Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos - and pac -Type Streptococcus thermophilus Bacteriophages

Abstract

The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described.Streptococcus thermophilus is a gram-positive thermophilic lactic acid bacterium used, along with Lactobacillus spp., as a starter culture for the manufacture of important fermented dairy foods, including yogurt and Swiss-or Italian-type hard cooked cheeses (5). Unfortunately, these bacteria are susceptible to infection by bacteriophages during the fermentation process, a phenomenon that ultimately results in fermentation failure. The common features of S. thermophilus phages include double-stranded DNA genomes that are 31 to 45 kb long, small isometric heads, long noncontractile tails, and affiliation with the Siphoviridae family, corresponding to Bradley's group B (3). They are currently divided into two groups (cos and pac types) based on the genome encapsidation machinery (11). In the case of yogurt isolates, these types are also related to host range and serotype (4).Fast detection methods are an important tool to avoid phage attacks in dairy factories. Detection of bacteriophages in milk is normally carried out using standard microbiological methods (plaque assays, activity tests, etc.) (8), but these methods are time-consuming. To speed up the analysis, PCR techniques have been used to detect phages in different kinds of dairy samples (1,4,6,7,10,12). Increasing demand for quantitative, more sensitive, and quicker procedures is prompting the development of real-time quantitative PCR (qPCR) methods. The objective of the present study was to develop a fast multiplex qPCR method that allows quantitative detection and identification of cos-and pac-type S. thermophilus bacteriophages in milk samples.Primer and probe design. In the first step, databases were screened to select the most conserved genes of cos-and pactype S. thermophilus phages. orf1510 encoding the putative minor tail protein of the Sfi11 bacteriophage (pac type) and orf18 encoding the antireceptor protein of the Sfi21 bacteriophage (cos type) were selected and aligned, using the CLUSTAL W algorithm (14), with the sequences of the orthologous genes available in the GenBank database. Highly similar sequences were selected to design primers qPac1, qPac2, qCos1, and qCos2 and probes mgbPac2 and mgbCos (Table 1) using Primer Express software (Applied Biosystems, Warrington, United Kingdom). The species specificity of the primers was assessed by using BLAST 2.2.15 (Basic Local Alignment Search Tool) to ensure that they amplify only the corresponding S. thermophilus bacteriophage sequences. Both the mgbPac2 and mgbCos probes were synthesized with a minor groove binder (MGB) nonfluorescent quencher attached to the 3Ј end and with a different reporter dye attached to the 5Ј end (VIC and 6-carboxyfluorescein [FAM], respectively) in order to combine them in the...

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Streptococcus thermophilus phage monitoring in a cheese factory: Phage characteristics and starter sensitivity

“…Nowadays, the knowledge of eight complete genomic sequences for S. thermophilus phages (four cos-and four pac-type) has allowed the development of different molecular methods to detect, identify, characterize and quantify them, facilitating the classification of S. thermophilus bacteriophages according to diverse criteria (Binetti et al, 2005;Brü ssow et al, 1994;del Río et al, 2007del Río et al, , 2008Duplessis and Moineau, 2001;Quiberoni et al, 2006). Moreover, some studies have addressed the biological function of phage gene products.…”

Detection of Airborne Lactococcal Bacteriophages in Cheese Manufacturing Plants

“…Every phage genome detected in qPCR analysis of the BioSampler samples represented 10 4 phage genomes/m 3 of air. Only 1 sample out of 18 allowed the detection of airborne 936-like phages, whereas 3 out of 18 qPCRs revealed c2-like phages.…”

“…For example, several strains of the low-GC Gram-positive bacterium Lactococcus lactis are extensively used for the transformation of milk into cheeses. The lactococcal starter culture population can reach levels of up to 10 9 CFU/ml of milk inside large industrial fermentation vats (24). This ecosystem is also ideal for the multiplication of virulent L. lactis phages, which are ubiquitous in dairy environments (4).…”

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