Folia Microbiol DOI 10.1007/s12223-014-0353-z

Evaluation of biochemical and molecular methods for Lactobacillus reuteri strains differentiation Bilková Andrea & Kiňová Sepová Hana & Dubničková Martina & Májeková Hyacinta & Bilka František

Received: 17 March 2014 / Accepted: 24 September 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014

Abstract Several biochemical and molecular methods were used for discrimination of four Lactobacillus reuteri strains isolated from goatling and lamb stomach mucosa. Internal transcribed spacer (ITS)-PCR method and protein analysis by SDS-PAGE and MALDI-TOF showed to be suitable for strain discrimination whereas ITS-PCR/RFLP and enterobacterial repetitive intergenic consensus (ERIC)-PCR were not strain specific. The used methods differentiated tested strains into distinct groups; however, the location of strains in groups varied. Consistency in results was observed in the case of L. reuteri E and L. reuteri KO4m that were clustered into the same groups using all techniques, except of MALDI-TOF MS. The last one grouped goatling strains and lamb isolate into separate clusters. All investigated methods, except of ITS-PCR/RFLP and ERIC-PCR, were assessed as appropriate for distinguishing of L. reuteri strains.

Introduction Lactobacilli have been widely used in fermentation and food conservation for centuries. Nowadays, there is focus on their beneficial effects on human and animal health. They are used for elimination of problems related to digestion and uptake of nutrition; for instance, they modulate the composition of gastrointestinal microbiota and function of host immune system; moderate the postantibiotic (Sepp et al. 2011) and traveler’s diarrhea (Lin et al. 2009), lactose intolerance (Ibarra et al. 2012), inflammatory bowel diseases (Crohn’s disease (Van Gossum et al. 2007), ulcerative colitis (Schultz et al. B. Andrea (*) : K. S. Hana : D. Martina : M. Hyacinta : B. František Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic e-mail: [email protected]

2004)), and hypercholesterolemia (Simons et al. 2006). The health benefits are strain dependent, and therefore, accurate identification and differentiation of each strain is essential prior to probiotic application. To date (May 2014), 201 species and 29 subspecies were identified in genus Lactobacillus (Euzéby 2014). Identification techniques have been markedly modified since the time when the first species was described (Beijerinck 1901). In the twentieth century, classical microbiological and biochemical methods were preferably used (cell morphology, API 50 CHL, enzymatic activities) (Zheng et al. 2013). Nowadays, mostly molecular methods are employed in bacterial identification, and additionally, classical methods are being used. Majority of the molecular methods are based on DNA analysis: DNADNA hybridization; various PCR-based techniques (e.g., PCR product sequencing, qPCR, PCR-DGGE, rep-PCR, ITS-PCR, ERIC-PCR, RAPD-PCR, AFLP), comparison of restriction fragments length (RFLP, ARDRA); or protein analysis (SDS-PAGE, MALDI-TOF MS, isoelectric focusing) (de Magalhães et al. 2008; Dušková et al. 2012; Herbel et al. 2013; Jurkovič et al. 2006; Mikasová et al. 2005; Mohania et al. 2008; Nováková et al. 2013). The 16S-23S rDNA sequence analysis is exploited very often as well. Many of the mentioned techniques are suitable even for closely related species differentiation, however, with different sensitivity and accuracy. L. reuteri strains are common in human and warm-blooded animal gut microflora, and many strains are also used in probiotic preparations, food or feed. In the present study, we compare the discriminative power of several methods employed in L. reuteri strain differentiation. Each technique has its specific advantages and limitations that we try to confront. Four studied L. reuteri isolates originated from lamb and goatling were identified on species level previously and are the subject of research for their potential probiotic properties. All strains assigned diverse phenotypical features

Folia Microbiol

(Bilková et al. 2011; Kiňová Sepová et al. 2011; Kiňová Sepová and Bilková 2013).

Material and methods Bacterial cultures and growth media Lactobacillus reuteri Strains were isolated from stomach mucosa of breast-fed lamb (strain E; breeding station Očová, Slovakia) and goatling (strains KO4b, KO4m, KO5; breeding station Teplý Vrch, Slovakia). They were already identified and partly characterized as published elsewhere (Kiňová Sepová et al. 2011; Kiňová Sepová and Bilková 2013). For comparison, collection strains were included: Lactobacillus reuteri CCM 3625, L. reuteri ATCC 55845, L. reuteri DSM 20056, L. reuteri DSM 20016T, L. reuteri DSM 20015, L. reuteri DSM 8534, L. reuteri DSM 20053, L. reuteri CCM 3642. Lactobacilli were grown overnight in de Man-Sharpe-Rogosa (MRS) broth (Oxoid, Great Britain) at 37 °C in anaerobic conditions. Isolation of bacterial DNA For DNA isolation, DNeasy Tissue Kit (Qiagen GmbH, Germany) was used. Procedure was performed according to manufacturer’s recommendations. ITS-PCR Combination of PCR primers ITSf (5′-CAAGGC ATCCACCGT-3′; Jensen et al. 1993) and ITSr (5′-GTGAAG TCGTAACAAGG-3′; Drahovská et al. 2002) was used, and PCR conditions were the following: denaturation (94 °C/60 s), 30 cycles of denaturation (90 °C/30 s), annealing (54 °C/30 s) and polymerization (72 °C/60 s), and final polymerization (72 °C/8 min). ITS-PCR/RFLP For PCR amplification, primers Cy5-16S 1500F (5′-AAGTCGTAACAAGGTA-3′) and 23S-32R (5′GCCA(A/G)GGCATGGACC-3′) and PCR conditions according to Jacobsen et al. (1999) were used. Obtained PCR products were digested with restriction enzyme TaqI (65 °C/ 16 h). ERIC-PCR ERIC-PCR was performed according to Ventura and Zink (2002) using primers ERIC-I (5′-ATGTAAGCTC

CTGGGGATTCAC-3′) and ERIC-II (5′-AAGTAAGTGACT GGGGTGAGCG-3′). Electrophoresis of PCR products PCR products were separated in 2 % agarose gel containing 1×TAE and visualized by GoldView stain (SBS Genetech, China) or ethidium bromide at 254 nm. SDS-PAGE of whole-cell proteins Isolation of whole-cell proteins was performed according to Piraino et al. (2002). For each strain, three independent extracts were derived and analyzed. Proteins were stored at −20 °C until analysis in SDSPAGE. Electrophoretic separation of proteins was performed in 10 % polyacrylamide gel according to Laemmli (1970). In each lane, 10 μg of proteins were loaded. MALDI-TOF MS Samples for analysis were gained from three independent cultivations. Procedure was accomplished according to Freiwald and Sauer (2009). Cells were suspended in water/ethanol mixture (1:3) and centrifuged afterwards (12,000g, 2 min). Supernatants were eliminated, and samples were sent for analysis to Central Laboratory of Special Techniques (Department of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Brno, Czech Republic). Statistical analysis Cluster analysis of data was done with Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Data were processed with GelCompar II/ BioNumerics (Applied Math, Belgium). All experiments were done in three independent replicates to provide reproducible results.

Results and discussion In the present work, we focused on the comparison of efficiency of several biochemical and molecular-based methods for L. reuteri strains differentiation. Methods evaluated in this study are based on comparison of DNA fragments (ITS-PCR, ITS-PCR/RFLP, ERIC-PCR)

Fig. 1 ITS-PCR. Dendrogram based on the UPGMA cluster analysis. Level of similarity was set at 92 %

Folia Microbiol

Fig. 2 Fingerprinting of whole-cell proteins. Level of similarity based on UPGMA analysis was set at 77 %

or proteins (SDS-PAGE of whole-cell proteins, MALDI-TOF MS). To get more complex outcomes, the results of previous study employing amplified fragment length polymorphism (AFLP) (Kiňová Sepová and Bilková 2013) are included. Intergenic transcribed spacers located between rRNA genes are usually variable in length through individual bacterial species, and several authors declare the possibility of strain discrimination using the study of their 16S-23S region (Berthier and Ehrlich 1998; Jensen et al. 1993). Our results of ITS-PCR are consistent with these data; used experimental conditions seemed to be suitable for L. reuteri differentiation. UPGMA analysis with level of similarity 92 % clustered studied strains into two groups (Fig. 1). However, several of strain-specific bands are weak, and therefore, they may be irreproducible after mild modification of PCR conditions. The

primer set L1/G1 (that corresponds to ITSf and at 5′ end two nucleotides longer ITSr, respectively) was successfully used for distinguishing of L. reuteri strains by Dec et al. (2014). This is in contrast to in silico analysis (Chebeňová-Turcovská et al. 2011), which uncovered that primer L1 contains mismatches with several lactic acid bacteria, including some species of lactobacilli. Both ITS-PCR/RFLP and ERIC-PCR did not provide satisfactory results. On the basis of obtained PCR product’s profiles, used software was not able to distinguish tested strains (data not shown). However, Ventura and Zink (2002), using the same conditions as were used in this study, recommend ERIC-PCR as a powerful method for discrimination of L. johnsonii strains. Other authors stated ITS-PCR/RFLP to be suitable for Lactobacilli species identification (Yavuz et al. 2004), while De las Rivas et al. (2006) do not recommend it

Fig. 3 MALDI-TOF MS dendrogram based on UPGMA analysis. Distance level was set at value 223 (corresponds to the level of similarity ca 77 %)

Folia Microbiol

for strain differentiation and propose to use more discriminating methods. SDS-PAGE of whole-cell (Ghazi et al. 2009) and cell-wall associated proteins (Dimitrov et al. 2005) are phenotypic methods widely used for lactic acid bacteria identification and taxonomic discrimination at species and subspecies level (Ghazi et al. 2009). When the standardized conditions of SDSPAGE are fulfilled, patterns of whole-cell proteins are extremely unfailing and may be correlated with DNA-DNA hybridization (Vandamme et al. 1996). SDS-PAGE of whole-cell proteins gave strain specific fingerprints of compared L. reuteri isolates, and according to their similarity, they were divided into two groups (level of similarity 77 %); goatling strain L. reuteri KO5 was clustered individually (Fig. 2). Since identification and differentiation of lactic acid bacteria require polyphasic approach (Ghazi et al. 2009), SDS-PAGE of whole-cell proteins represents one of the phenotypic methods that can be used. MALDI-TOF MS is a technique that analyzes exact molecular mass of peptides and small proteins mainly of ribosomal origin (Lartigue 2013). Nowadays, MALDI-TOF MS becomes widely used in bacterial identification and classification (Fenselau and Demirev 2001; Lay 2001), as well as in clinical practice (Wolters et al. 2011). Strain specific MS spectra were observed in all L. reuteri strains tested in this study (data not shown). UPGMA analysis at level of similarity ca 77 % grouped them into three main clusters: collection strains, goatling isolates, and lamb isolate were clustered separately (Fig. 3). Out of three independent cultivations of L. reuteri E, one (E II) was clustered in separate group probably due to the presence of unidentifiable impurity. In our previous study, ALFP analysis separated the investigated L. reuteri strains into two groups (I. group: L. reuteri E and L. reuteri KO4m; II. group: L. reuteri KO4b, L. reuteri KO5 and L. reuteri CCM 3625) on the basis of similarity level 77 % (Kiňová Sepová and Bilková 2013). Results present in our study showed that the clustering of strains was not cohesive in all methods used. In our conditions, two PCR-based methods, ERIC-PCR and ITS-PCR/ RFLP were not suitable for strain discrimination. All other methods showed unambiguous discriminative power with the SDS-PAGE and MALDI-TOF MS being more sensitive than ITS-PCR and AFLP. However, low degree of consistency in grouping was observed. L. reuteri E and L. reuteri KO4m were clustered in the same groups by all methods with exception of MALDI-TOF MS, but clustering of L. reuteri KO4b and L. reuteri KO5 demonstrated higher variability. The choice of certain method could depend on the possibilities and limits of laboratory. To obtain more precise results, we recommend to couple phenotypic and molecular approaches (polyphasic approach) for differentiation of Lactobacillus reuteri strains.

Acknowledgments We would like to kindly acknowledge Dr. Epp Songisepp (Institute of Microbiology, University of Tartu, Estonia) for providing L. reuteri ATCC 55845 used in ERIC-PCR analysis. Conflict of interest None

References Beijerinck MW (1901) Sur les ferments lactiques de l’industrie. Archives Néerlandaises des Sciences Exactes et Naturelles (Section 2) 6:212– 243 Berthier F, Ehrlich SD (1998) Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. FEMS Microbiol Lett 161:97– 106 Bilková A, Kiňová Sepová H, Bukovský M, Bezáková L (2011) Antibacterial potential of lactobacilli isolated from a lamb. Vet Med 56:319–324 Chebeňová-Turcovská V, Ženišová K, Kuchta T, Pangallo D, Brežná B (2011) Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese. Int J Food Microbiol 150:73–78 De las Rivas B, Marcobal A, Muñoz R (2006) Development of multilocus sequence typing method for analysis of Lactobacillus plantarum strains. Microbiology 152:85–93 de Magalhães JT, Uetanabaro APT, de Moraes CA (2008) Identification of Lactobacillus UFV H2B20 (probiotic strain) using DNA-DNA hybridization. Braz J Microbiol 39:542–546 Dec M, Urban-Chmiel R, Gnat S, Puchalski A, Wernicki A (2014) Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis. Res Microbiol 165:190–201 Dimitrov Z, Michaylova M, Mincova S (2005) Characterization of strains isolated from Bulgarian yoghurt, cheese, plants and human faecal samples by sodium dodecilsulfate polyacrylamide gel electrophoresis of cell-wall proteins, ribotyping and pulse filed gel fingerprinting. Int Dairy J 15:998–1005 Drahovská H, Kocíncová D, Seman M, Turňa J (2002) PCR-based methods for identification of Enterococcus species. Folia Microbiol 47:649–653 Dušková M, Šedo O, Kšicová K, Zdráhal Z, Karpíšková R (2012) Identification of lactobacilli isolated from food by genotypic methods and MALDI-TOF MS. Int J Food Microbiol 159:107–114 Euzéby F (2014) List of prokaryotic names with standing in nomenclature. http://www.bacterio.net/lactobacillus.html (5/May/2014) Fenselau C, Demirev PA (2001) Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 20:157– 171 Freiwald A, Sauer S (2009) Phylogenetic classification and identification of bacteria by mass spectrometry. Nat Prot 4:732–742 Ghazi F, Henni DE, Benmechernene Z, Kihal M (2009) Phenotypic and whole cell protein analysis by SDS-PAGE for identification of dominants lactic acid bacteria isolated from Algerian raw milk. World J Diary Food Sci 4:78–87 Herbel SR, Vahjen W, Wieler LH, Guenther S (2013) Timely approaches to identify probiotic species of the genus Lactobacillus. Gut Pathogens 5:http://www.gutpathogens.com/content/5/1/27 (17/ January/2014) Ibarra A, Acha R, Calleja MT, Chiral-Boix A, Witting E (2012) Optimization and shelf life of a low-lactose yogurt with Lactobacillus rhamnosus HN001. J Sci Dairy 95:3536–3548 Jacobsen CN, Rosenfeldt Nielsen V, Hayford AE, Møller PL, Michaelsen KF, Pærregaard A, Sandström B, Tvede M, Jakobsen M (1999) Screening of probiotic activities of forty-seven strains of

Folia Microbiol Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in human. Appl Environ Microbiol 65:4949–4956 Jensen MA, Webster JA, Straus N (1993) Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphism. Appl Environ Microbiol 59:945–952 Jurkovič D, Križková L, Sojka M, Belicová A, Dušinský R, Krajčovič J, Snauwaert C, Naser S, Vandamme P, Vancanneyt M (2006) Molecular identification and diversity of enterococci isolated from Slovak bryndza cheese. J Gen Appl Microbiol 52:329–337 Kiňová Sepová H, Bilková A (2013) Isolation and identification of new lactobacilli from goatling stomach and investigation of reuterin production in Lactobacillus reuteri strains. Folia Microbiol 58:33– 38 Kiňová Sepová H, Dubničková M, Bilková A, Bukovský M, Bezáková L (2011) Identification and biological activity of potential probiotic bacterium isolated from the stomach mucus of breast-fed lamb. Braz J Microbiol 42:1188–1196 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 Lartigue MF (2013) Matrix-assisted laser desorption ionization time-offlight mass spectrometry for bacterial strain characterization. Infect Genet Evol 13:230–235 Lay JO (2001) MALDI-TOF mass spectrometry of bacteria. Mass Spectrom Rev 20:172–194 Lin PP, Hsieh YM, Tsai CC (2009) Antagonistic activity of Lactobacillus acidophilus RY2 isolated from healthy infancy feces on the growth and adhesion characteristics of enteroaggregative Escherichia coli. Anaerobe 15:122–126 Mikasová E, Oravcová K, Kaclíková E, Kuchta T, Drahovská H (2005) Typing of food-borne Listeria monocytogenes by polymerase chain reaction—restriction enzyme analysis and amplified fragment length polymorphism. New Microbiol 28:265–270 Mohania D, Nagpal R, Kumar M, Bhardwaj A, Yadav M, Jain S, Marotta F, Singh V, Parkash O, Yadav H (2008) Molecular approaches for identification and characterization of lactic acid bacteria. J Dig Dis 9:190–198 Nováková D, Švec P, Kukletová M, Žáčková L, Sedláček I (2013) Evaluation of the strain identity between isolates from caries lesions

and root canals in early childhood caries cases. Folia Microbiol 58: 649–656 Piraino P, Ricciardi A, Lanorte MT, Malkhazova I, Parente E (2002) A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins. Biotechnol Lett 24:1477–1482 Schultz M, Timmer A, Herfarth HH, Sartor RB, Vanderhoof JA, Rath HC (2004) Lactobacillus GG in inducing and maintaining remission of Crohn’s disease. BMC Gastroenterol 4:doi: 10.1186/1471-230X-4-5 Sepp E, Štšepetova J, Smidt I, Rätsep M, Kõljalg S, Lõivukene K, Mändar R, Jaanimäe L, Löhr HI, Natås OB, Naaber P (2011) Intestinal lactoflora in Estonian and Norwegian patients with antibiotic associated diarrhea. Anaerobe 17:407–409 Simons LA, Amansec SG, Conway P (2006) Effect of Lactobacillus fermentum on serum lipids in subjects with elevated serum cholesterol. Nutr Metab Cardiovasc Dis 16:531–535 Van Gossum A, Dewit O, Louis E, de Hertogh G, Baert F, Fontaine F, de Vos M, Enslen M, Paintin M, Franchimont D (2007) Multicenter randomized-controlled clinical trial of probiotics (Lactobacillus johnsonii, LA1) on early endoscopic recurrence of Crohn’s disease after ileo-caecal resection. Inflamm Bowel Dis 13:135–142 Vandamme P, Pot B, Gillis M, de Vos P, Kersters K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Mol Biol Rev 60:407–438 Ventura M, Zink R (2002) Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis. FEMS Microbiol Lett 217: 141–154 Wolters M, Rohd H, Maier T, Belmar-Campos C, Franke G, Scherpe S, Aepfelbacher M, Christner M (2011) MALDI-TOF MS fingerprints allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages. Int J Med Microbiol 301:64–68 Yavuz E, Gunes H, Bulut C, Harsa S, Yenidunya AF (2004) RFLP of 16S-ITS rDNA region to differentiate Lactobacilli at species level. World J Microbiol Biotechnol 20:535–537 Zheng Y, Yingli L, Jinfeng W, Longfei Y, Pan C, Ying H (2013) Probiotic properties of Lactobacillus strains isolated from Tibetan kefir grains. PLoS ONE 8:e69868