Comparison of Statistical Methods for Identification of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium from Randomly Amplified Polymorphic DNA Patterns

Abstract

Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of Streptococcus thermophilus, Enterococcus faecium, and Enterococcus faecalis (G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25-36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of S. thermophilus but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of S. thermophilus, E. faecium, and E. faecalis using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.A large variety of genotypic and phenotypic methods are currently used for the identification and classification of microorganisms (32). Many of these techniques generate complex patterns whose interpretation for classification and identification purposes requires multivariate statistical techniques. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) is one of the most popular genotypic typing techniques. It was developed to reveal intra-and interspecific differences in bacterial genomes (33,35), and since it can be performed not only on purified DNA (35) but also on untreated (19) or lysed cells without DNA extraction (23), it can replace time-consuming restriction endonuclease analysis in strain typing and DNA-DNA hybridization techniques for species identification. In fact, RAPD-PCR has been used for the classification of a variety of food-borne microorganisms, including Saccharomyces spp. Statistical treatment of RAPD-PCR patterns usually involves calculation of a similarity matrix and use of hierarchical cluster analysis for grouping of the patterns. Similarity can be calculated using the formula of Nei and Li (24) when only the presence or the absence of bands is scored (21, 23), while Pearson's product-moment correlation coefficient is used when both the position and the intensity of bands are measured with image analysis software (3,28,31). Although unknown isolates can be assigned to a speci...

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

“…small plasmids could have as many amplicons as large plasmids). These ranges have also been reported for chromosomal DNA (Harrison et al, 1992 ;Roberts & Crawford, 2000 ;Moschetti et al, 2001). In addition, it is possible to distinguish between individual plasmids by noting polymorphisms, apparent among plasmids obtained from different diazotroph strains (Fig.…”

“…The genotypic typing of genomes by RAPD-PCR can reveal intra-and interspecific differences (Williams et al, 1990 ;Welsh & McClelland, 1990). RAPD analyses are increasingly being used to identify micro-organisms (Harrison et al, 1992 ;Moschetti et al, 2001) as well as determine clonal diversity between strains of the same bacterial species (Dye et al, 1995 ;Hyytia et al, 1999 ;Roberts & Crawford, 2000). In addition, RAPD-PCR has been used to compare microbial communities within aquatic (Franklin et al, 1999) and terrestrial (Picard et al, 2000) environments and to generate probes for the spatial and temporal comparison of marine virioplankton (Wommack et al, 1999).…”

Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy Streptococcus thermophilus Strains Reveals Four Different Fermentation Profiles

“…One microliter of the DNA dilution and 25 pmol of primer XD9 (5Ј-GAAGT CGTCC-3Ј; Isogen Bioscience BV, Maarssen, The Netherlands) were used in the PCR. RAPD-PCR amplification with this primer yields distinctive and reproducible patterns and allows the identification of S. thermophilus (15). The PCR was performed with a thermocycler (model 9700; Perkin-Elmer, Shelton, CT) by using 45 cycles of 1 min at 94°C, 1 min at 33°C, and 2 min at 72°C.…”

Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

“…identify the restriction enzyme profiles for E. coli O156:H7 (4), the pyrolysis mass spectra for Mycobacterium tuberculosis complex species (7), bacterial species from randomly amplified polymorphic DNA patterns (14), fatty acid profiles of microbial communities (17), stable low-molecular-weight rRNA from gel electrophoresis patterns (16), and T d from microarray data (25). However, to our knowledge, no study has used the method outlined in this paper to determine the relative importance of inputs to outputs.…”