Automated ribotyping and random ampli®ed polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy A. De Cesare 1 , G. Manfreda 1 , T.R. Dambaugh 3 , M.E. Guerzoni 2 and A. Franchini 1 1 Dipartimento di Scienze degli Alimenti, 2 Dipartimento di Protezione e Valorizzazione AgroAlimentare, University of Bologna, Italy, and 3 Qualicon Inc., Wilmington, DE, USA 758/1/01: received 31 January 2001, revised 3 April 2001 and accepted 2 May 2001 A. DE CESARE, G. MANFREDA, T.R. DAMBAUGH, M.E. GUERZONI AND A. FRANCHINI. 2001. Aims: The ability of automated ribotyping and random ampli®ed polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. Methods and Results: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identi®ed among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter Ò , and 25 subtypes using RAPD. Conclusions: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). Signi®cance and Impact of the Study: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a speci®c source and location, and provide powerful tools for epidemiological investigations. INTRODUCTION Epidemiological studies of Salmonella infections require the use of ef®cient markers to trace precisely the diffusion of strains. Since genetic differences between isolates may not encode differences in phenotypic markers (i.e. phage types, antigens, enzymes or isozymes, antimicrobial susceptibility, or metabolic pro®les), phenotypic techniques have limited utility for subtyping of bacterial isolates. As subtypes must, by de®nition, differ in DNA, genotype subtyping approa- ches are characterized by the greatest discriminatory power (Lin et al. 1996). A variety of DNA-based typing methods has been applied to characterize Salmonella species (Schmidt 1994; Farber 1996). Among these are ribotyping and random ampli®ed polymorphic DNA (RAPD). Automated ribotyping is possible using the RiboPrinter Ò system, which analyses genomic fragments generated by restriction digestion of the rRNA operons, highly conserved and present in several copies on the bacterial chromosome (Woese 1987). The most conserved regions of the operons allow identi®cation of a bacterium to genus and species level, whereas variable regions of rRNA operons and ¯anking regions permit discrimination between strains (Bruce 1996). Automated ribotyping has been used to characterize bacteria such as Pseudomonas aeruginosa (Blanc et al. 1994), Listeria monocytogenes (Allerberger et al. 1999) and Campylobacter jejuni (Lindstedt et al. 2000). Randomly ampli®ed polymorphic DNA (RAPD) analysis, also known as arbitrarily primed-polymerase chain reaction (AP-PCR), is a PCR-based assay that has been developed to detect polymorphisms in genomic DNA (Welsh and McClelland 1990; Williams et al. 1990). The technique relies on the presence of priming sites on the genome, close enough to permit PCR ampli®cation using single primers of arbitrary nucleotide sequence. RAPD analysis has been used for epidemiological subtyping of food hygiene-relevant species, such as Listeria (Mazurier and Werners 1992b; Levett et al. 1993; Czajka and Batt 1994; Farber and Addison 1994), Campylobacter (Mazurier et al. 1992a), Correspondence to: A. De Cesare, Dipartimento di Scienze degli Alimenti, University of Bologna, Via S. Giacomo 9, 40126 Bologna, Italy (e-mail: decesare@alma.unibo.it). ã 2001 The Society for Applied Microbiology Journal of Applied Microbiology 2001, 91, 780±785