16S–23S rDNA Intergenic Spacer Region Polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as Revealed by PCR and Nucleotide Sequence Analysis Giuseppe Blaiotta, Olimpia Pepe, Gianluigi Mauriello, Francesco Villani, Rosamaria Andolfi, and Giancarlo Moschetti Dipartimento di Scienza degli Alimenti, Sezione di Microbiologia Agraria, Alimentare ed Ambientale e di Igiene, Università degli Studi di Napoli “Federico II”, Portici, Italy Received: August 15, 2002 Summary The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differen- tiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 differ- ent species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leu- conostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of “Fior di Latte” cheese were analyzed. After 16S–23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nu- cleotide analysis show that the 16S–23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S–23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates. Key words: 16S–23S rDNA ISR – L. lactis – L. garvieae – L. raffinolactis – S. thermophilus – S. suis – S. parauberis Introduction Lactococcus (L.) garvieae and L. raffinolactis are com- monly isolated, as “non-dominant” species, in the dairy en- vironment such as natural starter cultures, raw milk, curd and cheese [23, 32, 39, 43]. However, L. garvieae is also considered an emerging zoognotic pathogen that has been isolated from cattle, fish and humans [6, 7, 16, 29, 48] even though their role as infection agents remains unclear [1]. L. garvieae, L. raffinolactis and L. lactis are genealogi- cally quite distinct but phenotypically closely related, making their differentiation on the basis of phenotypic criteria very difficult [13, 40]. Ribosomal RNA gene re- striction patterns and rRNA-targeted oligonucleotide probes allow the five currently recognized lactococcal species to be distinguished [28, 40]. It is well known that non-starter lactic acid bacterial floras, particularly raw milk microflora, increase the di- versity of cheese flavors and may also be involved in pro- ducing the typical organoleptic characteristics of cheeses during ripening [12, 31, 18]. 0723-2020/02/25/04-520 $ 15.00/0 System. Appl. Microbiol. 25, 520–527 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam Therefore, it is of primary importance to obtain a reli- able description of the physiologically active microbial community in order to understand the role that the differ- ent species of lactic acid bacteria play in dairy fermenta- tion. Classically, such questions are addressed through the enumeration of some microbial groups on a variety of culture media, followed by identification through tradi- tional microbiological methods. This approach is general- ly time-consuming, and only a limited number of isolates can be identified. Molecular identification methods are a powerful alter- native to the conventional differentiation of bacteria by plating especially when closely related species are ana- lyzed. Detecting and identifying various species with rapid methods is also important for in vivo monitoring fermentation processes and for quality control (especially in mixed cultures). At species level there are several reports on specific identification systems for LAB, mainly based on 16S ribo-