Application of Single Strand Conformation Polymorphism — PCR method for distinguishing cheese bacterial communities that inhibit Listeria monocytogenes

Abstract

The aim of this study was to compare the microbial communities of different cheeses where Listeria monocytogenes either grew or did not grow. For this purpose, (i) isolates from the most inhibitory cheese ecosystem were identified and their ability to produce anti-Listeria substances was determined, (ii) bacterial communities of cheeses with and without L. monocytogenes growth were compared using the Single Strand Conformation Polymorphism method. The study showed SSCP to be an effective tool for differentiating between the bacterial communities of different cheeses manufactured with the same technology. All the cheeses with the lowest L. monocytogenes counts on day 8 were distinguished by the dominance in their SSCP profiles, after amplification of the V2 region of the 16S rRNA gene, of 3 peaks whose nucleotide sequences comigrated with Enterococcus faecium and Enterococcus saccharominimus, Chryseobacterium sp and Corynebacterium flavescens, Lactococcus garvieae and Lactococcus lactis respectively. However, no anti-Listeria compounds were produced under our experimental conditions. These six bacterial species were inoculated, separately or together, into pasteurised milk and their anti-listerial activity in cheese was evaluated. The area of inhibition between the control and trial curves confirmed that L. monocytogenes is inhibited by E. saccharominimus, C. flavescens, L. lactis, L. garvieae and the mixture of all six bacterial strains. Further studies should be performed to determine the metabolites involved in L. monocytogenes inhibition.

Recent developments in cheese cultures with protective and probiotic functionalities

“…Naturally established cheese microflora were found to be more or less permissive for the growth of pathogenic or spoilage microorganisms [13,85,93,124]. The inhibitory activity was related to biodiversity and/or dynamics of the microflora because no antimicrobial activity was detected with pure cultures of the isolated strains.…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…Single-strand conformation polymorphism-PCR (SSCP-PCR) is another PCR-based method for microbial diversity investigation that has been applied to dairy products (Duthoit et al, 2003;Feurer et al, 2004a;Feurer et al, 2004b;Delbes and Montel, 2005;Duthoit et al, 2005;Callon et al, 2006;Delbes et al, 2007;Saubusse et al, 2007;Mounier et al, 2009). This technique is based on the sequence-dependent differential intra-molecular folding of single strand DNA, which alters the migration speed of the molecules under non-denaturing conditions.…”

“…The hypervariable regions are flanked by conserved sequences, which can serve for amplification with "universal" primers (Baker et al, 2003). The variable V1 Bonetta et al, 2008), V3 Ercolini et al, 2001;Ogier et al, 2002;Duthoit et al, 2003;Ercolini et al, 2003;Mauriello et al, 2003;Andrighetto et al, 2004;Ercolini et al, 2004;Feurer et al, 2004a;Feurer et al, 2004b;Lafarge et al, 2004;Ogier et al, 2004;Duthoit et al, 2005;Flórez and Mayo, 2006;Delbes et al, 2007;El-Baradei et al, 2007;Parayre et al, 2007;Abriouel et al, 2008;Ercolini et al, 2008;Gala et al, 2008;Van Hoorde et al, 2008;Alegría et al, 2009;Casalta et al, 2009;Dolci et al, 2009;Giannino et al, 2009;Mounier et al, 2009;Serhan et al, 2009;Dolci et al, 2010;Fontana et al, 2010;Van Hoorde et al, 2010;Masoud et al, 2011), V2 (Duthoit et al, 2003;Delbes and Montel, 2005;Saubusse et al, 2007), V4-V5 , V1-V3 (Randazzo et al, 2002), V4-V8 (Randazzo et al, 2006), V5-V6 (Le Bourhis et al, 2005;Le Bourhis et al, 2007) and V6-V8 …”

Probiotic Lactococcus lactis: A Review

“…During the last decades, lots of approaches have been introduced under culture independent molecular methods which are frequently used in the identification and characterization purposes of L. lactis such as Culture-Independent Polymerase Chain Reaction (Salbi et al, 2014;) 16S rDNA Sequencing (Aquilanti et al, 2007;Khemariya et al, 2013b), PCR-Denaturing Gradient Gel Electrophoresis (PDGE) (Mrkonjić Fuka et al, 2010;Pogačić et al, 2010;Marui et al, 2015;), PCR-Temporal Temperature Gradient Electrophoresis (TTGE) (ElBaradei et al, 2008), Single-Strand Conformation Polymorphism-PCR (SSCP-PCR) (Saubusse et al, 2007), Real Time PCR (qPCR) (Grattepanche et al, 2005;Ruggirello et al, 2014), Fluorescence in situ Hybridization (FISH) (Miks-Krajnik and Babuchowski, 2014), Amplified Ribosomal DNA Restriction Analysis (Partial ARDRA) (Delgado and Mayo, 2003), Length Heterogeneity PCR (LH-PCR) (Brusetti et al, 2006), Multilocus Sequence Analysis (MLSA) (Rademarker et al, 2007;Khemariya et al, 2012) (GTG) 5 -PCR Fingerprinting (Rademarker et al, 2007;Khemariya et al, 2014), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) (Deveau et al, 2003;Khemariya et al, 2013b), Multiple Locus Minisatelite Typing (MLVA) (Que'ne'e et al, 2005), RAPD-PCR (Randomly Amplified Polymorphic DNA-PCR) (Samaržija et al, 2002), Pulse Field Gel Electrophoresis (PFGE) (Campo et al, 2002), Repetitive Element Sequence based PCR (Rep-PCR) (Fernandez et al, 2010), Comparative Genomic Hybridization (CGH) (Taibi et al, 2010), Nested PCR (Khemariya et al, 2013c), Multiplex PCR (Pu et al, 2002) and Sau-PCR (Corich et al, 2005).…”