRNA gene according to L. casei numbering, and 23-1B (5’ GGG TTC CCC CAT TCG GA 3’), corresponding to nucleotides 123 to 113 of the 23S rRNA of L. casei, primers described by Tilsala-Timisjarvi and Alatossava (1997), PCR Master Mix containing 0.2 mM of each deoxyribonucleotide triphosphate, 1.5 mM of MgCl2, 1.5 units of Taq DNA polymerase (Promega Corporation, Madison, WI, USA) and 10 ng of template DNA. The amplification conditions were as follows: denaturation at 95 °C for 2 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, and a single, final extension at 72 °C for 5 min. The three amplified spacers were digested using the restriction endonucleases SphI, NcoI, NheI, SspI, Csp45I (an isoschizomer of SfuI), EcoRV, DraI, VspI, HincII, EcoRI, HindIII (Promega Corporation), and AvrII (New England Biolabs Inc., Ipswich, MA, USA), using buffer and temperature for optimal activity as recommended by manufacturers. AvrII endonuclease was included because it differentiates L. gasseri, L. johnsonii and Lactobacillus jensenii species, which was impossible in Moreira et al. (2005). Three μl of PCR reaction in 10 μl final volume were directly digested with each enzyme for 3h at 37 °C. The amplified and digested products (10 μl) were electrophoresed in a 1.4% agarose gel and visualised by UV transillumination at 302 nm after staining in 5 μg/ ml ethidium bromide solution using the digital photo documentation system T26M EasyDoc 100 (Bio Agency Biotecnologia LTDA, São Paulo, Brazil).
Grouping of restriction digestion patterns Digitalised images of gels were normalised in BioNumerics V6.5 software (Applied Math, Sint-Martens-Latem, Belgium) by using the molecular weights’ lane to name the reference positions and then the bands in the sample lanes were assigned with the software’s automatic band search followed by manual corrections. Cluster analysis was a two-step process: firstly calculating all pairwise similarity values with a similarity coefficient and then converting the resulting similarity matrix into a dendrogram by the unweighted pair group method using an arithmetical average.
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3. Results We verified the number and size of the 16S-23S rRNA spacer regions of several LAB often co-isolated with Lactobacillus in diversity studies and during prospecting for new probiotic strains. As described previously (Moreira et al., 2005), bacteria belonging to Pediococcus, Propionibacterium, Weissella, and Lactobacillus genera (Figure 1, lanes 6 to 9, respectively) present a pattern of three amplicons. The pattern for Enterococcus is composed of two amplicons (Figure 1, lane 5), and Lactococcus, Leuconostoc, Oenococcus, and Streptococcus show just a single amplicon (Figure 1, lanes 1 to 4, respectively). Next, the enzyme digestion patterns of several previously identified Lactobacillus species were established (Figure 2). Twenty-nine of the 45 evaluated species (64.4%) presented unique digestion patterns, and could therefore be precisely identified to the species level without further testing (Table 2). Other species showed identical patterns between two or more different species: (1) Lactobacillus antri DSM 16041, Lactobacillus panis DSM 6035, Lactobacillus pontis ATCC 51518, and Lactobacillus reuteri B6F-02 (Figure 2.3, Table 2); (2) L. reuteri 912 and Lactobacillus frumenti DSM 13145 (Figure 2.4, Table 2); (3) Lactobacillus paraplantarum ATCC 700211, Lactobacillus pentosus ATCC 8041, and Lactobacillus plantarum 1ANG4 (Figure 2.14, Table 2); (4) Lactobacillus kefiri ATCC 35411, Lactobacillus hilgardii ATCC 8290, and Lactobacillus parafarraginis 12P (Figure 2.16, Table 2); (5) Lactobacillus alimentarius ATCC 29643 and Lactobacillus paralimentarius DSM 13238 (Figure 2.18, Table 2); (6) Lactobacillus sakei ATCC 15521 and Lactobacillus curvatus 16 (Figure 2.22, Table 2); and (7) Lactobacillus crispatus 567 and Lactobacillus kefiranofaciens ATCC 43761 (Figure 2.28, Table 2). Moreover, the species L.
1
2
3
4
5
6
7
8
9
M (bp) 1000 750 500 250
Figure 1. The 16S-23S amplicon patterns of genera commonly co-isolated with Lactobacillus in MRS medium. The forward and reverse primers anneal in the 5’ region of the 16S gene and the 3’ region of the 23S gene, respectively, amplifying the entire spacer region and highlighting the size polymorphisms in each of the evaluated genera. 1, Lactococcus lactis 2U; 2, Leuconostoc mesenteroides 10U1; 3, Oenococcus oeni 4R2; 4, Streptococcus pyogenes ATCC 19615; 5, Enterococcus casseliflavus 14; 6, Pediococcus pentosaceus ATCC 33314; 7, Propionibacterium acnes P6; 8, Weissella confusa 01; 9, Lactobacillus johnsonii B1F-34 (control); M, size marker (1 Kb DNA Ladder). Beneficial Microbes 5(4)
Lactobacillus typing by ITS ARDRA
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1650 1000 850 650 500 400 300 200
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
2.1. L. gastricus DSM 16045
2.2. L. farciminis ATCC 29644
2.3. L. antri DSM 16041; L. panis DSM 6035; L. pontis ATCC 51518; L. reuteri B6F-02
2.4. L. reuteri 912; L. frumenti DSM 13145
2.5. L. vaginalis 25C
2.6. L. vaginalis 923
2.7. L. coleohominis 97
2.8. L. fermentum V3B-08
2.9. L. sanfranciscencis ATCC 27651
2.10. L. agilis 1AC41
2.11. L. satsumensis 2P3
2.12. L. fructivorans ATCC 8288
2.13. L. mali 21U2
2.14. L. paraplantarum ATCC 700211; L. pentosus ATCC 8041; L. plantarum 1ANG4
2.15. L. plantarum ATCC 14917
2.16. L. kefiri ATCC 35411; L. hilgardii ATCC 8290; L. parafarraginis 12P
2.17. L. camelliae 29(L3)
2.18. L. alimentarius ATCC 29643; L. paralimentarius DSM 13238
2.19. L. animalis 06
2.20. L. rossiae ATCC BAA-822
2.21. L. vini TR7.5.7
2.22. L. sakei ATCC 15521; L. curvatus 16
2.23. L. diolivorans 1Z
2.24. L. brevis ATCC 14869
Figure 2. Restriction patterns of the 16S-23S ITSs (long, medium, and short) of 45 Lactobacillus species previously identified to the species level. The amplified spacer regions were digested by 12 different restriction enzymes, all of which recognise six-base sites. Since these enzymes cut less often than four-base cutters, they generate simplified restriction patterns from the amplicons. For each image, from left to right, the restriction enzymes used were SphI, NcoI, NheI, SspI, Csp45I, EcoRV, DraI, VspI, HincII, EcoRI, HindIII, and AvrII; first lane (M) was 100 bp DNA ladder. Beneficial Microbes 5(4)
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1650 1000 850 650 500 400 300 200
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
2.25. L. kalixensis DSM 16043
2.26. L. delbrueckii UFV-H2B20
2.27. L. acidophilus ATCC 4356
2.28. L. crispatus 567; L. kefiranofaciens ATCC 43761
2.29. L. salivarius B2F-38
2.30. L. ruminis 32
2.31. L. casei 15U2
2.32. L. rhamnosus DSM 8745
2.33. L. iners 512
2.34. L. gasseri 452
2.35. L. johnsonii B1F-34
2.36. L. jensenii 1021
2.37. L. perolens 11P2
Figure 2. Continued.
reuteri (Figures 2.3 and 2.4, Table 2), Lactobacillus vaginalis (Figures 2.5 and 2.6, Table 2), and L. plantarum (Figures 2.14 and 2.15, Table 2) presented distinct digestion patterns for different strains. Clustering of the species based on the 16S-23S rRNA ARDRA patterns was compared with grouping in a phylogenetic tree based on 16S rRNA gene sequence analysis done by Salvetti et al. (2012). Eight well-defined groups were observed: L. reuteri, L. plantarum, L. salivarius, L. casei, L. alimentarius, L. sakei, Lactobacillus buchneri, and Lactobacillus delbrueckii groups (Figure 3). The 16S-23S rRNA ARDRA pattern similarity did not cluster some closely related species belonging to the same phylogenetic group. For example, Lactobacillus fructivorans and Lactobacillus sanfranciscensis, both belonging to the L. fructivorans group, and Lactobacillus gastricus and Lactobacillus animalis, belonging, respectively, to the L. reuteri and L. salivarius groups, were grouped with 16S rRNA phylogenetically unrelated species. Due to an insufficient number of related species, Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus perolens could not be classified into groups. 476
4. Discussion Evaluation of amplified DNA polymorphisms and partial sequences of the ribosomal RNA operon (rrn) are frequently used to identify bacteria at the species level. This operon is organised as follows: 16S – spacer region 1 – 23S – spacer region 2 – 5S, and is responsible for 16S, 23S, and 5S rRNA synthesis. Since it is highly conserved, homologous, and stable (without horizontal gene transfer), it is often used for evolutionary studies (Felis and Dellaglio, 2007). Nonetheless, the rrn copy number in the bacterial genome is variable (Tourova, 2003), and some non-coding regions, such as the 16S-23S rRNA ITS regions, can acquire and accumulate mutations without compromising the general bacterial metabolism. Therefore, the 16S-23S ITS is recommended for microdiversity analysis, which is used for groups of individuals with high genetic affinity. To address diversity among phylogenetically distant isolates, amplified 16S or 23S rRNA fragments can be used. These genes generate simple banding patterns, depending on the restriction enzyme used (Junior et al., 2004).
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Lactobacillus typing by ITS ARDRA
Table 2. 16S-23S intergenic spacers (long, medium and short) restriction patterns of 45 Lactobacillus species previously identified to species level by 16S rRNA gene sequencing.1
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Endonuclease SphI
NcoI
NheI
SspI
Csp45I EcoRV DraI
VspI
HincII
EcoRI HindIII AvrII
Lactobacillus species
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
––– +–– +++ +++ +++ +++ +++ ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– +–– +++ +++ –––
––– ––– ––– ––– ––– ––– +++ ––– ––– +++ +++
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
––– +++ ––– ––– ––– ––– ––– ––– +++ ––– +++
––– +++ +++ +++ +++ +++ +++ +++ +++ +++ –––
––– +–– ––– +–– +–– ––– ––– ––– ––– ––– +––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
––– +++ ––– ––– ––+ ––+ ––– ––– +++ ––– –––
––– +++ ––– ––– ––– ––– ––– ––– ––– ––– –––
L. gastricus L. farciminis L. antri, L. panis, L. pontis, L. reuteri 2 L. reuteri 2, L. frumenti L. vaginalis 2 L. vaginalis 2 L. coleohominis L. fermentum L. sanfranciscensis L. agilis L. satsumensis
+++ +++ +++ +++ +++ +++
––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– –––
+++ +++ +++ +++ +++ –––
––– ––– ––– +–– ––– +––
––– ––– ––– ––– ––– +++
+++ +++ ––– ––– ––– +++
+++ ––– +++ +++ +++ –––
+–– ––– +++ +++ +++ –––
––– ––– ––– ––– ––– –––
+++ ––– ––– ––– +++ +++
––– ––– ––– ––– ––– –––
L. fructivorans L. mali L. plantarum 2, L. paraplantarum, L. pentosus L. plantarum 2 L. parafarraginis, L. kefiri, L. hilgardii L. camelliae
+++ +++ +++ +++ +++ +++ +++ ––– ––– ––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– +++ +++ +++ +++ ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– ––+ ––– ––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– +++ +++ ––– –––
––– ––– ––– ––– ––– ––– ––– +–– +++ +–– +–– ––– +++ ––– –––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– +++ ––– ––– +++ +++
+++ +++ +++ +++ +++ ––– ––– +–– ––– ––– ––– +++ +++ +++ +++
+++ +++ ––– ––– ––– +++ +++ ––– ––– ––– ––– ––– ––– +++ –––
+–– +–– +–– ––– ––– +++ ––– ––– ––– ––– ––– ––– +–– ––– +––
––– ––– ––– –+– ––– ––– ––– ––– ––– +++ +++ ––– ––– ––– –––
+++ +++ +++ ––– +++ +++ +++ +++ ––– +++ +++ ––– ––– +++ +++
+++ ––– ––– ––– ––– ––– ––– ––– ––– +++ +++ ––– ––– ––– –––
L. alimentarius, L. paralimentarius L. animalis L. rossiae L. vini L. sakei, L. curvatus L. diolivorans L. brevis L. kalixensis L. delbrueckii L. acidophilus L. kefiranofaciens, L. crispatus L. salivarius L. ruminis L. casei, L. paracasei L. rhamnosus
––– ––– ––– –––
––– ––– ––– –––
––– ––– ––– –––
––– ––– ––– –––
––– ––– ––– –––
+++ +++ +++ +++
––– ––– ––– –––
––– ––– ––– –––
+–– ––– ––– –––
––– ––– ––– –––
––– +–– +–– –––
+++ +++ ––– –––
L. iners L. gasseri L. johnsonii L. jensenii
–––
–––
–––
–––
–––
–––
–––
–––
+––
–––
+++
+++
L. perolens
1 The 2
plus and minus signs refers, respectively, to positive and negative digestion for long, medium and short spacers for a specific enzyme. L. reuteri, L. vaginalis and L. plantarum present two distinct restriction patterns.
In Lactobacillus, Weissella, Propionibacterium, and Pediococcus genera, sequence insertions encoding tRNAs in some copies of the 16S-23S spacer region generate characteristic patterns of three amplicons: short (without insertion), medium (tRNA-Ala insertion), and long (tRNAAla and tRNA-Ile insertions). These patterns, along with cultivation in MRS medium and morphological and Beneficial Microbes 5(4)
colorimetric analyses, such as Gram staining, can be used to isolate microorganisms within these genera in a specific and reproducible way. They are therefore of use in both studies of bacterial diversity and in prospective study for new probiotic strains (Moreira et al., 2005).
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60
70
80
90
100 L. antri DSM 16041 L. panis DSM 6035 L. pontis ATCC 51518 L. reuteri B6F-02 L. vaginalis 25C L. vaginalis 923 L. reuteri 912 L. frumenti DSM 13145 L. coleohominis 97 L. fermentum V3B-08 L. brevis ATCC 14869
L. reuteri group
L. brevis group
L. plantarum ATCC 14917 L. plantarum 1ANG4 L. paraplantarum ATCC 700211 L. pentosus ATCC 8041
L. plantarum group
L. gastricus DSM 16045
L. reuteri group
L. vini TR7.5.7 L. ruminis 032 L. agilis 1AC41 L. satsumensis 2P3 L. mali 21U2 L. salivarius B2F-38
L. salivarius group
L. casei 15U2 L. rhamnosus DSM 8745
L. casei group
L. camelliae 29(L3)
Single species group
L. alimentarius ATCC 29643 L. paralimentarius DSM 13238 L. farciminis ATCC 29644
L. alimentarius group
L. sanfranciscencis ATCC 27651 L. animalis 06 L. rossiae ATCC BAA-822
L. fructivorans group L. salivarius group Couples group
L. sakei ATCC 15521 L. curvatus 16
L. sakei group
L. fructivorans ATCC 8288 L. diolivorans 1Z L. kefiri ATCC 35411 L. hilgardii ATCC 8290 L. parafarraginis 12P
L. fructivorans group L. buchneri group
L. perolens 11P2
L. perolens group
L. jensenii 1021 L. johnsonii B1F-34 L. gasseri 452 L. iners 512 L. delbrueckii UFV-H2B20 L. kalixensis DSM 16043 L. acidophilus ATCC 4356 L. kefiranofaciens ATCC 43761 L. crispatus 567
L. delbrueckii group
Figure 3. The amplified ribosomal DNA restriction analysis patterns of Lactobacillus species for the 16S-23S ITS regions were grouped by similarity and compared with a phylogenetic tree based on 16S rRNA gene sequence analysis. The dotted frames denote grouping of species based on phylogenetic analysis of 16S rRNA gene sequences. 478
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The main study focus of our working group is the selection of new probiotic strains, especially Lactobacillus, for use as food supplements or vaccine vehicles for livestock (Mota et al., 2006; Souza et al., 2007). Accurate specieslevel identification is a fundamental requirement (FAO/ WHO, 2002) that is hindered by the great diversity of this genus, including a large number of closely related species (Felis and Dellaglio, 2007). Lactobacillus is composed of 190 species and 28 subspecies (Euzéby, 2013), some of which may share more than 99% similarity between 16S rRNA sequences (Mañes-Lázaro et al., 2009), necessitating more discriminative methods of genomic analysis (Felis and Dellaglio, 2007). Moreira et al. (2005) were able to distinguish 43 strains of only 17 Lactobacillus species (almost 1/3 of species used in the present work). Over the past 8 years, our group has used analysis based on 16S-23S rRNA spacer restriction patterns to identify, to the species level, more than five hundred strains of 45 species of Lactobacillus from different animals, humans, and food sources. To accomplish this, we used 12 different six-base cutter endonucleases. This approach allowed us to establish a simplified restriction pattern for each species evaluated, compared with four-base cutters. As already noted, a significant number of Lactobacillus species (29/45) can be effectively typed on the basis of the restriction patterns of their amplified 16S-23S ITS regions, including closely related species that share more than 97% similarity between their 16S rRNA sequences, such as L. casei and L. rhamnosus, and L. gasseri and L. johnsonii (Felis and Dellaglio, 2007; Salvetti et al., 2012). Compared with the method described by Moreira et al. (2005), simultaneous digestion of the three ITSs was able to increase discriminative capacity, allowing increased accuracy in species-level identification through the formation of unique patterns. This was observed for Lactobacillus farciminis, L. vaginalis, Lactobacillus satsumensis, Lactobacillus mali, L. plantarum, L. rossiae, Lactobacillus iners, L. gasseri, L. johnsonii, and L. jensenii. Nevertheless, for some species, restriction patterns were not sufficiently informative for accurate identification; they could only indicate the general group to which the species belonged. For these cases, species distinction can be established using other approaches. For example, (1) L. reuteri can be differentiated from L. antri, L. panis, L. pontis, and L. frumenti by 16S rRNA gene sequence analysis, and the remaining four species may be differentiated from each other by PCR using species-specific primers in the 16S-23S ITS region (Ferchichi et al., 2008); (2) L. paraplantarum, L. pentosus, and L. plantarum can be differentiated by multiplex PCR assay with recA gene-derived primers (Torriani et al., 2001); (3) L. kefiri, L. hilgardii, and L. parafarraginis can be differentiated by fermentation profiles analysis using L-arabinose and D-xylose (Endo and Okada, 2007); (4) L. alimentarius and L. paralimentarius can be Beneficial Microbes 5(4)
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differentiated by two-step multiplex PCR using speciesspecific primers in the 16S-23S ITS, or 16S and 23S coding regions (Settanni et al., 2005); (5) L. sakei and L. curvatus can be differentiated by multiplex PCR-based restriction enzyme analysis (Lee et al., 2004); and (6) L. crispatus and L. kefiranofaciens can be differentiated by 16S rRNA coding sequence analysis. According to Salvetti et al. (2012), Lactobacillus shows a phylogenetic structure that is quite complicated. It is paraphyletic with Pediococcus, and includes 15 phyletic groups: (1) L. delbrueckii, (2) L. salivarius, (3) L. reuteri, (4) L. buchneri, (5) L. alimentarius, (6) L. casei, (7) L. sakei, (8) L. fructivorans, (9) Lactobacillus coryniformis, (10) L. plantarum, (11) L. perolens, (12) L. brevis, (13) Lactobacillus collinoides, (14) Lactobacillus manihotivorans and (15) Lactobacillus vaccinostercus groups. In addition, the genus includes two ‘artificial’ groups: (1) couple species (represented by the pairs L. rossiae/Lactobacillus siliginis, L. concavus/Lactobacillus dextrinicus, Lactobacillus kunkeei/Lactobacillus ozensis, and Lactobacillus pantheris/ Lactobacillus thailandensis) and (2) single species (represented by Lactobacillus algidus, Lactobacillus brantae, Lactobacillus camelliae, Lactobacillus composti, Lactobacillus floricola, Lactobacillus malefermentans, Lactobacillus saniviri, Lactobacillus selangorensis, Lactobacillus senioris, and Lactobacillus sharpeae). Based on this, we propose to group several species according to similarity of 16S-23S rRNA restriction patterns. Coding regions of the rrn operon, such as that encoding 16S rRNA, are highly homologous between species. By contrast, the ITS regions are able to accumulate mutations, and are therefore more variable. We observed that the restriction analysis based on the ITS region formed groups similar to those based on 16S rRNA sequences for most of the species, indicating that ARDRA of this region may be appropriate for evolutionary studies. However, L. gastricus clustered far away from the L. reuteri group. L. gastricus is a poorly characterised species at the molecular level. Few strains had their rrn genes sequenced and only recently the genome has been sequenced for one strain. We tried to get the 1623 rRNA sequences from this genome project but were unsuccessful. We had sequenced the 16S rRNA gene before the start of ARDRA, so we are sure that it is L. gastricus DSM 16045 from the DSMZ culture collection. Therefore, the reason that L. gastricus does not group in the L. reuteri clade might be because the initial grouping was based on the 16S rRNA (a conserved gene), while here we are dealing with the three ITS-1 regions of all rrn operons (which are probably more polymorphic between the operons than the 16S rRNA genes).
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5. Conclusions Restriction patterns generated from the 16S-23S ITS regions enabled the identification of 29 species of Lactobacillus. For an additional 16 species, identity could be established to the level of groups of closely related species. This approach, besides being reproducible, produced simplified patterns that grouped the species into clusters that reflected, to a large extent, their phylogenetic relationships as determined by 16S rRNA sequences. Our results supported the well-established advantages of using this region for evolutionary studies.
Acknowledgments The following Brazilian financing programs or institutions supported our work: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). CAPES supported S.H.C. Sandes, L.B. Alvim, and B.C. Silva; FAPEMIG granted L.R.C. Jung and D.F. Zanirati.
References Aragon, G., Graham, D.B., Borum, M. and Doman D.B., 2010. Probiotic therapy for irritable bowel syndrome. Gastroenterology and Hepatology 6: 39-44. Chenoll, E., Macián, M.C. and Aznar, R., 2003. Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rRNA-based techniques. Systematic and Applied Microbiology 26: 546-556. Endo, A. and Okada, S., 2007. Lactobacillus farraginis sp. nov. and Lactobacillus parafarraginis sp. nov., heterofermentative lactobacilli isolated from a compost of distilled shochu residue. International Journal of Systematic and Evolutionary Microbiology 57: 708-712. Euzéby, J.P., 2013. List of bacterial names with standing in nomenclature (LPSN): a folder available on the internet. International Journal of Systematic Bacteriology 47: 590-592. Ferchichi, M., Valcheva, R., Prévost, H., Onno, B. and Dousset, X., 2008. A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences. Journal of Applied Microbiology 104: 1797-1807. Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO), 2002. Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. Available at: ftp://ftp.fao.org/es/esn/food/wgreport2.pdf. Felis, G.E and Dellaglio, F., 2007. Taxonomy of lactobacilli and bifidobacteria. Current Issues in Intestinal Microbiology 8: 44-61.
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Florou-Paneri, P., Christaki E. and Bonos E., 2013. Lactic acid bacteria as source of functional ingredients. In: Kongo, J. (ed.) Lactic acid bacteria – R&D for food, health and livestock purposes. Intech, Rijeka, Croatia, pp. 589-614. Gareau, G.M., Sherman, P.M, and Walker, W.A., 2010. Probiotics and the gut microbiota in intestinal health and disease. Nature Reviews Gastroenterology and Hepatology 7: 503-514. Gevers, D., Huys, G. and Swings, J., 2001. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiology Letters 205: 31-36. Junior, F.B.F., Teixeira, K.R.S. and Reis, V.M., 2004. Análise de restrição do DNA ribossomal amplificado (ARDRA) em estudos de diversidade intra-específica de Azospirillum amazonense isolado de diferentes espécies de Brachiaria. Documentos Embrapa Cerrados 117: 1-41. Lee, J., Jang, J., Kim, B., Kim, J., Jeong, G. and Han, H., 2004. Identification of Lactobacillus sakei and Lactobacillus curvatus by multiplex PCR-based restriction enzyme analysis. Journal of Microbiological Methods 59: 1-6. Li, Q., Chen Q., Ruan, H., Zhu, D., and He, G., 2010. Isolation and characterisation of an oxygen, acid and bile resistant Bifidobacterium animalis subsp. lactis Qq08. Journal of the Science of Food and Agriculture 90: 1340-1346. Maldonado, N.C., De Ruiz, C.S., Otero, M.C., Sesma, F. and NaderMacías, M.E., 2012. Lactic acid bacteria isolated from young calves – characterization and potential as probiotics. Research in Veterinary Science 92: 342-349. Mañes-Lázaro, R., Ferrer, S., Rosselló-Mora, R. and Pardo, I., 2009. Lactobacillus oeni sp. nov., from wine. International Journal of Systematic and Evolutionary Microbiology 59: 2010-2014. Markiewicz, L.H., Biedrzycka, E., Wasilewska, E. and Bielecka, M., 2010. Rapid molecular identification and characteristics of Lactobacillus strains. Folia Microbiologica 55: 481-488. Moreira, J.L.S., Mota, R.M., Horta, M.F., Teixeira, M.R.S., Neumann, E., Nicoli, J.R. and Nunes, A.C., 2005. Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling. BMC Microbiology 5: 15. Mota, R.M., Moreira, J.L.S., Souza, M.R., Horta, M.F., Teixeira, S.M.R., Neumann, E., Nicoli, J.R. and Nunes, A.C., 2006. Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines. BMC Biotechnology 6: 2. Naser, S.M., Dawyndt, P., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Vancanneyt, M. and Swings J., 2007. Identification of lactobacilli by pheS and rpoA gene sequence analyses. International Journal of Systematic and Evolutionary Microbiology 57: 2777-2789. Rabot, S., Rafter, J., Rijkers, J.T., Watzl, B. and Antoine J.M., 2010. Guidance for substantiating the evidence for beneficial effects of probiotics: impact of probiotics on digestive system metabolism. Journal of Nutrition 140: 677S-689S. Ripamonti, B., Agazzi, A., Bersani, C., De Dea, P., Pecorini, C., Pirani, S., Rebucci, R., Savoini, G., Stella, S., Stenico, A., Tirloni, E. and Domeneghini, C., 2011. Screening of species-specific lactic acid bacteria for veal calves multi-strain probiotic adjunct. Anaerobe 17: 97-105.
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Salvetti, E., Torriani, S. and Felis, G.E, 2012. The Genus Lactobacillus: a taxonomic update. Probiotics and Antimicrobial Proteins 4: 217-226. Settanni, L., Sinderen, D.V., Rossi, J. and Corsetti, A., 2005. Rapid differentiation and in situ detection of 16 sourdough Lactobacillus species by multiplex PCR. Applied and Environmental Microbiology 71: 3049-3059. Shevtsov, A.B., Kushugulova, A.R., Tynybaeva, I.K., Kozhakhmetov, S.S., Abzhalelov A.B., Momynaliev K.T. and Stoyanova, L.G., 2011. Identification of phenotypically and genotypically related Lactobacillus strains based on nucleotide sequence analysis of the groEL, rpoB, rplB, and 16S rRNA genes. Microbiology 80: 672-681. Silva, B.C., Jung, L.R.C., Sandes, S.H.C., Alvim, L.B., Bomfim, M.R.Q., Nicoli, J.R., Neumann, E. and Nunes A.C., 2013. In vitro assessment of functional properties of lactic acid bacteria isolated from faecal microbiota of healthy dogs for potential use as probiotics. Beneficial Microbes 4: 267-275. Singh, V.P., Pathak, V. and Verma, A.K., 2012. Fermented meat products: organoleptic qualities and biogenic amines – a review.
Tannock, G.W., Tilsala-Timisjarvi, A., Rodtong, S., Ng, J., Munro, K. and Alatossava, T., 1999. Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons. Applied and Environmental Microbiology 65: 4264-4267. Thirabunyanon, M., and Hongwittayakorn, P., 2013. Potential probiotic lactic acid bacteria of human origin induce antiproliferation of colon cancer cells via synergic actions in adhesion to cancer cells and short-chain fatty acid bioproduction. Applied Biochemistry and Biotechnology 169: 511-525. Tilsala-Timisjarvi, A. and Alatossava, T., 1997. Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. International Journal of Food Microbiology 35: 49-56. Torriani, S., Felis, G.E. and Dellaglio, F., 2001. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA
American Journal of Food Technology 7: 278-288. Souza, M.R., Moreira, J.L., Barbosa, F.H., Cerqueira, M.M., Nunes, A.C. and Nicoli J.R., 2007. Influence of intensive and extensive breeding on lactic acid bacteria isolated from Gallus gallus domesticus ceca. Veterinary Microbiology 120: 142-50.
gene-derived primers. Applied and Environmental Microbiology 67: 3450-3454. Tourova, T.P., 2003. Copy number of ribosomal operons in prokaryotes and its effect on phylogenetic analyses. Microbiology 72: 389-402.
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Lactobacillus species identification by amplified ribosomal 16S-23S rRNA restriction fragment length polymorphism analysis S.H.C. Sandes1#, L.B. Alvim1#, B.C. Silva1, D.F. Zanirati1, L.R.C. Jung1, J.R. Nicoli2, E. Neumann2 and A.C. Nunes1* 1Departamento
de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Campus Pampulha, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil; 2Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Campus Pampulha, Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil; [email protected]; #These authors contibuted equally to this work Received: 18 December 2013 / Accepted: 2 April 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies. Keywords: Lactobacillus, species-level identification, ARDRA, 16S-23S rRNA intergenic region
1. Introduction Lactic acid bacteria (LAB) are often used for food production, preservation, and the generation of desirable organoleptic properties of certain foods (Florou-Paneri et al., 2013; Singh et al., 2012). Many strains have probiotic properties, promoting homeostasis of host intestinal microbiota, assisting in the digestion and assimilation of nutrients (Rabot et al., 2010), protecting against colonisation by pathogens (Gareau et al., 2010), and helping in the treatment of irritable bowel syndrome (Aragon et al., 2010). Among the main LAB genera, Lactobacillus, Lactococcus, Enterococcus and Leuconostoc are frequently isolated and characterised in terms of their physiological and probiotic properties with regard to consumption by animals (Maldonado et al., 2012; Ripamonti et al., 2011; Silva et al., 2013) and humans (Li
et al., 2010; Thirabunyanon and Hongwittayakorn, 2013). Species-level identification is a fundamental prerequisite of such characterisation (FAO/WHO, 2002), but is hindered by the absence of adequate selective media, which allows the co-isolation of different genera. The Lactobacillus genus is extremely diverse, with 190 known species (Euzéby, 2013) that have been identified and characterised by phenotypic and/or genotypic methods. Identification of Lactobacillus by phenotypic methods is difficult because it can require up to 17 phenotypic tests to identify a Lactobacillus isolate accurately to the species level (Tannock et al. 1999). Furthermore, misidentification of probiotic cultures employed in commercial products is frequently seen. For example, species of Lactobacillus gasseri and Lactobacillus johnsonii reported as Lactobacillus
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acidophilus and Lactobacillus casei strains described as Lactobacillus paracasei. Therefore, molecular approaches are regularly used for identification in prospective studies that seek to select new LAB with probiotic properties. Such approaches are not only more accurate but are also less laborious (Moreira et al., 2005). Molecular methods commonly applied include analysis of conserved gene sequences, such as 16S rRNA and rpo (Naser et al., 2007; Shevtsov et al., 2011); PCR amplification using repetitive sequences, such as GTG5 PCR, enterobacterial repetitive intergenic consensus sequence-based PCR, and BOX PCR (Gevers et al., 2001); and amplified ribosomal DNA restriction analysis (ARDRA) (Chenoll et al., 2003; Markiewicz et al., 2010). The use of ARDRA of the 16S-23S internal transcribed spacers (ITS) to identify LAB to the species level is one of the areas of focus of our working group. Microorganisms belonging to Lactobacillus typically exhibit three-length polymorphisms in the region (long, medium, and short spacers) due to the presence or absence of tRNA-encoding sequences (Moreira et al., 2005). Moreira et al. (2005) conducted in silico and in situ analyses of restriction sites in the short spacers of several Lactobacillus isolates, generating a simplified panel that allowed the identification of most of the evaluated microorganisms. However, the evaluation of only a single spacer is not sufficiently informative for the accurate distinction of species belonging to very diverse genera such as Lactobacillus. Therefore, the aim of this study was to describe restriction patterns for all three ITS types (long, medium, and short) in different Lactobacillus strains for application in species-level identification.
2. Materials and methods Bacterial strains, growth conditions, and DNA extraction The LAB strains used in this study and their isolation sources are listed in Table 1. All strains had their taxonomy affiliation confirmed by 16S rRNA gene sequencing as described further. The bacteria were stored at -80 °C in De Man Rogosa Sharpe broth (MRS) (Difco Laboratories Inc., Detroit, MI, USA) or M17 broth (Difco Laboratories Inc.) with 30% glycerol. Before experimental use, cultures were grown for 18 h in accordance with the specific metabolism of each strain, in anaerobic (85% N2, 10% H2 and 5% CO2) or in aerobic conditions, at 28 °C or 37 °C, and in MRS or M17 broth. They were then subcultured twice under the same conditions. Chromosomal DNAs were isolated from LAB cultures in 10 ml MRS or M17 broth. The entire cultures were centrifuged at 8,000×g for 10 min, the bacterial pellets washed with 1 ml of deionised water, suspended in 1 ml of 5 M LiCl, and incubated for 1 h under constant shaking. Cells were washed once more with 1 ml of deionised water and each pellet was resuspended in 1 ml of protoplasting buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 25 mM sucrose) supplemented with lysozyme (10 mg/ml), before incubating for 1 h at 37 °C. The samples were centrifuged once again and the genomic DNA isolated using a NucleoSpin Tissue XS kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s recommendations. DNA yield was quantified by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) at 260 nm, and DNA purity by 260/230 nm and 260/280 nm ratios. DNA
Table 1. Strains and sources of the bacterial species used in this study. Lactobacillus species Culture collection sources L. acidophilus L. alimentarius L. antri L. brevis L. farciminis L. fructivorans L. frumenti L. gastricus L. hilgardii L. kalixensis L. kefiranofaciens L. kefiri L. panis L. paralimentarius L. paraplantarum
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Strain1
Source (origin)
ATCC 4356T ATCC 29643T DSM 16041T ATCC 14869T ATCC 29644T ATCC 8288T DSM 13145T DSM 16045T ATCC 8290T DSM 16043 T ATCC 43761T ATCC 35411T DSM 6035T DSM 13238T ATCC 700211T
human marinated fish product stomach mucosa, human faeces, human sausage spoiled salad dressing rye-bran sourdough stomach mucosa, human wine stomach mucosa, human kefir grains kefir grains sourdough sourdough beer contaminant
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Table 1. Continued. Lactobacillus species
Strain1
Source (origin)
L. pentosus L. plantarum L. pontis L. rhamnosus L. rossiae L. sakei L. sanfranciscensis Human sources L. coleohominis L. crispatus L. gasseri L. iners L. jensenii L. reuteri L. ruminis L. vaginalis L. vaginalis L. delbrueckii Kefir sources L. casei L. diolivorans L. mali L. parafarraginis L. perolens L. satsumensis Animal and industrial sources L. agilis L. animalis L. camelliae L. curvatus L. fermentum L. johnsonii L. plantarum L. reuteri L. salivarius L. vini Other lactic acid bacteria Culture collection sources Pediococcus pentosaceus Streptococcus pyogenes Kefir sources Lactococcus lactis Leuconostoc mesenteroides Oenococcus oeni Human sources Propionibacterium acnes Animal sources Weissella confusa Enterococcus casseliflavus
ATCC 8041T ATCC 14917 T ATCC 51518T DSM 8745 ATCC BAA-822T ATCC 15521T ATCC 27651T
corn silage pickled cabbage rye sourdough endocarditis, human wheat sourdough moto, starter of sake San Francisco sourdough
97 567 452 512 1021 912 032 25C 923 UFV-H2B20
vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human vaginal mucosa, human newborn faeces, human
15U2 1Z 21U2 12P 11P2 2P3
sugar water kefir sugar water kefir sugar water kefir sugar water kefir sugar water kefir milk kefir
1AC41 06 29(L3) 16 V3B-08 B1F-34 1ANG4 B6F-02 B2F-38 TR7.5.7
caecum, chicken faeces, canine bioethanol contaminant mouth, maned wolves mouth, bovine faeces, bovine mouth, porcine faeces, bovine faeces, bovine bioethanol contaminant
ATCC 33314T ATCC 12344T
sake mash throat, human
2U 10U1 4R2
milk kefir milk kefir sugar water kefir
P6
prostate, human
01 14
faeces, equine faeces, equine
1 ATCC
= American Type Culture Collection; DSM = German collection of microorganisms and cell cultures; T = type strain.
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integrity was verified by 0.8% agarose gel electrophoresis as described further.
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PCR amplification of 16S-23S rRNA ITS regions and restriction enzyme digestion The present analysis was based on the ARDRA of Moreira et al. (2005), making use of the three-length polymorphism observed for the 16S-23S rRNA ITS in Lactobacillus species. The amplification was performed using 10 ρmols of 16-1A (5’ GAA TCG CTA GTA ATC G 3’), corresponding to nucleotides 1361 to 1380 of the 16S rRNA gene according to L. casei numbering, and 23-1B (5’ GGG TTC CCC CAT TCG GA 3’), corresponding to nucleotides 123 to 113 of the 23S rRNA of L. casei, primers described by Tilsala-Timisjarvi and Alatossava (1997), PCR Master Mix containing 0.2 mM of each deoxyribonucleotide triphosphate, 1.5 mM of MgCl2, 1.5 units of Taq DNA polymerase (Promega Corporation, Madison, WI, USA) and 10 ng of template DNA. The amplification conditions were as follows: denaturation at 95 °C for 2 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, and a single, final extension at 72 °C for 5 min. The three amplified spacers were digested using the restriction endonucleases SphI, NcoI, NheI, SspI, Csp45I (an isoschizomer of SfuI), EcoRV, DraI, VspI, HincII, EcoRI, HindIII (Promega Corporation), and AvrII (New England Biolabs Inc., Ipswich, MA, USA), using buffer and temperature for optimal activity as recommended by manufacturers. AvrII endonuclease was included because it differentiates L. gasseri, L. johnsonii and Lactobacillus jensenii species, which was impossible in Moreira et al. (2005). Three μl of PCR reaction in 10 μl final volume were directly digested with each enzyme for 3h at 37 °C. The amplified and digested products (10 μl) were electrophoresed in a 1.4% agarose gel and visualised by UV transillumination at 302 nm after staining in 5 μg/ ml ethidium bromide solution using the digital photo documentation system T26M EasyDoc 100 (Bio Agency Biotecnologia LTDA, São Paulo, Brazil).
Grouping of restriction digestion patterns Digitalised images of gels were normalised in BioNumerics V6.5 software (Applied Math, Sint-Martens-Latem, Belgium) by using the molecular weights’ lane to name the reference positions and then the bands in the sample lanes were assigned with the software’s automatic band search followed by manual corrections. Cluster analysis was a two-step process: firstly calculating all pairwise similarity values with a similarity coefficient and then converting the resulting similarity matrix into a dendrogram by the unweighted pair group method using an arithmetical average.
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3. Results We verified the number and size of the 16S-23S rRNA spacer regions of several LAB often co-isolated with Lactobacillus in diversity studies and during prospecting for new probiotic strains. As described previously (Moreira et al., 2005), bacteria belonging to Pediococcus, Propionibacterium, Weissella, and Lactobacillus genera (Figure 1, lanes 6 to 9, respectively) present a pattern of three amplicons. The pattern for Enterococcus is composed of two amplicons (Figure 1, lane 5), and Lactococcus, Leuconostoc, Oenococcus, and Streptococcus show just a single amplicon (Figure 1, lanes 1 to 4, respectively). Next, the enzyme digestion patterns of several previously identified Lactobacillus species were established (Figure 2). Twenty-nine of the 45 evaluated species (64.4%) presented unique digestion patterns, and could therefore be precisely identified to the species level without further testing (Table 2). Other species showed identical patterns between two or more different species: (1) Lactobacillus antri DSM 16041, Lactobacillus panis DSM 6035, Lactobacillus pontis ATCC 51518, and Lactobacillus reuteri B6F-02 (Figure 2.3, Table 2); (2) L. reuteri 912 and Lactobacillus frumenti DSM 13145 (Figure 2.4, Table 2); (3) Lactobacillus paraplantarum ATCC 700211, Lactobacillus pentosus ATCC 8041, and Lactobacillus plantarum 1ANG4 (Figure 2.14, Table 2); (4) Lactobacillus kefiri ATCC 35411, Lactobacillus hilgardii ATCC 8290, and Lactobacillus parafarraginis 12P (Figure 2.16, Table 2); (5) Lactobacillus alimentarius ATCC 29643 and Lactobacillus paralimentarius DSM 13238 (Figure 2.18, Table 2); (6) Lactobacillus sakei ATCC 15521 and Lactobacillus curvatus 16 (Figure 2.22, Table 2); and (7) Lactobacillus crispatus 567 and Lactobacillus kefiranofaciens ATCC 43761 (Figure 2.28, Table 2). Moreover, the species L.
1
2
3
4
5
6
7
8
9
M (bp) 1000 750 500 250
Figure 1. The 16S-23S amplicon patterns of genera commonly co-isolated with Lactobacillus in MRS medium. The forward and reverse primers anneal in the 5’ region of the 16S gene and the 3’ region of the 23S gene, respectively, amplifying the entire spacer region and highlighting the size polymorphisms in each of the evaluated genera. 1, Lactococcus lactis 2U; 2, Leuconostoc mesenteroides 10U1; 3, Oenococcus oeni 4R2; 4, Streptococcus pyogenes ATCC 19615; 5, Enterococcus casseliflavus 14; 6, Pediococcus pentosaceus ATCC 33314; 7, Propionibacterium acnes P6; 8, Weissella confusa 01; 9, Lactobacillus johnsonii B1F-34 (control); M, size marker (1 Kb DNA Ladder). Beneficial Microbes 5(4)
Lactobacillus typing by ITS ARDRA
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1650 1000 850 650 500 400 300 200
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
2.1. L. gastricus DSM 16045
2.2. L. farciminis ATCC 29644
2.3. L. antri DSM 16041; L. panis DSM 6035; L. pontis ATCC 51518; L. reuteri B6F-02
2.4. L. reuteri 912; L. frumenti DSM 13145
2.5. L. vaginalis 25C
2.6. L. vaginalis 923
2.7. L. coleohominis 97
2.8. L. fermentum V3B-08
2.9. L. sanfranciscencis ATCC 27651
2.10. L. agilis 1AC41
2.11. L. satsumensis 2P3
2.12. L. fructivorans ATCC 8288
2.13. L. mali 21U2
2.14. L. paraplantarum ATCC 700211; L. pentosus ATCC 8041; L. plantarum 1ANG4
2.15. L. plantarum ATCC 14917
2.16. L. kefiri ATCC 35411; L. hilgardii ATCC 8290; L. parafarraginis 12P
2.17. L. camelliae 29(L3)
2.18. L. alimentarius ATCC 29643; L. paralimentarius DSM 13238
2.19. L. animalis 06
2.20. L. rossiae ATCC BAA-822
2.21. L. vini TR7.5.7
2.22. L. sakei ATCC 15521; L. curvatus 16
2.23. L. diolivorans 1Z
2.24. L. brevis ATCC 14869
Figure 2. Restriction patterns of the 16S-23S ITSs (long, medium, and short) of 45 Lactobacillus species previously identified to the species level. The amplified spacer regions were digested by 12 different restriction enzymes, all of which recognise six-base sites. Since these enzymes cut less often than four-base cutters, they generate simplified restriction patterns from the amplicons. For each image, from left to right, the restriction enzymes used were SphI, NcoI, NheI, SspI, Csp45I, EcoRV, DraI, VspI, HincII, EcoRI, HindIII, and AvrII; first lane (M) was 100 bp DNA ladder. Beneficial Microbes 5(4)
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1650 1000 850 650 500 400 300 200
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
M 1 2 3 4 5 6 7 8 9 10 11 12
2.25. L. kalixensis DSM 16043
2.26. L. delbrueckii UFV-H2B20
2.27. L. acidophilus ATCC 4356
2.28. L. crispatus 567; L. kefiranofaciens ATCC 43761
2.29. L. salivarius B2F-38
2.30. L. ruminis 32
2.31. L. casei 15U2
2.32. L. rhamnosus DSM 8745
2.33. L. iners 512
2.34. L. gasseri 452
2.35. L. johnsonii B1F-34
2.36. L. jensenii 1021
2.37. L. perolens 11P2
Figure 2. Continued.
reuteri (Figures 2.3 and 2.4, Table 2), Lactobacillus vaginalis (Figures 2.5 and 2.6, Table 2), and L. plantarum (Figures 2.14 and 2.15, Table 2) presented distinct digestion patterns for different strains. Clustering of the species based on the 16S-23S rRNA ARDRA patterns was compared with grouping in a phylogenetic tree based on 16S rRNA gene sequence analysis done by Salvetti et al. (2012). Eight well-defined groups were observed: L. reuteri, L. plantarum, L. salivarius, L. casei, L. alimentarius, L. sakei, Lactobacillus buchneri, and Lactobacillus delbrueckii groups (Figure 3). The 16S-23S rRNA ARDRA pattern similarity did not cluster some closely related species belonging to the same phylogenetic group. For example, Lactobacillus fructivorans and Lactobacillus sanfranciscensis, both belonging to the L. fructivorans group, and Lactobacillus gastricus and Lactobacillus animalis, belonging, respectively, to the L. reuteri and L. salivarius groups, were grouped with 16S rRNA phylogenetically unrelated species. Due to an insufficient number of related species, Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus perolens could not be classified into groups. 476
4. Discussion Evaluation of amplified DNA polymorphisms and partial sequences of the ribosomal RNA operon (rrn) are frequently used to identify bacteria at the species level. This operon is organised as follows: 16S – spacer region 1 – 23S – spacer region 2 – 5S, and is responsible for 16S, 23S, and 5S rRNA synthesis. Since it is highly conserved, homologous, and stable (without horizontal gene transfer), it is often used for evolutionary studies (Felis and Dellaglio, 2007). Nonetheless, the rrn copy number in the bacterial genome is variable (Tourova, 2003), and some non-coding regions, such as the 16S-23S rRNA ITS regions, can acquire and accumulate mutations without compromising the general bacterial metabolism. Therefore, the 16S-23S ITS is recommended for microdiversity analysis, which is used for groups of individuals with high genetic affinity. To address diversity among phylogenetically distant isolates, amplified 16S or 23S rRNA fragments can be used. These genes generate simple banding patterns, depending on the restriction enzyme used (Junior et al., 2004).
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Lactobacillus typing by ITS ARDRA
Table 2. 16S-23S intergenic spacers (long, medium and short) restriction patterns of 45 Lactobacillus species previously identified to species level by 16S rRNA gene sequencing.1
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Endonuclease SphI
NcoI
NheI
SspI
Csp45I EcoRV DraI
VspI
HincII
EcoRI HindIII AvrII
Lactobacillus species
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
––– +–– +++ +++ +++ +++ +++ ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– +–– +++ +++ –––
––– ––– ––– ––– ––– ––– +++ ––– ––– +++ +++
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
––– +++ ––– ––– ––– ––– ––– ––– +++ ––– +++
––– +++ +++ +++ +++ +++ +++ +++ +++ +++ –––
––– +–– ––– +–– +–– ––– ––– ––– ––– ––– +––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
––– +++ ––– ––– ––+ ––+ ––– ––– +++ ––– –––
––– +++ ––– ––– ––– ––– ––– ––– ––– ––– –––
L. gastricus L. farciminis L. antri, L. panis, L. pontis, L. reuteri 2 L. reuteri 2, L. frumenti L. vaginalis 2 L. vaginalis 2 L. coleohominis L. fermentum L. sanfranciscensis L. agilis L. satsumensis
+++ +++ +++ +++ +++ +++
––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– –––
+++ +++ +++ +++ +++ –––
––– ––– ––– +–– ––– +––
––– ––– ––– ––– ––– +++
+++ +++ ––– ––– ––– +++
+++ ––– +++ +++ +++ –––
+–– ––– +++ +++ +++ –––
––– ––– ––– ––– ––– –––
+++ ––– ––– ––– +++ +++
––– ––– ––– ––– ––– –––
L. fructivorans L. mali L. plantarum 2, L. paraplantarum, L. pentosus L. plantarum 2 L. parafarraginis, L. kefiri, L. hilgardii L. camelliae
+++ +++ +++ +++ +++ +++ +++ ––– ––– ––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– +++ +++ +++ +++ ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– ––+ ––– ––– ––– ––– ––– ––– –––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– +++ +++ ––– –––
––– ––– ––– ––– ––– ––– ––– +–– +++ +–– +–– ––– +++ ––– –––
––– ––– ––– ––– ––– ––– ––– ––– ––– ––– +++ ––– ––– +++ +++
+++ +++ +++ +++ +++ ––– ––– +–– ––– ––– ––– +++ +++ +++ +++
+++ +++ ––– ––– ––– +++ +++ ––– ––– ––– ––– ––– ––– +++ –––
+–– +–– +–– ––– ––– +++ ––– ––– ––– ––– ––– ––– +–– ––– +––
––– ––– ––– –+– ––– ––– ––– ––– ––– +++ +++ ––– ––– ––– –––
+++ +++ +++ ––– +++ +++ +++ +++ ––– +++ +++ ––– ––– +++ +++
+++ ––– ––– ––– ––– ––– ––– ––– ––– +++ +++ ––– ––– ––– –––
L. alimentarius, L. paralimentarius L. animalis L. rossiae L. vini L. sakei, L. curvatus L. diolivorans L. brevis L. kalixensis L. delbrueckii L. acidophilus L. kefiranofaciens, L. crispatus L. salivarius L. ruminis L. casei, L. paracasei L. rhamnosus
––– ––– ––– –––
––– ––– ––– –––
––– ––– ––– –––
––– ––– ––– –––
––– ––– ––– –––
+++ +++ +++ +++
––– ––– ––– –––
––– ––– ––– –––
+–– ––– ––– –––
––– ––– ––– –––
––– +–– +–– –––
+++ +++ ––– –––
L. iners L. gasseri L. johnsonii L. jensenii
–––
–––
–––
–––
–––
–––
–––
–––
+––
–––
+++
+++
L. perolens
1 The 2
plus and minus signs refers, respectively, to positive and negative digestion for long, medium and short spacers for a specific enzyme. L. reuteri, L. vaginalis and L. plantarum present two distinct restriction patterns.
In Lactobacillus, Weissella, Propionibacterium, and Pediococcus genera, sequence insertions encoding tRNAs in some copies of the 16S-23S spacer region generate characteristic patterns of three amplicons: short (without insertion), medium (tRNA-Ala insertion), and long (tRNAAla and tRNA-Ile insertions). These patterns, along with cultivation in MRS medium and morphological and Beneficial Microbes 5(4)
colorimetric analyses, such as Gram staining, can be used to isolate microorganisms within these genera in a specific and reproducible way. They are therefore of use in both studies of bacterial diversity and in prospective study for new probiotic strains (Moreira et al., 2005).
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60
70
80
90
100 L. antri DSM 16041 L. panis DSM 6035 L. pontis ATCC 51518 L. reuteri B6F-02 L. vaginalis 25C L. vaginalis 923 L. reuteri 912 L. frumenti DSM 13145 L. coleohominis 97 L. fermentum V3B-08 L. brevis ATCC 14869
L. reuteri group
L. brevis group
L. plantarum ATCC 14917 L. plantarum 1ANG4 L. paraplantarum ATCC 700211 L. pentosus ATCC 8041
L. plantarum group
L. gastricus DSM 16045
L. reuteri group
L. vini TR7.5.7 L. ruminis 032 L. agilis 1AC41 L. satsumensis 2P3 L. mali 21U2 L. salivarius B2F-38
L. salivarius group
L. casei 15U2 L. rhamnosus DSM 8745
L. casei group
L. camelliae 29(L3)
Single species group
L. alimentarius ATCC 29643 L. paralimentarius DSM 13238 L. farciminis ATCC 29644
L. alimentarius group
L. sanfranciscencis ATCC 27651 L. animalis 06 L. rossiae ATCC BAA-822
L. fructivorans group L. salivarius group Couples group
L. sakei ATCC 15521 L. curvatus 16
L. sakei group
L. fructivorans ATCC 8288 L. diolivorans 1Z L. kefiri ATCC 35411 L. hilgardii ATCC 8290 L. parafarraginis 12P
L. fructivorans group L. buchneri group
L. perolens 11P2
L. perolens group
L. jensenii 1021 L. johnsonii B1F-34 L. gasseri 452 L. iners 512 L. delbrueckii UFV-H2B20 L. kalixensis DSM 16043 L. acidophilus ATCC 4356 L. kefiranofaciens ATCC 43761 L. crispatus 567
L. delbrueckii group
Figure 3. The amplified ribosomal DNA restriction analysis patterns of Lactobacillus species for the 16S-23S ITS regions were grouped by similarity and compared with a phylogenetic tree based on 16S rRNA gene sequence analysis. The dotted frames denote grouping of species based on phylogenetic analysis of 16S rRNA gene sequences. 478
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The main study focus of our working group is the selection of new probiotic strains, especially Lactobacillus, for use as food supplements or vaccine vehicles for livestock (Mota et al., 2006; Souza et al., 2007). Accurate specieslevel identification is a fundamental requirement (FAO/ WHO, 2002) that is hindered by the great diversity of this genus, including a large number of closely related species (Felis and Dellaglio, 2007). Lactobacillus is composed of 190 species and 28 subspecies (Euzéby, 2013), some of which may share more than 99% similarity between 16S rRNA sequences (Mañes-Lázaro et al., 2009), necessitating more discriminative methods of genomic analysis (Felis and Dellaglio, 2007). Moreira et al. (2005) were able to distinguish 43 strains of only 17 Lactobacillus species (almost 1/3 of species used in the present work). Over the past 8 years, our group has used analysis based on 16S-23S rRNA spacer restriction patterns to identify, to the species level, more than five hundred strains of 45 species of Lactobacillus from different animals, humans, and food sources. To accomplish this, we used 12 different six-base cutter endonucleases. This approach allowed us to establish a simplified restriction pattern for each species evaluated, compared with four-base cutters. As already noted, a significant number of Lactobacillus species (29/45) can be effectively typed on the basis of the restriction patterns of their amplified 16S-23S ITS regions, including closely related species that share more than 97% similarity between their 16S rRNA sequences, such as L. casei and L. rhamnosus, and L. gasseri and L. johnsonii (Felis and Dellaglio, 2007; Salvetti et al., 2012). Compared with the method described by Moreira et al. (2005), simultaneous digestion of the three ITSs was able to increase discriminative capacity, allowing increased accuracy in species-level identification through the formation of unique patterns. This was observed for Lactobacillus farciminis, L. vaginalis, Lactobacillus satsumensis, Lactobacillus mali, L. plantarum, L. rossiae, Lactobacillus iners, L. gasseri, L. johnsonii, and L. jensenii. Nevertheless, for some species, restriction patterns were not sufficiently informative for accurate identification; they could only indicate the general group to which the species belonged. For these cases, species distinction can be established using other approaches. For example, (1) L. reuteri can be differentiated from L. antri, L. panis, L. pontis, and L. frumenti by 16S rRNA gene sequence analysis, and the remaining four species may be differentiated from each other by PCR using species-specific primers in the 16S-23S ITS region (Ferchichi et al., 2008); (2) L. paraplantarum, L. pentosus, and L. plantarum can be differentiated by multiplex PCR assay with recA gene-derived primers (Torriani et al., 2001); (3) L. kefiri, L. hilgardii, and L. parafarraginis can be differentiated by fermentation profiles analysis using L-arabinose and D-xylose (Endo and Okada, 2007); (4) L. alimentarius and L. paralimentarius can be Beneficial Microbes 5(4)
Lactobacillus typing by ITS ARDRA
differentiated by two-step multiplex PCR using speciesspecific primers in the 16S-23S ITS, or 16S and 23S coding regions (Settanni et al., 2005); (5) L. sakei and L. curvatus can be differentiated by multiplex PCR-based restriction enzyme analysis (Lee et al., 2004); and (6) L. crispatus and L. kefiranofaciens can be differentiated by 16S rRNA coding sequence analysis. According to Salvetti et al. (2012), Lactobacillus shows a phylogenetic structure that is quite complicated. It is paraphyletic with Pediococcus, and includes 15 phyletic groups: (1) L. delbrueckii, (2) L. salivarius, (3) L. reuteri, (4) L. buchneri, (5) L. alimentarius, (6) L. casei, (7) L. sakei, (8) L. fructivorans, (9) Lactobacillus coryniformis, (10) L. plantarum, (11) L. perolens, (12) L. brevis, (13) Lactobacillus collinoides, (14) Lactobacillus manihotivorans and (15) Lactobacillus vaccinostercus groups. In addition, the genus includes two ‘artificial’ groups: (1) couple species (represented by the pairs L. rossiae/Lactobacillus siliginis, L. concavus/Lactobacillus dextrinicus, Lactobacillus kunkeei/Lactobacillus ozensis, and Lactobacillus pantheris/ Lactobacillus thailandensis) and (2) single species (represented by Lactobacillus algidus, Lactobacillus brantae, Lactobacillus camelliae, Lactobacillus composti, Lactobacillus floricola, Lactobacillus malefermentans, Lactobacillus saniviri, Lactobacillus selangorensis, Lactobacillus senioris, and Lactobacillus sharpeae). Based on this, we propose to group several species according to similarity of 16S-23S rRNA restriction patterns. Coding regions of the rrn operon, such as that encoding 16S rRNA, are highly homologous between species. By contrast, the ITS regions are able to accumulate mutations, and are therefore more variable. We observed that the restriction analysis based on the ITS region formed groups similar to those based on 16S rRNA sequences for most of the species, indicating that ARDRA of this region may be appropriate for evolutionary studies. However, L. gastricus clustered far away from the L. reuteri group. L. gastricus is a poorly characterised species at the molecular level. Few strains had their rrn genes sequenced and only recently the genome has been sequenced for one strain. We tried to get the 1623 rRNA sequences from this genome project but were unsuccessful. We had sequenced the 16S rRNA gene before the start of ARDRA, so we are sure that it is L. gastricus DSM 16045 from the DSMZ culture collection. Therefore, the reason that L. gastricus does not group in the L. reuteri clade might be because the initial grouping was based on the 16S rRNA (a conserved gene), while here we are dealing with the three ITS-1 regions of all rrn operons (which are probably more polymorphic between the operons than the 16S rRNA genes).
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5. Conclusions Restriction patterns generated from the 16S-23S ITS regions enabled the identification of 29 species of Lactobacillus. For an additional 16 species, identity could be established to the level of groups of closely related species. This approach, besides being reproducible, produced simplified patterns that grouped the species into clusters that reflected, to a large extent, their phylogenetic relationships as determined by 16S rRNA sequences. Our results supported the well-established advantages of using this region for evolutionary studies.
Acknowledgments The following Brazilian financing programs or institutions supported our work: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). CAPES supported S.H.C. Sandes, L.B. Alvim, and B.C. Silva; FAPEMIG granted L.R.C. Jung and D.F. Zanirati.
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