A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of theseLactic acid bacteria (LAB) have been the focus of extensive research due to their value in the food-processing industry (14,27). One of the most recent taxonomic additions to the LAB group is the genus Carnobacterium. Members of this genus are widespread in nature, and the habitats of these organisms range from foods, such as poultry, meat, cheese, and seafood, to fish intestines and anoxic Antarctic lake water (2,10,16,26,29). The four species of Carnobacterium isolated from food are Carnobacterium divergens, C. piscicola, C. gallinarum, and C. mobile (6). Most research on the genus Carnobacterium has been focused on the production of bacteriocins and the regulation of metabolic enzymes (9, 28). In the last few years, many ways to differentiate Carnobacterium species have been developed. The phylogenetic interrelationships of species in the genus Carnobacterium have been investigated by using numerical taxonomic matrices and sequencing of the 16S rrn segments encoding mature rRNAs (19,41). The Carnobacterium species form a phylogenetically coherent group, which is quite distinct from all other LAB. However, the number of polymorphic sites in the 16S rRNA of Carnobacterium species is rather low, since some species have the same sequence (the sequences of C. piscicola and C. gallinarum are 98% identical) and other species exhibit very high degrees of sequence similarity (the sequences of C. divergens and C. mobile are 96% similar) (41). Thus, it is difficult to define specific 16S RNA rrn sequences that can be used for differentiation of these closely related species. Carnobacteria have been identified at the genus level by nucleic acid hybridization by using 16S rRNA-targeted genus-specific probes (32). Species-specific primers have been designed by using the domains that exhibit low homology in the 16S ribosomal DNA (rDNA) sequences of species of Carnobacterium (1, 4). However, the PCR primers used in these studies were not specific enough to differentiate Carnobacterium spp. from other genera of LAB. Data obtained with the randomly amplified polymorphic DNA PCR technique enable only C. divergens to be differentiated from other Carnobacterium species (20).In view of this, a study of the more variable sequences in the rRNA operon of phylogenetically closely related Carnobacterium species could be useful. In most prokaryotes, the ribosomal genes form an operon with the order 5Ј-16S-23S-5S-3Ј, and the genes are separated by two intergenic spacer regions (ISRs). ISRs, especially those located between the 16S and 23S rDNAs, are thought to be under less evolutionary pressure and, therefore, to provide great...

Aims:  The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy‐to‐perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S‐23S rDNA intergenic spacer region (ISR). Methods and Results:  PCR amplification of the 16S‐23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro‐organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L‐ISR), containing tRNAIle and tRNAAla genes; small spacer (S‐ISR) without tRNA genes. The third described spacer (medium, M‐ISR), original for L. sanfranciscensis, harboured a tRNA‐like structure. An oligonucleotide sequence targeting the variable region between tDNAIle and tDNAAla of L. sanfranciscensis L‐ISR was approved to be suitable in species‐specific identification procedure. Analysis by pulse‐field gel electrophoresis of the chromosomal digest with the enzyme I‐CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1·3 Mb. Conclusions:  Direct amplification of 16S‐23S ISRs or PCR with specific primer derived from L‐ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. Significance and Impact of the Study:  In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.

Aims:The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. Methods and Results: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR Ò 2AE1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. Results and Conclusions: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. Significance and Impact of the Study: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA Ala and tRNA Ile , which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.