A PCR-based method for identification of lactobacilli at the genus level (original) (raw)

Journal Article

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Laboratoire de Microbiologie Alimentaire, EA 3213, USC INRA, Université de Caen - Basse Normandie, Esplanade de la Paix, 14032 Caen cedex, France

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Laboratoire de Microbiologie Alimentaire, EA 3213, USC INRA, Université de Caen - Basse Normandie, Esplanade de la Paix, 14032 Caen cedex, France

Search for other works by this author on:

Laboratoire de Microbiologie Alimentaire, EA 3213, USC INRA, Université de Caen - Basse Normandie, Esplanade de la Paix, 14032 Caen cedex, France

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Revision received:

15 July 2002

Published:

01 September 2002

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Abstract

We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

1 Introduction

Lactic acid bacteria (LAB) are extensively used in the food industry. Lactobacillus species are essential to the dairy industry, where they are used in the preparation of cheeses, yoghurt and other fermented milk products. As many LAB have similar nutritional and growth requirements, it is often difficult to use classical microbiological methods to identify them even to genus level. Research has focused on the application of molecular biology techniques for the rapid detection and differentiation of LAB. The use of primers and probes that target genes encoding ribosomal ribonucleic acid (rRNA) is promising. Due to the high interspecies variability of this region, rDNA sequences coding for the 16S and 23S rRNA has been validated as a means of identification [[[,,,].

However, the data in the literature concern the identification of strains known to be lactobacilli to the species level [[,[[,,,,,,,,,,,,,8]. The objective of this study was to find a specific primer for the identification of lactobacilli at the genus level.

2 Materials and methods

2.1 Bacterial strains and growth conditions

The strains used in this study were obtained from the Collection de l'Institut Pasteur (CIP), the Centre National de Recherche Zootechnique (CNRZ) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Table 1). In this study, the choice of lactobacilli strain was carried out according to their various origin by taking account of their distribution within fermentation types and to the three phylogenetic subgroups (the Lactobacillus delbrueckii group, the Lactobacillus casei-Pediococcus group and the Leuconostoc group)[19]. The strains were maintained at −80°C in 15% glycerol. Lactobacilli strains were subcultured in MRS medium[20] (AES Laboratoire, Combourg, France) and all other strains were subcultured in brain heart infusion agar (BHI; AES Laboratoire). The optimal growth temperature of each strain is indicated in Table 1.

1

Strains used in this study

Species Strain designationa Origin Phylogenetic groupb Fermentative groupc Optimal temperatured
L. acidophilus CNRZ 204T human a I 37–45°C
L. amylovorus CIP 102989T cattle (waste-corn fermentation) a I 37–45°C
L. brevis CIP 102806T human faeces b III 30–37°C
L. buchneri CIP 103023T tomato purée b III 30–37°C
L. casei subsp. casei CNRZ 313T cheese b II 30–37°C
L. coryniformis subsp. torquens CIP 103134T air contaminant (dairy barn) b II 30–37°C
L. delbrueckii subsp. bulgaricus CIP 101027T Bulgarian yoghurt a I 37–45°C
L. delbrueckii subsp. delbrueckii CNRZ 225T sour grain mash a I 37–45°C
L. delbrueckii subsp. lactis CNRZ 207T cheese (Emmental swiss) a I 37–45°C
L. farciminis CIP 103136 sausage b I 30°C
L. fermentum CNRZ 209T fermented beets b III 37–45°C
L. fructosus CIP 102985T flowers c III 30°C
L. gallinarum CIP 103611T normal chicken crop a I 37°C
L. graminis CIP 105164T grass silage a II 30–35°C
L. helveticus CNRZ 223T cheese (Emmental swiss) a I 37°C
L. johnsonii CIP 103653 ND a I 37°C
L. paracasei subsp. paracasei CNRZ 763 ND b II 37–40°C
L. pentosus CIP 103156T ND b II 37–40°C
L. plantarum CNRZ 211T vinegar cabbage b II 30–37°C
L. reuteri CIP 101887T human faeces b III 37°C
L. rhamnosus CIP A157T ND b II 37°C
L. ruminis CIP 103153T bovine rumen b I 30°C
L. suebicus CIP 103411T apple mash b III 37°C
L. zeae CIP 103253 corn steep liquor ND II 37°C
E. coli CIP 54.127 ND 30°C
L. lactis subsp. cremoris CNRZ 105T ND 30°C
L. lactis subsp. lactis CNRZ 142T ND 30°C
L. mesenteroides subsp. cremoris CNRZ 361T lactic starter 25°C
S. thermophilus CNRZ 1358T pasteurised milk 37°C
C. pissicola CIP 103158T kidney 30°C
P. pentosaceus CIP 102260T dried American beer yeast 30°C
B. bifidum CIP 56.7T child faeces 37°C
W. confusa DSMZ 20196 ND 30°C
Enterococcus hirae CIP 58.55 ND 30°C
S. aureus CIP 53.154 ND 37°C
L. monocytogenes CIP 105457 ovine (brain) 37°C
Species Strain designationa Origin Phylogenetic groupb Fermentative groupc Optimal temperatured
L. acidophilus CNRZ 204T human a I 37–45°C
L. amylovorus CIP 102989T cattle (waste-corn fermentation) a I 37–45°C
L. brevis CIP 102806T human faeces b III 30–37°C
L. buchneri CIP 103023T tomato purée b III 30–37°C
L. casei subsp. casei CNRZ 313T cheese b II 30–37°C
L. coryniformis subsp. torquens CIP 103134T air contaminant (dairy barn) b II 30–37°C
L. delbrueckii subsp. bulgaricus CIP 101027T Bulgarian yoghurt a I 37–45°C
L. delbrueckii subsp. delbrueckii CNRZ 225T sour grain mash a I 37–45°C
L. delbrueckii subsp. lactis CNRZ 207T cheese (Emmental swiss) a I 37–45°C
L. farciminis CIP 103136 sausage b I 30°C
L. fermentum CNRZ 209T fermented beets b III 37–45°C
L. fructosus CIP 102985T flowers c III 30°C
L. gallinarum CIP 103611T normal chicken crop a I 37°C
L. graminis CIP 105164T grass silage a II 30–35°C
L. helveticus CNRZ 223T cheese (Emmental swiss) a I 37°C
L. johnsonii CIP 103653 ND a I 37°C
L. paracasei subsp. paracasei CNRZ 763 ND b II 37–40°C
L. pentosus CIP 103156T ND b II 37–40°C
L. plantarum CNRZ 211T vinegar cabbage b II 30–37°C
L. reuteri CIP 101887T human faeces b III 37°C
L. rhamnosus CIP A157T ND b II 37°C
L. ruminis CIP 103153T bovine rumen b I 30°C
L. suebicus CIP 103411T apple mash b III 37°C
L. zeae CIP 103253 corn steep liquor ND II 37°C
E. coli CIP 54.127 ND 30°C
L. lactis subsp. cremoris CNRZ 105T ND 30°C
L. lactis subsp. lactis CNRZ 142T ND 30°C
L. mesenteroides subsp. cremoris CNRZ 361T lactic starter 25°C
S. thermophilus CNRZ 1358T pasteurised milk 37°C
C. pissicola CIP 103158T kidney 30°C
P. pentosaceus CIP 102260T dried American beer yeast 30°C
B. bifidum CIP 56.7T child faeces 37°C
W. confusa DSMZ 20196 ND 30°C
Enterococcus hirae CIP 58.55 ND 30°C
S. aureus CIP 53.154 ND 37°C
L. monocytogenes CIP 105457 ovine (brain) 37°C

ND, not determined.

aCIP, Collection de l'Institut Pasteur, Paris, France; CNRZ, Centre National de Recherches Zootechniques, Jouy-en-Josas, France; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.

bPhylogenetic group: a, L. delbrueckii group; b, L. casei-Pediococcus group; c, Leuconostoc group[19].

cFermentative group: I, obligatory homofermentative; II, facultatively heterofermentative; III, obligatory heterofermentative.

1

Strains used in this study

Species Strain designationa Origin Phylogenetic groupb Fermentative groupc Optimal temperatured
L. acidophilus CNRZ 204T human a I 37–45°C
L. amylovorus CIP 102989T cattle (waste-corn fermentation) a I 37–45°C
L. brevis CIP 102806T human faeces b III 30–37°C
L. buchneri CIP 103023T tomato purée b III 30–37°C
L. casei subsp. casei CNRZ 313T cheese b II 30–37°C
L. coryniformis subsp. torquens CIP 103134T air contaminant (dairy barn) b II 30–37°C
L. delbrueckii subsp. bulgaricus CIP 101027T Bulgarian yoghurt a I 37–45°C
L. delbrueckii subsp. delbrueckii CNRZ 225T sour grain mash a I 37–45°C
L. delbrueckii subsp. lactis CNRZ 207T cheese (Emmental swiss) a I 37–45°C
L. farciminis CIP 103136 sausage b I 30°C
L. fermentum CNRZ 209T fermented beets b III 37–45°C
L. fructosus CIP 102985T flowers c III 30°C
L. gallinarum CIP 103611T normal chicken crop a I 37°C
L. graminis CIP 105164T grass silage a II 30–35°C
L. helveticus CNRZ 223T cheese (Emmental swiss) a I 37°C
L. johnsonii CIP 103653 ND a I 37°C
L. paracasei subsp. paracasei CNRZ 763 ND b II 37–40°C
L. pentosus CIP 103156T ND b II 37–40°C
L. plantarum CNRZ 211T vinegar cabbage b II 30–37°C
L. reuteri CIP 101887T human faeces b III 37°C
L. rhamnosus CIP A157T ND b II 37°C
L. ruminis CIP 103153T bovine rumen b I 30°C
L. suebicus CIP 103411T apple mash b III 37°C
L. zeae CIP 103253 corn steep liquor ND II 37°C
E. coli CIP 54.127 ND 30°C
L. lactis subsp. cremoris CNRZ 105T ND 30°C
L. lactis subsp. lactis CNRZ 142T ND 30°C
L. mesenteroides subsp. cremoris CNRZ 361T lactic starter 25°C
S. thermophilus CNRZ 1358T pasteurised milk 37°C
C. pissicola CIP 103158T kidney 30°C
P. pentosaceus CIP 102260T dried American beer yeast 30°C
B. bifidum CIP 56.7T child faeces 37°C
W. confusa DSMZ 20196 ND 30°C
Enterococcus hirae CIP 58.55 ND 30°C
S. aureus CIP 53.154 ND 37°C
L. monocytogenes CIP 105457 ovine (brain) 37°C
Species Strain designationa Origin Phylogenetic groupb Fermentative groupc Optimal temperatured
L. acidophilus CNRZ 204T human a I 37–45°C
L. amylovorus CIP 102989T cattle (waste-corn fermentation) a I 37–45°C
L. brevis CIP 102806T human faeces b III 30–37°C
L. buchneri CIP 103023T tomato purée b III 30–37°C
L. casei subsp. casei CNRZ 313T cheese b II 30–37°C
L. coryniformis subsp. torquens CIP 103134T air contaminant (dairy barn) b II 30–37°C
L. delbrueckii subsp. bulgaricus CIP 101027T Bulgarian yoghurt a I 37–45°C
L. delbrueckii subsp. delbrueckii CNRZ 225T sour grain mash a I 37–45°C
L. delbrueckii subsp. lactis CNRZ 207T cheese (Emmental swiss) a I 37–45°C
L. farciminis CIP 103136 sausage b I 30°C
L. fermentum CNRZ 209T fermented beets b III 37–45°C
L. fructosus CIP 102985T flowers c III 30°C
L. gallinarum CIP 103611T normal chicken crop a I 37°C
L. graminis CIP 105164T grass silage a II 30–35°C
L. helveticus CNRZ 223T cheese (Emmental swiss) a I 37°C
L. johnsonii CIP 103653 ND a I 37°C
L. paracasei subsp. paracasei CNRZ 763 ND b II 37–40°C
L. pentosus CIP 103156T ND b II 37–40°C
L. plantarum CNRZ 211T vinegar cabbage b II 30–37°C
L. reuteri CIP 101887T human faeces b III 37°C
L. rhamnosus CIP A157T ND b II 37°C
L. ruminis CIP 103153T bovine rumen b I 30°C
L. suebicus CIP 103411T apple mash b III 37°C
L. zeae CIP 103253 corn steep liquor ND II 37°C
E. coli CIP 54.127 ND 30°C
L. lactis subsp. cremoris CNRZ 105T ND 30°C
L. lactis subsp. lactis CNRZ 142T ND 30°C
L. mesenteroides subsp. cremoris CNRZ 361T lactic starter 25°C
S. thermophilus CNRZ 1358T pasteurised milk 37°C
C. pissicola CIP 103158T kidney 30°C
P. pentosaceus CIP 102260T dried American beer yeast 30°C
B. bifidum CIP 56.7T child faeces 37°C
W. confusa DSMZ 20196 ND 30°C
Enterococcus hirae CIP 58.55 ND 30°C
S. aureus CIP 53.154 ND 37°C
L. monocytogenes CIP 105457 ovine (brain) 37°C

ND, not determined.

aCIP, Collection de l'Institut Pasteur, Paris, France; CNRZ, Centre National de Recherches Zootechniques, Jouy-en-Josas, France; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.

bPhylogenetic group: a, L. delbrueckii group; b, L. casei-Pediococcus group; c, Leuconostoc group[19].

cFermentative group: I, obligatory homofermentative; II, facultatively heterofermentative; III, obligatory heterofermentative.

2.2 DNA isolation

Bacteria cells were grown in 10 ml MRS broth or BHI broth for 18 h at their optimal temperature. A 1.5 ml aliquot of each culture was centrifuged at 6000×g (4°C, 10 min; Sigma Laborzentrifugen 3K10, Bioblock Scientific, Illkirsch, France). Cells were then washed with 1 ml of sodium chloride solution (0.9% w/v) and further centrifuged (4°C, 6000×g, 10 min). The supernatant was discarded and the pellet was washed once again. The biomass pellet was stored at −20°C until further use. It was resuspended in 567 μl of freshly prepared lysis solution (3 mg ml−1 lysozyme (Fluka, St Quentin Fallavier, France), in sterile TE buffer (Tris 10 mM, EDTA 1 mM) pH 8.5) and 30 μl of proteinase K solution (20 mg ml−1; Sigma Aldrich) and incubated for 1 h at 37°C. Cell debris was removed by precipitation with 150 μl of 5 M NaCl and 80 μl of a hot (65°C) cetyltrimethylammonium bromide/NaCl solution (10% CTAB/0.7 M NaCl; Sigma Aldrich). After 10 min at 65°C, 50 μl of RNase solution (3 mg ml−1 in sterile TE; Sigma Aldrich) was added and the mixture was further incubated for 1 h at 37°C. DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1; Fluka), precipitated with isopropanol, washed with ethanol (70%) and dried under vacuum[21]. The DNA pellet was resuspended in 25 μl of sterile distilled water and stored at −20°C.

2.3 Oligonucleotide primers

All primers used in this study were obtained from Invitrogen (Cergy Pontoise, France). The sequences of the 16S/23S ribosomal RNA intergenic spacer region of phylogenetically related species were retrieved from GenBank (http://www.ncbi.nlm.nih.gov) and used to perform multiple alignments using Clustal W[22]. A potential target site, starting at position 70 of Lactobacillus acidophilus (U32971), was selected. A 21-mer polymerase chain reaction (PCR) primer was designed based on this site and called LbLMA1-rev (5′-CTC AAA ACT AAA CAA AGT TTC-3′) (Fig. 1). A second primer R16–1 (5′-CTT GTA CAC ACC GCC CGT CA-3′)[3] corresponding to the flanking terminal sequence of the 16S rRNA gene and conserved among various bacteria, including lactobacilli, was used.

Nucleotide sequences of the 16S to 23S spacer region of L. acidophilus (U32971), L. amylovorus (AF182732), L. brevis (X74221), L. casei subsp. casei (Z75478), L. crispatus (AF074857), L. curvatus (U97135), L. delbrueckii subsp. bulgaricus (Z75475), L. fermentum (AF080099), L. gasseri (AF074859), L. graminis (U97136), L. hamsteri (AF113601), L. helveticus (Z75482), L. heterohiochii (X74223), L. japonicus (X74222), L. jensenii (AB035486), L. johnsonii (AF074860), L. paracasei subsp. paracasei (AF182724), L. paraplantarum (U97138), L. pentosus (U97134), L. plantarum (U97139), L. reuteri (AF080100), L. rhamnosus (AF121201), L. ruminis (AF080103), L. sakei (U97137), L. salivarius (AF113600), L. sharpeae (AF074861), L. vaginalis (AF182731), L. zeae (AF074862), L. mesenteroides subsp. cremoris (Z75494), S. thermophilus (U32965), L. lactis subsp. lactis (AF284218), E. coli (U55299), E. faecalis (X87182) and L. monocytogenes (U44063). Sequences were aligned with Clustal W to a maximum homology[22]. Gaps introduced to maintain alignment are indicated by ‘-’.

1

Nucleotide sequences of the 16S to 23S spacer region of L. acidophilus (U32971), L. amylovorus (AF182732), L. brevis (X74221), L. casei subsp. casei (Z75478), L. crispatus (AF074857), L. curvatus (U97135), L. delbrueckii subsp. bulgaricus (Z75475), L. fermentum (AF080099), L. gasseri (AF074859), L. graminis (U97136), L. hamsteri (AF113601), L. helveticus (Z75482), L. heterohiochii (X74223), L. japonicus (X74222), L. jensenii (AB035486), L. johnsonii (AF074860), L. paracasei subsp. paracasei (AF182724), L. paraplantarum (U97138), L. pentosus (U97134), L. plantarum (U97139), L. reuteri (AF080100), L. rhamnosus (AF121201), L. ruminis (AF080103), L. sakei (U97137), L. salivarius (AF113600), L. sharpeae (AF074861), L. vaginalis (AF182731), L. zeae (AF074862), L. mesenteroides subsp. cremoris (Z75494), S. thermophilus (U32965), L. lactis subsp. lactis (AF284218), E. coli (U55299), E. faecalis (X87182) and L. monocytogenes (U44063). Sequences were aligned with Clustal W to a maximum homology[22]. Gaps introduced to maintain alignment are indicated by ‘-’.

2.4 PCR conditions

Amplification was carried out in a thermal cycler PTC 200 (MJC research, Waltham, USA). The reaction mixture (50 μl) contained 25 pmol of each primer, 0.2 mM of each deoxyribonucleotide triphosphate (Invitrogen), 1×PCR buffer without MgCl2, 1.5, 2.0, 2.5 or 3.0 mM MgCl2 depending on experiment, 50–100 ng of bacterial DNA and 2.5 U of Taq DNA Polymerase (Qbiogene, Illkirch, France).

DNA fragments were amplified as follows: initial denaturation at 95°C for 5 min, followed by 20 or 30 cycles consisting of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a 7 min final extension step at 72°C. The products were stored at 4°C until analysis. Aliquots (10 μl) of the amplified products were subjected to electrophoresis in 1% agarose gels (Electrophoresis grade, Invitrogen) in TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.2). Gels were stained with ethidium bromide (5 μg ml−1) and visualised under UV light. A 123 bp polymer was used as a molecular mass marker (Invitrogen).

2.5 Nucleotide sequence accession number

The DDBJ-EMBL GenBank accession numbers of the spacer sequences used in this study are as follows: L. acidophilus (U32971), Lactobacillus amylovorus (AF182732), Lactobacillus brevis (X74221), Lactobacillus casei subsp. casei (Z75478), Lactobacillus crispatus (AF074857), Lactobacillus curvatus (U97135), Lactobacillus delbrueckii subsp. bulgaricus (Z75475), Lactobacillus fermentum (AF080099), Lactobacillus gasseri (AF074859), Lactobacillus graminis (U97136), Lactobacillus hamsteri (AF113601), Lactobacillus helveticus (Z75482), Lactobacillus heterohiochii (X74223), Lactobacillus japonicus (X74222), Lactobacillus jensenii (AB035486), Lactobacillus johnsonii (AF074860), Lactobacillus paracasei subsp. paracasei (AF182724), Lactobacillus paraplantarum (U97138), Lactobacillus pentosus (U97134), Lactobacillus plantarum (U97139), Lactobacillus reuteri (AF080100), Lactobacillus rhamnosus (AF121201), Lactobacillus ruminis (AF080103), Lactobacillus sakei (U97137), Lactobacillus salivarius (AF113600), Lactobacillus sharpeae (AF074861), Lactobacillus vaginalis (AF182731), Lactobacillus zeae (AF074862), Leuconostoc mesenteroides subsp. cremoris (Z75494), Streptococcus thermophilus (U32965), Lactococcus lactis subsp. lactis (AF284218), Escherichia coli (U55299), Enterococcus faecalis (X87182) and Listeria monocytogenes (U44063).

3 Results and discussion

Multiple alignments, carried out with all sequences (28 currently available) of lactobacilli in GenBank, enabled us to design a 21-mer PCR primer, LbLMA1-rev. This was the only lactobacilli-specific sequence identified. We thus chose a universal sequence, R16-1, from the flanking terminal region of the 16S rRNA gene that was conserved among various bacteria, including lactobacilli, as the second primer[3]. Genomic DNA from 24 strains of lactobacilli (Table 1) was amplified using LbLMA1-rev and R16-1. The PCR product obtained was approximately 250 bp long and encompassed the 16S–23S spacer region rDNA with a slight polymorphism (Fig. 2).

PCR amplification products from lactobacilli. 1. 123 bp ladder, 2. L. acidophilus CNRZ 204, 3. L. amylovorus CIP 102989, 4. L. delbrueckii subsp. bulgaricus CIP 101027, 5. L. delbrueckii subsp. delbrueckii CNRZ 225, 6. L. delbrueckii subsp. lactis CNRZ 207, 7. Lactobacillus farciminis CIP 103136, 8. Lactobacillus gallinarum CIP 103611, 9. L. helveticus CNRZ 223, 10. L. johnsonii CIP 103653, 11. L. ruminis CIP 103153, 12. L. casei subsp. casei CNRZ 313, 13. Lactobacillus coryniformis subsp. torquens CIP 103134, 14. L. graminis CIP 105164, 15. L. paracasei subsp. paracasei CNRZ 763, 16. L. pentosus CIP 103156, 17. L. plantarum CNRZ 211, 18. L. rhamnosus CIP A157, 19. L. zeae CIP 103253, 20. Lactobacillus buchneri CIP 103023, 21. L. fermentum CNRZ 209, 22. L. reuteri CIP 101887, 23. Lactobacillus suebicus CIP 103411, 24. no DNA: control.

2

PCR amplification products from lactobacilli. 1. 123 bp ladder, 2. L. acidophilus CNRZ 204, 3. L. amylovorus CIP 102989, 4. L. delbrueckii subsp. bulgaricus CIP 101027, 5. L. delbrueckii subsp. delbrueckii CNRZ 225, 6. L. delbrueckii subsp. lactis CNRZ 207, 7. Lactobacillus farciminis CIP 103136, 8. Lactobacillus gallinarum CIP 103611, 9. L. helveticus CNRZ 223, 10. L. johnsonii CIP 103653, 11. L. ruminis CIP 103153, 12. L. casei subsp. casei CNRZ 313, 13. Lactobacillus coryniformis subsp. torquens CIP 103134, 14. L. graminis CIP 105164, 15. L. paracasei subsp. paracasei CNRZ 763, 16. L. pentosus CIP 103156, 17. L. plantarum CNRZ 211, 18. L. rhamnosus CIP A157, 19. L. zeae CIP 103253, 20. Lactobacillus buchneri CIP 103023, 21. L. fermentum CNRZ 209, 22. L. reuteri CIP 101887, 23. Lactobacillus suebicus CIP 103411, 24. no DNA: control.

The specificity of the primers was evaluated using DNA extracted from the other strains. The presence of non-specific and non-reproducible products (data not shown) led us to modify the PCR conditions. When the concentration of MgCl2 was altered to 2.5 mM and the number of cycles decreased to 20 cycles, the 12 reference strains belonging to the following species: E. coli, L. lactis subsp. lactis, L. lactis subsp. cremoris, L. mesenteroides subsp. cremoris, S. thermophilus, E. hirae, L. monocytogenes, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa and Staphylococcus aureus did not generate an amplicon (Fig. 3). Interestingly, L. mesenteroides subsp. cremoris generated a fragment of approximately 740 bp, although given the similarity between its target sequence (Fig. 1) and that of Lactobacillus a ∼250 bp amplicon could have been expected. When DNA from the Lactobacillus species was used as a template (Fig. 2, data not shown for L. brevis), a ∼250 bp PCR product was obtained for all the tested species except for Lactobacillus fructosus which did not give any product (data not shown). It is interesting to note that this species was the only Lactobacillus species classified in the Leuconostoc group by Vandamme et al. (Table 1)[19]. Moreover, Antunes et al. recently proposed that L. fructosus could be reclassified in the genus Leuconostoc as _Leuconostoc fructosum_[23].

PCR amplification. 1. 123 bp ladder, 2. E. coli CIP 54.127, 3. L. lactis subsp. cremoris CNRZ 105, 4. L. lactis subsp. lactis CNRZ 142, 5. L. mesenteroides subsp. cremoris CNRZ 361, 6. S. thermophilus CNRZ 1358, 7. C. pissicola CIP 103158, 8. P. pentosaceus CIP 102260, 9. B. bifidum CIP 56.7, 10. W. confusa DSMZ 20196, 11. E. hirae CIP 58.55, 12. S. aureus CIP 53.154, 13. L. monocytogenes CIP 105457, 14. L. fermentum CNRZ 209: positive control, 15. no DNA: control.

3

PCR amplification. 1. 123 bp ladder, 2. E. coli CIP 54.127, 3. L. lactis subsp. cremoris CNRZ 105, 4. L. lactis subsp. lactis CNRZ 142, 5. L. mesenteroides subsp. cremoris CNRZ 361, 6. S. thermophilus CNRZ 1358, 7. C. pissicola CIP 103158, 8. P. pentosaceus CIP 102260, 9. B. bifidum CIP 56.7, 10. W. confusa DSMZ 20196, 11. E. hirae CIP 58.55, 12. S. aureus CIP 53.154, 13. L. monocytogenes CIP 105457, 14. L. fermentum CNRZ 209: positive control, 15. no DNA: control.

We defined a couple of PCR primers that amplified DNA only from bacteria belonging to the Lactobacillus group of LAB and did not amplify other strains closely related to lactobacilli. Although not all the species from the Lactobacillus genus, which now includes 85 species and 15 subspecies (http://www.bacterio.cict.fr/index.html, June 18, 2002), were tested in this study, the primers proved to be useful in the analysis of 23 lactobacilli strains, corresponding to 21 species, from various origins, representing the three phylogenetic groups and fermentative groups, respectively [[9,[4]. Thus, we showed that a LbLMA1-rev/R16-1-based PCR could be a useful tool for identification of the members of the Lactobacillus genus.

Acknowledgements

Dr S. Gente, V. Coeuret and S. Larpin are gratefully acknowledged for helpful discussions and for critical reading of the manuscript. Our thanks also go to Dr V. Pichereau for helpful discussions. This study was supported in part by the Syndicat Normand des Fabricants de Camembert (SNFC), the Institut National de la Recherche Agronomique (INRA) and the Conseil Régional de Basse-Normandie, France.

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