Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: A review

LWT - Food Science and Technology 42 (2009) 448–457

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Review

Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: A review Smita Singh a, *, Pawas Goswami a, Rameshwar Singh b, Knut J. Heller c a

Department of Microbiology, Maharishi Dayanand Saraswati University, Pushkar Bypass, Ajmer 305001, Rajasthan, India National Collection of Dairy Cultures, Dairy Microbiology Division, National Dairy Research Institute, Karnal, Haryana, India c Department of Microbiology and Biotechnology, Max Rubner-Institut, Federal Research Institute for Nutrition and Food, Kiel, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 October 2007 Received in revised form 3 May 2008 Accepted 26 May 2008

Lactobacillus is among the most important GRAS food lactic acid bacteria, with nearly 140 species at present, mostly of industrial importance. Being part of the natural flora of a range of food products like raw milk, fermented dairy products, fruits, vegetables, meat products they also serve as starters for a number of fermented food products either to enhance the quality or to add health benefits. These groups of economically important species are often alike in phenotypic and physiological characteristics, probably due to their co-evolution in the same ecological niches; hence they are difficult to be differentiated. This demands advanced methods for their proper identification and characterization. With the advancement of molecular biology, a range of DNA-based molecular techniques has replaced the largely cumbersome phenotypic methods. This review summarizes the various molecular techniques available for detection and identification within the genus Lactobacillus, with special emphasis on the four groups of closely resembling species: L. casei group, L. acidophilus group, L. delbrueckii subspecies, and L. plantarum group. This review also provides insights into current trends for alternative molecular markers other than 16S rRNA to resolve the ambiguity within phylogenetically close species in the genus Lactobacillus. Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Lactobacillus Molecular techniques Ribotyping ARDRA PFGE AFLP Taxonomy

1. Introduction The genus Lactobacillus contains a diverse assemblage of 140 species (Euze´by, 1997) and includes gram-positive, catalase negative, non-motile, non-sporulating, facultative anaerobes, growing under microaerophilic to strictly anaerobic conditions (Klein, Pack, Bonaparte, & Reuter, 1998). Lactobacilli are usually thin slender rods, although they can also attain spiral or coccobacillary forms under certain conditions. They are genetically diverse. With their G þ C content ranging from 32% for L. mali to 54 mol% for L. pontis and L. fermentum, they surpass the required threshold limit for a genus, hence giving the impression that Lactobacillus is not a well defined genus (Vandamme et al., 1996). Particular interest inflicted in lactobacilli is largely due to (i) the association of these organisms with health promoting properties; (ii) their inclusion in numerous food products from nutritional or quality improvement aspects and (iii) the requirement of legislative and industrial bodies, as well as consumer, with respect to safety, labeling, patenting and strain integrity (Charteris, Kelly, Morelli, & Collins, 1997; Holzapfel et al., 2001; Prassad, Gill, Smart, & Gopal, 1998). The most studied and accepted probiotic strains include L. acidophilus LA1, L. acidophilus * Corresponding author. E-mail address: [email protected] (S. Singh).

NCFB 1748, L. rhamnosus GG, L. casei Shirota, L. gasseri ADH and L. reuteri. Benefits from their consumption, like immune enhancements, reduction in fecal enzyme activity, prevention of intestinal disorders, viral diarrhoea, suggest their use as probiotic agents for the treatment of GI infections and inflammatory bowel disease (Macfarlane & Cummings, 2002; Madsen, 2001). They are also known to produce an important group of natural antibiotics i.e. bacteriocins like Lactacin B, Lactacin F, Brevicin 37, Buchnericin LB, Lacticin A, Helveticin J, Sakacin A, Plantaricin A, Gassericin A (Barefoot & Nettles, 1992; Klaenhammer, 1993; Muriana & Klaenhammer, 1991; Yildirim & Yildirim, 2001) which are being used as natural preservatives for food products. Owing to their vast range of beneficial properties, 10 draft genome sequences for major Lactobacillus species including strains of probiotic potential like L. acidophilus NCFB 1748 have been generated by 2006 and at least 11 more sequencing projects are ongoing (Claesson, van Sinderen, & O’Toole, 2007). The resulting information will help to determine the genetic basis for the taxonomy in genus Lactobacillus, and more specifically to eliminate inconsistencies in the Lactobacillus casei – Pediococcus group. Although a number of review articles have been published on molecular identification and characterization of lactobacilli (Charteris et al., 1997; Coeuret, Dubernet, Bernardeau, Gueguen, & Vernoux, 2003; Giraffa & Neviani, 2000; Lick, 2003; McCartney, 2002),

0023-6438/$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.05.019

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their focus is generally limited to probiotic species, or this issue has been addressed in brief. This present review deals with the discussion of various molecular methods used in the differentiation within the genus Lactobacillus and their rate of success especially within the group of closely resembling species. 2. Taxonomy of the genus Lactobacillus The large number of species within this genus and their similar phenotype and physiology along with horizontal transfer of plasmid linked characteristics put the taxonomy of this genus largely in confusion, and many times leads to misidentifications (Dalezios & Siebert, 2001; Hammes & Hertel, 2006; Hammes & Vogel, 1995). In order to make identification and characterization of a range of Lactobacillus species easier and comprehensive, various schemes have been proposed, based on distinguishable features/characteristics, such as phenotype, physiological, biochemical characteristics and sequence comparisons of 16S rRNA gene (Collins et al., 1991; Hammes & Vogel, 1995; Kandler & Weiss, 1986; Orla-Jensen, 1919; Stiles & Holzapfel, 1997). Hence, along with these taxonomic classification schemes the taxonomy of the various species within the genus Lactobacillus has undergone major changes and various species have been moved in and out of the Lactobacillus. Some atypical Lactobacillus species like L. confuses, L. halotolerans, L. kandleri, L. minor, L. viridescens, L. minutus, L. rimae and L. uli have been reclassified and the new genus Atopobium has been proposed (Collins, Metaxopoulus, & Wallbanks, 1993; Collins & Wallbanks, 1992). L. maltaromicus, L. carnis and L. divergens have been shifted into the new genus Carnobacterium (Collins, Farrow, Phillips, Ferusu, & Jones, 1987). In addition, there are issues of taxonomic dispute and the problem of very minute differences at nucleotide level in the 16S rRNA gene, creating ambiguity among the four prominent Lactobacillus groups namely, L. acidophilus, L. casei, L. plantarum and L. delbrueckii, well recognized for their use in dairy products as well as neutraceuticals. Before describing the various molecular methods and their potential in general, we will focus on the taxonomic status of these mentioned groups. The L. casei and L. acidophilus groups are of special relevance for the pharmaceutical industry due to their important role in promoting human health (Holzapfel, Haberer, Geisen, Bjorkroth, & Schillinger, 2001; Klein et al., 1998; Roy, Ward, Vincent, & Mondou, 2000). A single species casei with five subspecies namely casei, alactosus, pseudoplantarum, tolerans and rhamnosus was reclassified into three species: (i) L. casei including the reference strain of previous L. casei ssp. casei, (ii) L. paracasei with two subspecies paracasei and tolerans including the former subspecies alactosus and pseudoplantarum in subspecies paracasei and ssp. tolerans for the former L. casei ssp. tolerans, (iii) species L. rhamnosus as a replacement of L. casei ssp. rhamnosus (Collins, Phillips, & Zanoni, 1989). This classification, however, initiated a new stream of controversial results, largely due to the failure of differentiation between the newly created L. paracasei and former L. casei strains even by molecular techniques (Chavagnat, Haueter, Jimeno, & Casey, 2002; Vasquez, Ahrne, Pettersson, & Molin, 2001). Various researchers have produced reasonable evidences for the replacement of type strain of L. casei ATCC 393 by ATCC 334 and rejection of name L. paracasei by using different molecular methods (Chavagnat, Haueter, Jimeno, & Casey, 2002; Chen, Lim, Lee, & Chan, 2000; Dellaglio, Dicks, du Toit, & Torriani, 1991; Dicks, Du Plessis, Dellaglio, & Lauer, 1996; Felis, Dellaglio, Mizzi, & Torriani, 2001; Ryu, Czajka, Sakamoto, & Benno, 2001; Ward & Timmins, 1999). Moreover, a new species L. zeae has been proposed for the group of the former L. rhamnosus strains with type strain ATCC 15820, and inclusion of ATCC 393 has been suggested in L. zeae. Although these proposals have been strongly endorsed by the International

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Committee on Systematic Bacteriology (Biavati, 2001; Klein, 2001), a final decision has not been made. It is likely that the taxonomy of this group will undergo further changes with more extensive studies providing more evidence in the coming years. Heterogeneity among L. acidophilus strains was first recognized in the 1960s by Lerche and Reuter, who suggested four different biotypes for this species. Subsequently, DNA–DNA hybridization studies (Johnson, Phelps, Cummins, London, & Gasser, 1980; Lauer, Helming, & Kandler, 1980) confirmed this heterogeneity and evidenced the presence of six different species within the group namely, L. acidophilus, L. crispatus, L. amylovorous, L. gasseri, L. gallinarum and L. johnsonii (Cato, Moore, & Johnson, 1983; Fujisawa, Benno, Yaeshima, & Mitsuoka, 1992). However, it is difficult to differentiate unambiguously among some of these species (Holzapfel, Schillinger, Du Toit, & Dicks 1997; Klaenhammer, 1998; Song et al., 1999). There are several reports, regarding the misidentification of a number of strains belonging to this group (Schillinger, 1999; Song et al., 2000; Yeung, Sanders, Kitts, Cano, & Tong, 2002). In fact L. gasseri and L. johnsonii are difficult to be distinguished from each other sometimes even by molecular techniques (Walter et al., 2000). The third major group under this category is represented by L. delbrueckii species containing three highly resembling subspecies, namely: L. delbrueckii ssp. delbrueckii, L. delbrueckii ssp. bulgaricus, L. delbrueckii ssp. lactis, which are of special relevance in food fermentations. The subspecies bulgaricus and lactis are common starters used most often in the dairy industry, whereas L. delbrueckii ssp. delbrueckii is found mainly in vegetable fermentations. These three subspecies are known to share more than 80% of DNA–DNA homology (Weiss, Schillinger, & Kandler, 1983) along with 16S rRNA sequence homology reaching 90.8–99. 3% (Collins et al., 1991; Vandamme et al., 1996). The fourth group of closely resembling Lactobacillus species is the L. plantarum group consisting of L. plantarum, L. paraplantarum, and L. pentosus species. They exhibit very high levels of DNA homology, with L. plantarum and L. pentosus sharing even greater than 99% similarity with only a minute 0.3% difference in their 16S rRNA sequence (Collins et al., 1991; Quere, Deschamps, & Urdaci, 1997). Despite this, Zanoni, Farrow, Phillips, and Collins (1987) demonstrated that they are separate species on the basis of DNA–DNA hybridization studies. Likewise, many attempts to discriminate these species succumbed to failure in the past and only limited success could be achieved (Curk, Peladan, & Hubert, 1994; Van Reenen & Dicks, 1996). However lately, some success has been obtained in discrimination among these species with the use of alternative molecular markers (Berthier & Ehrlich, 1998; Torriani, Felis, & Dellaglio, 2001). 3. Molecular identification methods The identification of lactobacilli using biochemical methods is notoriously difficult largely due to the need for plenty of cumbersome biochemical tests along with the problems of highly resembling large number of species groups that are prone to transfer of plasmids among them. Hence, they alone are not sufficient for inter- and intra-species differentiation and need to be supplemented with sensitive molecular methods to obtain more reliable identification. Contrary to the phenotypic methods, molecular identification and characterization tools are far more consistent, rapid, reliable and reproducible and can discriminate even between closely related groups of species, which are otherwise indistinguishable on the basis of phenotype. In fact, many Lactobacillus species have been reclassified on the basis of fresh information from advanced molecular techniques and their correct taxonomic status has been determined, such as L. cellobiosus, L. pastorianus, L. arizonensis have

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been reassigned to L. fermentum (Dellaglio, Torriani, & Felis, 2004), L. paracollinoides (Ehrmann & Vogel, 2005), and L. plantarum (Kostinek et al., 2005), respectively. The most commonly employed molecular techniques for identification of lactobacilli can be divided into four groups: Non-PCR based, PCR based approaches, methods based on the combination of these two approaches and sequencing based approach. For an overview, the details regarding the capability of successful methods discussed in the following sections are listed in Table 1. 3.1. Non-PCR based approaches 3.1.1. Genetic probes/DNA dot blot techniques Probes are synthetically prepared oligonucleotides meant to bind to the specific complimentary recognition sequence on the target bacterial genomic DNA. Most often, probes have been designed either against 16S or 23S rRNA genes (Ehrmann, Ludwig, & Schleifer, 1994; Hensiek, Krupp, & Stackebrandt, 1992; Hertel et al., 1991; Hertel, Ludwig, Pot, Kersters, & Schleifer, 1993; Sghir, Antonopoulos, & Mackie, 1998), but 16S rRNA based probes remain more popular due to the smaller size of 16S rRNA (1.5 knt) in comparison to the 2.3 knt of the 23S rRNA gene. Species-specific oligonucleotide probes have been used for the successful identification of a number of important Lactobacillus species (Alander et al., 1999; Ampe, 2000; Chagnaud, Machinis, Coutte, Marecat, & Mercenier, 2001; Drake, Small, Spence, & Swanson, 1996; Lucchini et al., 1998; Nakagawa et al., 1994; Park & Itoh, 2005; Pot et al., 1993; Tilsala-Timisjarvi & Alatossava, 1997; Yasui, Okamoto, & Taguchi, 1997). However, probes based on other genes such as pyrDFE have also been successfully applied to resolve the differences among some closely related species (Briengel, Curk, & Hubert, 1996). Sometimes, species-specific fragments obtained from randomly amplified polymorphic DNA (RAPD) (Hayford, Petersen, Vogensen, & Jacobsen, 1999; Quere et al., 1997) or DNA fragments obtained from restriction digests of plasmids or genomic DNA, have also been utilized to obtain specific probes for some Lactobacillus species (De los Reyes-Gevilan, 1992; Giraffa & Neviani, 2000). 3.1.2. Ribotyping The method involves the separation and identification of the rRNA genes present within the bacterial genome. The detection of rDNA cistrons is accomplished by hybridization with a labeled probe, within the restriction endonuclease pattern. The resultant profile, ribopattern is simpler, since only DNA fragments complimentary to the rDNA probe are visualized. Additionally, complications due to plasmid bands are also reduced. Ribotyping affords good subspecies differentiation and has successfully been used for both species and strain discrimination within Lactobacillus (Kitahara, Sakata, & Benno, 2005; McCartney, Wang, & Tannock, 1996). It has successfully been applied to detect individual species or strains within the L. acidophilus complex, L. casei, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L. reuteri, L. rhamnosus, L. sakei, L. crispatus and L. gasseri species (Charteris et al., 1997; Giraffa & Neviani, 2000; Holzapfel et al., 2001; Ryu et al., 2001; Yansanjav, Svec, Sedlacek, Hollerova, & Nemec, 2003). The strongholds of ribotyping are: it allows typing of all isolates, it is reproducible, it has a high discriminating power, and it generates simple patterns. However, it is also time consuming and costly. The use of an automated riboprinter may provide an easier and faster alternative, as this system is able to allow identification in a shorter time span of 8 hours; thus it can substantially reduce assay time. In a study demonstrated by Ryu, Czajka, Sakamoto, and Benno (2001), automated ribotyping was evaluated to characterize 91 type and reference strains of L. casei and L. acidophilus groups and most of the strains could be discriminated at the species level. Later on in

another relevant study, strain-specific automated ribotyping was also successfully implicated to track the probiotic lactobacilli strains among isolates of fecal and vaginal origin, from the subjects fed on two probiotic products (Massi, Vitali, Federici, Matteuzzi, & Brigidi, 2004). 3.1.3. Macrorestriction enzyme analysis/PFGE Restriction enzyme analysis (REA) has been used in association with PCR or pulsed field gel lectrophoresis (PFGE) (Lee et al. 2004; Pepe et al., 2004). The problem of a large number of bands encountered in REA of chromosomal DNA can be overcome by the use of rare cutters, hence leading to relatively few large DNA fragments (up to 5000 kb), which are separated by PFGE. Strain typing has been successfully achieved by PFGE for the L. acidophilus complex, L. casei, L. delbrueckii and its three subspecies (bulgaricus, delbrueckii and lactis), L. fermentum, L. helveticus, L. plantarum, L. rhamnosus and L. sakei (Giraffa & Neviani, 2000; Klein et al., 1998; Roussel, Colmin, Simonet, & Decaris, 1993; Sanchez et al., 2004). However, in some cases, PFGE has also allowed species discrimination and identification at the group level. Various studies have demonstrated the success of PFGE in monitoring the predominant Lactobacillus population in the human gastrointestinal tract (Kimura, McCartney, McConnell, & Tannock, 1997; McCartney et al., 1996). Other workers have shown the ability of PFGE to differentiate probiotic strains. In a comparative study, PFGE was shown to be superior to both ribotyping as well as RAPD in discriminating strains of closely related species L. casei and L. rhamnosus (Tynkkynen, Satokari, Saarela, Mattila-Sandholm, & Saxelin, 1999). PFGE in association with PCR based methods is commonly used for strain monitoring (Bouton, Guyot, Beuvier, Tailliez, & Grappin, 2002; Ventura & Zink, 2002). Vancanneyt et al. (2006) compared the Not I and Asc I PFGE patterns of probiotic Lactobacillus strains along with other Lactobacillus isolates with potential probiotic attributes. They reported that some potentially probiotic strains were indistinguishable from the other probiotic cultures. Besides the superior discriminatory power and high reproducibility, PFGE is labor-intensive, requires expensive equipment not generally available in most microbiology and molecular biology laboratories , and can screen only a limited number of samples at a time. 3.2. PCR-based methodologies PCR based techniques, including multiplex PCR using specific primers and randomly amplified polymorphic DNA (RAPD), have been used to detect lactobacilli in various ecosystems. The V1–V3 region of 16S rRNA has been found sufficiently variable to provide species-specific patterns in PCR and, also in combination with denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) or single strand conformation polymorphism (SSCP) (Cocolin, Manzano, Cantoni, & Comi, 2001; Duthoit, Godon, & Montel, 2003; Kullen, Sanozky-Dawes, Crowell, & Klaenhammer, 2000; Vasquez et al., 2001; Walter et al., 2000; Ward & Timmins, 1999). Even the V1 region alone has also been reported successful for discrimination below the species level (Chagnaud et al., 2001). Besides the 16S rRNA gene, the 16S–23S spacer region has also been targeted successfully in PCR for a number of species (Berthier & Ehrlich, 1998; Chagnaud et al., 2001; Massi, Vitali, Federici, Matteuzzi, & Brigidi, 2004; Song et al., 2000; Tannock et al., 1999; Tilsala-Timisjarvi & Alatossava, 1997). Besides the 16S–23S spacer, the interspacer region in between tRNA genes has also been successfully employed for discrimination among Lactobacillus species on the basis of a unique tDNA fingerprint pattern in capillary electrophoresis; however it failed to discriminate some of the closely resembling species groups (Baele et al., 2002).

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Table 1 Comparative evaluation of molecular approaches used in discrimination among closely resembling Lactobacillus species groups Species identified and source

Methods used

L. casei group (L. casei, L. paracasei, L. rhamnosus, L. zeae) L. casei, L. paracasei, L. rhamnosus of Species-specific PCR (16S rRNA V1 cheese origin region), RAPD L. casei, L. rhamnosus, L. zeae isolates DGGE of 16S rRNA (V1–V2) amplicons, human and porcine gastrointestinal Species-specific PCR (16S–23S spacer), tract Sequencing (V2–V3 region of 16S rRNA) L. casei, L. paracasei (both subspecies), Comparative sequence analysis of L. rhamnosus, L. zeae, type and 318 bp. stretches of rec A gene reference strains, strain from a commercial probiotic product. L. casei, L. paracasei, L. rhamnosus, Eco RI ribotyping L. zeae, type and reference strains

L. casei, L. paracasei, L. rhamnosus, L. zeae, reference strains, L. rhamnosus strains from human intestinal mucosa L. casei, L. paracasei, L. rhamnosus, L. zeae, type and reference strains

TTGE of 16S rRNA amplicons from region (U1–U2) (Position 8-357, E. coli numbering)

L. casei, L. paracasei, L. rhamnosus, L. zeae, type and reference strains

Comparative sequence analysis of V1– V3 regions of 16S rRNA, ITS, cpn 60

L. casei/L. paracasei from wine

16S ARDRA (Bfa I, Mse I), RAPD, Eco RI ribotyping

Comparative analysis of 761 bp. stretches of tuf nucleotide and corresponding amino acid sequence.

Comments

References

Good discrimination at species level by both techniques. Identification of individual species by speciesspecific PCR only; otherwise only group level identification. Successful in clear separation of all type strains in distinct branches; identification of L. casei ATCC 393 and L. casei ATCC 334 as L. zeae and L. paracasei, respectively. Good discrimination among all type strains, but L. paracasei ssp. paracasei JCM 1172 characterized as L. paracasei ssp. tolerans and L. casei ATCC 334 as L. paracasei strains. Good discrimination with exception of L. casei ATCC 393 and L. casei NCFB 173; no discrimination between L. casei and L. paracasei strains. Good discrimination on the basis of three distinct consensus amino acid signature sequences; no discrimination between L. casei and L. paracasei; L. casei strains ATCC 393 and NCDO 173 identified as L. zeae. Three separate operational taxonomic units recognized for L. casei/paracasei, L. rhamnosus, L. zeae; L. casei ATCC 393 identified as L. zeae. 13 wine strains typed as L. paracasei/casei, based on similar band pattern as L. paracasei type strain and L. casei ATCC 334.

Ward and Timmins (1999)

L. acidophilus group (L. acidophilus, L. crispatus, L. amylovorous, L. helveticus, L. gasseri, L. johnsonii) L. acidophilus, L. crispatus, Multiplex PCR with species-specific Good discrimination, among all species. L. amylovorous, L. helveticus, L. gasseri, primers, targeting 16S–23S ITS and L. johnsonii within isolates from flanking 23S rRNA gene region. human stool, previously identified as Lactobacillus species Sequence comparison from V1 and Both V1 alone as well as V1–V2 regions found Type and reference strains of combined V1–V2 region in 16S rRNA satisfactory for discrimination among all Type L. acidophilus, L. amylovorus, gene. strains and clearly identified three groups of L. crispatus, L. gallinarum, L. gasseri, probable L. acidophilus human isolates up to L. johnsonii along with 17 human isolates. species level. L. acidophilus, L. crispatus, L. gasseri, DGGE of V2–V3 amplicons, SpeciesSpecies designation by specific primers only, L. johnsonii within Lactobacillus specific PCR (16S rRNA gene or 16S–23S while DGGE and V2–V3 sequencing achieved isolates of human and pig spacer), Sequencing of V2–V3 region group level discrimination. gastrointestinal region Type and reference strains of EcoRI ribotyping Failed to differentiate among strains of different L. acidophilus, L. gallinarum, species within this group except L. gallinarum L. amylovorus, L. crispatus, L. gasseri, L. johnsonii Type and reference strains of Amino acid sequence comparison of the Successfully confirmed the 40 strains of L. acidophilus, L. amylovorus, corresponding tuf gene sequences of L. acidophilus group and also categorized all 18 L. crispatus, L. gasseri, L. johnsonii, reference and unidentified strains unidentified strains into species. Moreover along with 18 unidentified strains suggested reclassification of L. gasseri CIP 103614 and L. crispatus CIP 103605 as L. johnsonii and L. gasseri, respectively L. plantarum group (L. plantarum, L. paraplantarum, L. pentosus) Type and reference strains of Species-specific PCR, primers based on L. plantarum, L. paraplantarum, 16S–23S spacer. L. pentosus species Type and reference strains of AFLP, RAPD L. plantarum, L. paraplantarum, L. pentosus Type and reference strains of L. plantarum, L. paraplantarum, L. pentosus L. plantarum, L. pentosus, Wine isolates

Walter et al. (2000)

Felis et al. (2001)

Ryu et al. (2001)

Vasquez et al. (2001)

Chavagnat et al. (2002)

Dobson et al. (2004)

Rodas et al. (2005)

Song et al. (2000)

Kullen et al. (2000)

Walter et al. (2000)

Ryu et al. (2001)

Chavagnat et al. (2002)

Specificity of primers not so reliable in the case of L. plantarum and L. paraplantarum

Berthier and Ehrlich (1998)

Torriani et al. (2001)

Species-specific PCR, primers based on rec A gene

Successful discrimination of 30 strains at a similarity level of 30% and 40% by AFLP and RAPD, respectively. L. plantarum like strain LMG 18404, identified as L. plantarum Good species level discrimination, also suitable for multiplex PCR

RFLP-PFGE, 16S ARDRA, Ribotyping, RAPD

Only RAPD and Ribotyping could discriminate between the type strains of both species

Rodas et al. (2005)

Could differentiate L. delbrueckii ssp. bulgaricus from L. delbrueckii ssp. lactis and ssp. delbrueckii, but latter two subspecies could not be discriminated from each other

Giraffa et al. (1998)

L. delbrueckii subspecies (delbrueckii, lactis and bulgaricus) L. delbrueckii dairy isolates including PCR-ARDRA (EcoRI) three type strains of all three subspecies

Torriani et al. (2001)

(continued on next page)

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Table 1 (continued) Species identified and source

Methods used

Comments

References

L. delbrueckii strains

PCR primers (34/2, 37/1, and 33, 37A) based on EcoRI/Pst I addA fragment specific for L. d. ssp. bulgaricus and L. d. ssp. lactis/delbrueckii, respectively Probes 34B and 33/2 derived from EcoRI/Pst I addA fragment specific for L. d. ssp. bulgaricus and L. d. ssp. lactis/ delbrueckii, respectively 23S rDNA-based probes Lbdb and Lbdl for L. d. ssp. bulgaricus and L. d. ssp. lactis/delbrueckii, respectively EcoRI ribotyping, rrn operon-ARDRA (EcoRI) Box PCR

Good discrimination by PCR between L. d. bulgaricus and L. d. lactis/delbrueckii

Lick et al., 2000

Collection strains belonging to three subspecies Raw milk isolates

Isolates from dairy products

L. curvatus, L. sakei, L graminis species LAB isolates from Pork loin and Bologna (meat) Type and reference strain of L. sakei, L. curvatus and L. bavaricus

Reference and type strains of L. curvatus, L. graminis, L. sake Reference and type strains of L. sakei, L. curvatus and L. graminis

PCR-RFLP of three protein-coding genes (b-galactosidase, lactose permease and proline dipeptidase) and 16S rDNA sequencing of variable regions 23S rDNA probe DNA–DNA hybridization and RAPD-PCR profiles

16S–23S rDNA-based specific primers RAPD-PCR, Species- and subgroupspecific PCR primers derived from RAPD fragments

Isolates from fermented sausage and artisanal meat plants

RAPD-PCR with primers M13 and D8635

Isolates from Kimchi (fermented cabbage)

Multiplex PCR-REA (Hind III) with 16S RDNA-based primers

A simple but very beneficial advancement of the classic PCR technique is multiplex PCR, which can detect a number of individual species simultaneously in a single common PCR step, with multiple primer sets; thus it can save plenty of time and labor. However, selection of primer sets with similar reaction specifications, especially annealing temperature is the utmost requirement. It has been successfully evaluated within genus Lactobacillus by many workers (Dubernet, Desmasures, & Gueguen, 2002; Heilig et al., 2002; Song et al., 2000; Tilsala-Timisjarvi & Alatosava, 1997). Kwon, Yang, Yeon, Kang, and Kim (2004) reported rapid detection of seven common probiotic Lactobacillus species i.e. L. acidophilus, L. delbrueckii, L. casei, L. gasseri, L. plantarum, L. reuteri and L. rhamnosus in a single step. However, multiplex PCR also suffers from the limitations of insufficiency of priming targets resulting from few variable regions on rRNA genes (Berthier & Ehrlich, 1998; Gurtler & Stanisich, 1996), difficulty in optimization of PCR conditions due to the high number of primer pairs, and generation of unanticipated PCR products caused by mispairing of forward or reverse primers. A number of developments have been raised in basic PCR to add specificity and to improve speed such as choice of alternative target selection as well as variations like multiplexing and nesting. Sideby-side efforts also have been made to improve electrophoresis as an associative technique of PCR to generate a more powerful combination of techniques for discrete differentiation of amplified products. Efforts on these lines led to the emergence of the techniques of DGGE and TGGE, in which DNA fragments of the same length but different sequences can also be separated based on

Problem of non specific signals with Probe 34B, Lbdb/Lbdbl, though in combination with species-specific 23S rDNA-based probe satisfactory subspecies discrimination

16S rDNA ARDRA as well as ribotyping were able to differentiate among all three subspecies Successful differentiation among L. d. lactis and L. d. bulgaricus at species, subspecies and strain level PCR-RFLP of protein-coding genes was found more effective than 16S rDNA sequencing in grouping L. delbrueckii strains into the two subspecies lactis and bulgaricus Probes unequivocally differentiated L. sakei and L. curvatus isolates Besides good distinction between two species, two subspecies within each species i.e. L. curvatus ssp. curvatus and L. curvatus ssp. melibiosus, L. sakei ssp. carnosus and L. sakei ssp. sakei were proposed on the basis of RAPD results. L. bavaricus reclassified as L. sakei ssp. sakei Good discrimination among the type strains of all species Species-specific primers for L. curvatus and subgroup specific primers for L. sakei were found satisfactory. RAPD efficient for typing at strain level Clear differentiation between L. curvatus and L. sakei species. No differentiation among subspecies Simple, rapid and reliable identification for both species by PCR-REA

Miteva et al. (2001) De Urraza, Mez-Zavaglia, Lozano, Romanowski, and De Antoni (2000) Giraffa et al. (2003)

Nissen and Dainty (1995) Torriani et al. (1996)

Berthier and Ehrlich (1998) Berthier and Ehrlich (1999)

Andrighetto et al. (2001)

Lee et al. (2004)

decreased electrophoretic mobility of partially melted double stranded molecules, in polyacrylamide gels containing a linear denaturing chemical (formamide or urea) or temperature gradient, respectively. The discriminatory power of these techniques is so high that even single nucleotide differences can also be spotted. Randazzo, Torriani, Akkermans, de Vos, and Vaughan (2002) demonstrated the presence of L. delbrueckii species in ripening Ragusano cheese by PCR, RT-PCR and DGGE of 16S rRNA genes, which could not be detected by cultivation. Very recently, rpoB has been used in PCR-DGGE analysis to evaluate the L. plantarum population present in red wine (Spano, Lonvaud-Funel, Claisse, & Massa, 2007). Although PCR is routinely executed for genus and species level discrimination, its specificity can even be extended up to strain level. It seems an ideal approach to ensure the detection of particular labeled probiotic strains with ease and reliability, and has been successfully used for L. gasseri 4B2 and three L. rhamnosus strains namely LC-705, GG, E-97800 (Brandt & Alatossava, 2003; Lucchini et al., 1998). Another popular variation in simple PCR is randomly amplified polymorphic DNA (RAPD), which is a rapid fingerprinting method, generally used for intra- and inter-species differentiation within the genus Lactobacillus (Bjorkroth, Ridell, & Korkeala, 1996; Du Plessis & Dicks, 1995; Elegado, Guerra, Macayan, Mendoza, & Lizaran, 2004; Hayford et al., 1999; Johansson, Quednau, Molin, & Ahrne, 1995; Khaled, Neilan, Henricsson, & Conway, 1997; Oneca, Irigoyen, Ortigosa, & Torre, 2003; Tynkkynen et al., 1999). The major

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attractions behind its wide popularity are: it is rapid, simple, easy and does not require any prior knowledge of the target sequence; also it is universally applicable to any genome. In principle, RAPD involves random amplification from the target bacterial genome with the aid of a single primer under less stringent conditions. Thus, a complex pattern of multiple amplification products from loci distributed throughout the genome is generated. Along with 16S rRNA gene sequencing, it is considered to be the most widely used technique for characterizing food-associated lactobacilli, and can distinguish between strains of L. pentosus and L. plantarum and between L. graminis and L. curvatus species (Charteris et al., 1997; Nigatu, 2000; Nigatu, Ahrne, & Molin, 2001). Specific RAPD bands have also been used to design species- and strain-specific probes or PCR primers for some lactobacilli including L. plantarum, L. gasseri and L. rhamnosus (Lucchini et al., 1998; Quere et al., 1997; TilsalaTimisjarvi & Alatossava, 1998). In a comparative study documented by Roy et al. (2000), RAPD was found more suitable for discrimination between strains of L. gallinarum and L. helveticus compared with rRNA-targeted probes, which cross-hybridized with both species. Moreover, strain ATCC 53673, earlier identified as L. helveticus with the Lbh probe, could be classified as L. gallinarum according to its RAPD profile. It has also been used to differentiate between the type and reference strains of lactobacilli (Nigatu et al., 2001). Dicks, Silvester, Lawson, and Collins (2000) defined a new Lactobacillus species from the posterior fornix of the human vagina on the basis of RAPD. Although a simple and rapid method, RAPD is prone to poor reproducibility in the band pattern, due to even small changes in reaction conditions. The time saved by the direct application of RAPD is often lost in achieving consistency and in confirming the reproducibility of the results. Thus, standardization of best resolving conditions and maintenance of very high consistency in all parameters are considered the most important aspects in applying RAPD. Recently, PCR amplification of repetitive bacterial DNA elements (REP-PCR) has also emerged as a powerful PCR based strain differentiation technique with the advantages of high discriminatory power, low cost and suitability for high throughput of strains. Gevers, Huys, and Swings (2001) employed REP-PCR to differentiate among L. pentosus/L plantarum/L. paraplantarum and also between L. alimentarius and L. paralimentarius species of sausage origin. Berthier, Beuvier, Dasen, and Grappin (2001) also demonstrated the success of this method to reveal the diversity of mesophilic lactobacilli in Comte cheese. Besides REP-PCR, ERIC-PCR is another potential typing technique, which in principle is alike to REP-PCR and is based on the Enterobacterial Repetitive Intergenic Consensus sequence amplification. Although REP and ERIC-PCR are multilocus techniques like RAPD, they use primers against known universal sequences in bacterial genome and provide more robust alternative approaches for strain level differentiation in genus Lactobacillus in comparison of RAPD. Both of these methods were also found highly reliable in the rapid characterization of L. johnsonii strains (Ventura & Zink, 2002). More recently, a very sensitive and quantitative form of PCR, Real Time PCR has been used for successful identification and quantification of few Lactobacillus species from variable sources like fermented sausages (Martin, Jofre, Garriga, Pla, & Aymerich, 2006), from human (Haarman & Knol, 2006) and from chicken faeces (Selim et al., 2005). 3.3. Techniques based on combinations of the two approaches 3.3.1. Amplified rDNA restriction analysis (ARDRA) ARDRA is essentially a technical variation of ribotyping, i.e. the restriction enzyme analysis of 16S rDNA PCR amplicons. It is equally efficient for species level identification among Lactobacillus species

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of importance. 16S–23S rRNA spacer ARDRA was also reported for differentiating closely related L. alimentarius and L. farciminis (Rachman, Kabadjova, Pre´vost, & Dousset, 2003) or other Lactobacillus species including L. casei and L. acidophilus groups (Hans et al., 2005; Moreira et al., 2005). However, the conservative nature of 16S rRNA genes limits the discriminatory power of techniques such as ARDRA compared to methods, which utilize differences in the whole genome, such as PFGE and RAPD (O’Sullivan, 1999). 3.3.2. Amplified fragment length polymorphism (AFLP) AFLP is basically a combination of PCR and RFLP, based on selective PCR amplification of restriction fragments from digested DNA; the DNA fragments are then separated by polyacrylamide gel electrophoresis. AFLP offers a number of advantages as it does not require any prior sequence information and does not need probe generation also. It is highly reproducible and allows a quick scan of the whole genome for polymorphisms. AFLP was confirmed recently as a powerful technique for the delineation of strains related beyond species level for several bacterial genera (Gancheva, Pot, Vanhonacker, Hoste, & Kersters, 1999). AFLP, although used to a lesser extent for Lactobacillus identification, was found good enough to discriminate among L. pentosus, L. plantarum and L. paraplantarum (Torriani et al., 2001). In another study, it was used to type L. johnsonii strains along with a number of other techniques (Ventura & Zink, 2002). Very recently, Vancanneyt et al. (2006) used fluorescent AFLP for intra-specific differentiation of a collection of Lactobacillus strains.

3.4. Sequencing based approach 3.4.1. Sequencing of variable regions within the 16S rRNA gene or 16S–23S rRNA intergenic spacer region With the increasing availability and declining cost of high throughput sequencing operations, determination of 16S rRNA gene nucleotide sequences in variable region V1–V3 has emerged as a viable option for strain identification and phylogenetic analysis (Amann, Ludwig, & Schleifer, 1995; Vandamme et al., 1996). However, the flaw in this approach is that as the evolutionary distance decreases, the diversity level in the 16S rRNA is often insufficient and genetic relationships of closely related species cannot be accurately defined on the basis of 16S rRNA gene sequence. As an alternative, the intergenic spacer region between 16S and 23S rRNA genes containing a variable number of tRNA genes can also be a suitable target for sequencing (Gurtler & Stanisich, 1996). It is smaller (200 bp) and due to the various copy numbers of rrn operons in bacterial genomes, multiple alleles of 16S rRNA genes further add to spacer variations between species, strains and genera and may be used for identification and typing purpose. Sequencing of this region allowed better discrimination between L. casei and L. rhamnosus strains in comparison to V2–V3 sequencing (Tannock et al., 1999). 3.4.2. Multilocus sequence typing Multilocus sequence typing (MLST) is a more powerful advancement to the conventional 16S rRNA gene sequencing technique for bacterial typing. MLST makes use of automated DNA sequencing to characterize the alleles present at different housekeeping gene loci. As it makes use of functional housekeeping genes, it is highly discriminatory and provides unambiguous results that are directly comparable between laboratories. It has been used mainly for pathogenic bacteria (Spratt, 1999) and has been introduced recently for species and strain discrimination within the genus Lactobacillus. In a recent study, De las Rivas, Marcobal, and Munoz (2006) successfully documented the sequence typing of six housekeeping genes namely, pgm, ddl, gyr B, purK1, gdh and mut S to

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determine the genetic relatedness as well as discrimination between L. plantarum strains. 3.5. 16S rRNA gene versus protein-coding phylogenetic markers Although the 16S rRNA gene based taxonomic evaluation is undoubtedly useful in species differentiation, the slow rate of 16S rDNA evolution is thought to be responsible for its failure to provide multiple diagnostic sites for closely related but ecologically distinct taxa. On the contrary, in the case of protein-coding genes, rates of evolutionary substitutions are one order of magnitude greater than those for 16S rRNA genes. Thus, some pairs of ecologically distinct taxa may have had time to accumulate neutral sequence divergence at rapidly evolving loci but not at the 16S rDNA level (Fox, Wisotzkey, & Jurtshuk, 1992; Palys, Berger, Mitrica, Nakamura, & Cohan, 2000). Many studies have tried alternative phylogenetic markers such as proline iminopeptidase (Torriani et al., 1999) and FBPase gene coding for fructose 1,6-bisphosphatase, (Roy & Ward, 2004). Other conserved functional genes such as rec A coding for Rec A protein, tuf for elongation factor Tu and hsp 60 for heat shock protein are being increasingly used by many researchers. These genes have an added advantage, since during evolution they have sustained the basic amino acid structure more or less conserved without modifying the product of translation substantially, by tolerating silent point mutations; thus leading to a greater degree of variability at the nucleotide level. Thus, these loci can be compared at nucleotide sequence as well as amino acid sequence for inference of phylogenetic/taxonomic relationships between closely related species and between distant taxa, respectively. Furthermore, these are free from the likely overestimation of the relatedness of the taxa with similar nucleotide differences, non-independence of substitution patterns at different sites, and bias derived from different G þ C contents of microorganisms (Eisen, 1995). To get a clearer picture, we will discuss the research carried out so far. rec A gene based primers have been found successful to differentiate among L. plantarum, L. paraplantarum and L. pentosus species (Torriani et al., 2001), which could otherwise not be differentiated on the basis of the 16S rRNA gene. Felis, Dellaglio, Mizzi, and Torriani (2001) studied the phylogenetic relationship among nucleotide as well as amino acid sequence of 277 bp of the rec A gene sequence of type strains of L. casei, L. paracasei ssp. paracasei, L. paracasei ssp. tolerans, L. rhamnosus, L. zeae and L. casei ATCC 334 in the neighbour-joining tree. They proposed that L. casei ATCC 393T and L. zeae LMG 17315 belong to same species, and that L. paracasei strains and L. casei ATCC 334 are indistinguishable. Moreover, they also reported 30-fold greater Knue values than those calculated for 16S rRNA sequences earlier by Mori et al. (1997). Hence, the study supported the denial of strain ATCC 393 as the type strain of L. casei and also the rejection of the name paracasei and inclusion of the strains belonging to this species into L. casei, as proposed earlier (Dellaglio, Felis, & Torriani, 2002). Besides recA, tuf gene based phylogenetic analysis also has gained momentum recently. In a study based on the tuf gene as well as Tuf protein sequence comparisons, valuable information could be obtained to determine the true taxonomic status of various disputed Lactobacillus species and subspecies such as discrimination between L. animalis and L. murinus, three subspecies of L. delbrueckii and between two subspecies of L. coryneformis (Chavagnat et al., 2002). Also, on the basis of the presence of conserved amino acid signatures two important conclusions were drawn: the name L. casei for all four former subspecies of L. casei and L. paracasei, with the rejection of species name paracasei, in support of the previous claims by Dellaglio et al. (1991, 2002) and Dicks et al. (1996). In addition, the type strain of the former L. casei ssp. rhamnosus ATCC 7469 was also suggested as type strain of newly

formed L. rhamnosus species. In the same study, 761-nucleotide sequence comparison of the tuf gene from 37 species of Lactobacillus on over 200 strains led to the conclusion that the tuf gene is slightly less conserved than the 16S rRNA gene in lactobacilli and distribution into three phylogenetic groups based on 16S rRNA gene (Collins et al., 1991) i.e. L. delbrueckii, L. casei-Pediococcus and Leuconostoc groups is not maintained with the tuf gene. The authors explained that the differences might be due to synonymous nucleotide substitutions, which may have occurred in the tuf gene without modifying the protein, nevertheless conferring a higher degree of variability of tuf nucleotide sequences between species. Ventura, Canchaya, Meylan, Klaenhammer, and Zink (2003) in a comparative tuf sequence based study of lactobacilli and bifidobacteria studied the location of tuf genes in both of these genera. They suggested a novel tuf operon specific for genus Lactobacillus and designed tuf gene based species-specific forward primers for L. paracasei, L. casei and L. rhamnosus (PAR, CAS and RHA along with a single reverse primer CPR). The phylogenetic relationship evaluated in general was found highly in accordance with 16S rRNA derived information. However, the tuf gene sequence analysis was found inadequate for strain level typing. In another study conducted by Cachat and Priest (2005) the phylogenetic evaluation of three malt whisky distillery isolates belonging to the newly isolated Lactobacillus species L. syntoryeus, based on tuf gene sequence comparisons, was found highly in accordance with partial 16S rRNA profiles and RAPD patterns. Surface (S) layer gene coding for cell surface proteins has also been reported as a potential candidate phylogenetic marker in genus Lactobacillus. The presence of S layer has been reported in several species of lactobacilli like L. brevis, L. acidophilus, and L. crispatus (Boot, Kolen, Pot, Kersters, & Pouwels, 1996; Masuda & Kawata, 1983; Palva, 1997; Toba et al., 1995). Ventura, Casas, Morelli, and Callegari (2000) designed L. helveticus specific primers as well as hybridization probes from the conserved region in S layer genes and reported their validity on 50 L. helveticus strains isolated from natural whey starters, previously identified by phenotypic tests as well as restriction analysis of species-specific amplification products (Drake et al., 1996) and hybridization with a 23S rRNA probe (Hertel et al., 1993). Horie, Kajikawa, and Toba (2002) demonstrated the superior specificity of S layer gene based L. crispatus primers with KOD polymerase over a16S rRNA-specific primer set, as the latter could not give strict specificity and produced non specific amplification from L. amylovorus. 4. Conclusions Besides being largely successful in species differentiation between various Lactobacillus species, the techniques discussed have been able to differentiate between closely resembling groups of L. casei, L. acidophilus, L. plantarum, L. delbrueckii and L. graminis, L. curvatus and L. sakei upto more or less extent. From the above discussion, it is evident that a polyphasic strategy essentially based on one or more molecular techniques is necessary for accurate species designation within this genus. In fact, polyphasic taxonomy has been recognized by the International Committee on Systematic Bacteriology as a reliable approach for description of species and for revision of the present nomenclature of some bacterial groups. Moreover, polyphasic taxonomic approaches become more important for classification of novel species from the taxonomic point of view, since they consider mutual validation of results based on various techniques and thus provide most reliable information. References Alander, M., Satokari, R., Korpela, R., Saxelin, M., Vilpponen-Salmela, T., MattilaSandholm, T., et al. (1999). Persistence of colonization of human colonic mucosa

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