Use of a species-specific multiplex PCR for the identification of pediococci

Abstract

In this study, the 23S rRNA genes of nine different Pediococcus type strains were sequenced. By using a multiple sequence alignment with 23S rDNA sequences of related lactic acid bacteria two primer pairs were constructed, one for the general identification of the genus Pediococcus and one for the identification of the atypical species, P. dextrinicus. Furthermore, a primer set for a rapid multiplex PCR identification of the eight typical Pediococcus species was developed. With this technique, the species P. damnosus, P. parvulus, P. inopinatus, P. cellicola, P. pentosaceus, P. acidilactici, P. claussenii, and P. stilesii could be discriminated simultaneously in a single PCR. Experiments with inoculated grape musts showed that the detection limit was 10 cells ml^− 1. The multiplex PCR assay was tested by the usage of 62 Pediococcus strains from different culture collections and 47 strains recently isolated from German wines and musts. In addition, contaminations with P. parvulus and P. damnosus could be detected after purification of DNA from spoilt wine samples. The method demonstrates a rapid and easy to handle tool for the species affiliation of pediococci in beverages and food samples.

Introduction

Pediococci are Gram-positive lactic acid bacteria (LAB). They are homofermentative as regards their glucose metabolism and grow under facultative aerobic to microaerophilic conditions. Most of the species can be found on plants and particularly occur during fermentation processes (Garvie, 1986). Currently ten species are recognized, comprising P. damnosus, P. parvulus, P. inopinatus, P. cellicola, P. ethanolidurans, P. claussenii, P. stilesii, P. acidilactici, P. pentosaceus and P. dextrinicus. On the basis of its 16S rDNA sequence, P. dextrinicus appears to be only distantly related to the genuine pediococci (Dobson et al., 2002). It has been proposed that this species should be reclassified in the genus Lactobacillus (Collins et al., 1991, Stiles and Holzapfel, 1997).

The occurrence of Pediococcus species during the fermentation of grapes (Weiller and Radler, 1970, Back, 1978, Beneduce et al., 2004), and in spoilt beer (Back, 1978, Dobson et al., 2002), has often been reported. Particularly strains of the species P. parvulus, P. damnosus and P. inopinatus have been isolated from grape musts and wines (Peynaud and Domercq, 1967, Back, 1978, Weiller and Radler, 1970, Edwards and Jensen, 1992, Rodas et al., 2003, Beneduce et al., 2004). Although they can be involved in malolactic fermentation in wine, pediococci are mostly undesirable because of their formation of metabolic compounds such as diacetyl and acetoin (Wibowo et al., 1985). Furthermore, the possibility of exopolysaccharide synthesis by some strains can cause ropiness, characterized by a viscous and thick texture of the beverage (Walling et al., 2005). Another detrimental influence on wine quality is the formation of biogenic amines such as histamine (Landete et al., 2005) as a result of amino acid decarboxylase activity of some strains (Bover-Cid and Holzapfel, 1999). P. acidilactici and P. pentosaceus are widely used as starter cultures in the fermentation processes of meat (Raccach, 1987, Luchansky et al., 1992, Kang and Fung, 1999), milk (Bhowmik and Marth, 1990, Back, 1999) and plant products (Gibbs, 1987). Besides the production of lactic acid, some strains of P. acidilactici produce antibacterial compounds like bacteriocins (Stiles, 1996, Albano et al., 2007). Even though pediococci can mostly be found on plants and plant products, they have already been isolated from animals (Juven et al., 1991, Kurzak et al., 1998, Simpson et al., 2002) and human sources (Barros et al., 2001, Walter et al., 2001).

There are various molecular identification methods for Pediococcus species, such as sequence analyses of the 16S rDNA, the internal transcribed spacer (ITS) regions and the heat-shock protein 60 gene (Dobson et al., 2002). Fingerprinting methods like ribotyping (Satokari et al., 2000, Barney et al., 2001), restriction analysis of the amplified 16S rDNA (16S-ARDRA) (Rodas et al., 2003), randomly amplified polymorphic DNA (RAPD)-PCR (Nigatu et al., 1998, Mora et al., 2000, Simpson et al., 2002), and pulsed-field gel electrophoresis (PFGE) (Luchansky et al., 1992, Barros et al., 2001, Simpson et al., 2002) were useful for the discrimination of strains at the species and intra-species level.

Up to date, no molecular method has been described that provides distinct PCR identification of all Pediococcus species and which is suitable for routine application in the food industry. In the present study we sequenced and analyzed the large subunit (LSU) rRNA gene sequences from nine Pediococcus type strains to construct genus- and species-specific PCR primers. The primers could be applied successfully in a multiplex PCR assay for a rapid and simultaneous identification. To validate the results of species affiliation based on 23S rDNA multiplex PCR, all Pediococcus strains examined in this study were tested with a high-resolution genotypic technique, the specifically amplified polymorphic DNA (SAPD)-PCR (Fröhlich and Pfannebecker, 2006). With both methods, strains of the nine examined Pediococcus species could be successfully identified and distinguished from each other.