Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

Abstract

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

Sources of Clostridia in Raw Milk on Farms

“…On the basis of 16S rRNA gene sequence analyses, 73 out of 152 validly described species fall within cluster I, often referred to as Clostridium sensu stricto (9,39). Spores of clostridia belonging to cluster I are responsible for spoilage of cheeses with long ripening times, causing the so-called late blowing defect (17,22,38). Specifically, species able to convert lactic acid to butyric acid, carbon dioxide, and hydrogen at relatively low pH are detrimental (22,38).…”

“…Spores of clostridia belonging to cluster I are responsible for spoilage of cheeses with long ripening times, causing the so-called late blowing defect (17,22,38). Specifically, species able to convert lactic acid to butyric acid, carbon dioxide, and hydrogen at relatively low pH are detrimental (22,38). Clostridium tyrobutyricum is considered the primary cause of late blowing.…”

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

“…In a study of the bacterial community from an artisanal Sicilian cheese, Randazzo et al (2002) compared the intensity of bands from DNA and RNA-derived DGGE profiles and concluded that some species of the samples were not very metabolically active. Other studies of RNA profiles involving either DGGE (Rantsiou et al, 2008b;Dolci et al, 2010;Masoud et al, 2011), TTGE (Le Bourhis et al, 2007), SSCP (Le Bourhis et al, 2005), T-RFLP (Sanchez et al, 2006), clone library sequencing (Carraro et al, 2011) or pyrosequencing (Masoud et al, 2011) have been published.…”

“…Denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and temporal temperature gradient gel electrophoresis (TTGE) are widely used to study cheese microbial communities Ercolini et al, 2001;Ogier et al, 2002;Randazzo et al, 2002;Ercolini et al, 2003;Mauriello et al, 2003;Andrighetto et al, 2004;Cocolin et al, 2004;Ercolini et al, 2004;Henri-Dubernet et al, 2004;Lafarge et al, 2004;Ogier et al, 2004;Rantsiou et al, 2004;Le Bourhis et al, 2005;Flórez and Mayo, 2006;Randazzo et al, 2006;Cocolin et al, 2007;El-Baradei et al, 2007;Le Bourhis et al, 2007;Parayre et al, 2007;Abriouel et al, 2008;Bonetta et al, 2008;Ercolini et al, 2008;Gala et al, 2008;Henri-Dubernet et al, 2008;Nikolic et al, 2008;Rantsiou et al, 2008b;Van Hoorde et al, 2008;Alegría et al, 2009;Casalta et al, 2009;Dolci et al, 2009;Giannino et al, 2009;Serhan et al, 2009;Dolci et al, 2010;Fontana et al, 2010;Fuka et al, 2010;Randazzo et al, 2010;Van Hoorde et al, 2010;Masoud et al, 2011). Target sequences from rRNA or housek...…”

“…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 …”