Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese

Abstract

Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.Staphylococcus aureus is a significant bacterial pathogen producing a variety of proteins and toxins that contribute to its ability to colonize and cause diseases (15). Some S. aureus strains are able to produce staphylococcal enterotoxins (SEs) in food matrices and are responsible for food poisoning, characterized by such symptoms as nausea, vomiting, abdominal cramps, and diarrhea (2). In France S. aureus is reported as the most frequent pathogen involved in food-borne diseases associated with dairy products (12) and especially with raw milk cheeses (9). It is generally accepted that SE production constitutes a risk when S. aureus bacteria exceed a threshold of 10 5 S. aureus CFU per gram of cheese during manufacture. Numerous studies have reported on S. aureus behavior during cheese manufacturing, focusing only on S. aureus growth and enterotoxin production (1, 8, 11, 18, 21, 22, 31, 32, 34-36, 42, 43, 49). To our knowledge, no study has investigated the expression of the genes encoding SEs in foods or during food production. Numerous parameters, such as pH, aeration, or temperature, could, indeed, affect the expression of these genes. During the cheese-making process, natural staphylococcal contamination is minor in the total microbial population. The initial S. aureus contamination is usually below 10 3 CFU/ml of raw milk, while bacterial starters are inoculated at least at 10 6 CFU/ml of milk. Analysis of SE gene expression in situ thus requires an efficient method of extracting bacterial RNA from cheese to ensure recovery of quantifiable amounts of staphylococcal RNA. A precise method is also needed to quantify minor transcripts in the extracted bact...

The formation ofStaphylococcus aureusenterotoxin in food environments and advances in risk assessment

“…129 This indicates that quantitative details for the population sensing mechanisms, and its relationship with the environment and with toxin types, will be necessary to inform improved risk assessments. Molecular methods have been used to identify differential expression of the staphylococcal enterotoxin genes during cell growth 58 and at explicit points during cheese making; 138 the details of transcription for SE genes can potentially inform improved models for toxin production and, hence, improved risk assessments.…”

Expression of toxic shock syndrome toxin-1 gene of Staphylococcus aureus in milk: Proof of concept

“…In contrast, the increase in storage temperature and storage time of both, pasteurized and UHT milk, resulted in increased number of S. aureus, as also observed by Janštová et al (2012). The number of S. aureus of pasteurized and UHT milk cultured at 15 °C and 22 °C for 24 h, exceeded the threshold of 10 5 CFU mL -1 , which represents the risk for enterotoxin production (Duquenne et. al., 2010).…”

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

“…This decrease probably reflected the global decrease of mRNA transcription in the cheese matrix, after the end of growth of L. lactis. Duquenne et al (2010) were able to quantify the expression of Staphyloccus aureus enterotoxins genes in model cheeses using a set of three stably expressed reference genes. A similar approach was applied for the study of the growth of L. lactis subsp.…”

“…RNA extraction methods involving prior separation of the cells from cheeses and other dairy products have been used in several studies (Randazzo et al, 2002;Bleve et al, 2003;Sanchez et al, 2006;Bogovic Matijasic et al, 2007;Smeianov et al, 2007;Makhzami et al, 2008;Rantsiou et al, 2008a;Rantsiou et al, 2008b;Ulvé et al, 2008;Duquenne et al, 2010;Falentin et al, 2010;Cretenet et al, 2011;La Gioia et al, 2011;Masoud et al, 2011;Taïbi et al, 2011). The recovery of microbial cells is done following similar protocols than for DNA extraction methods (see above).…”

“…Sanchez et al (2006) recovered lactic acid bacteria cells from milk cultures after dispersion of caseins with EDTA, and extracted RNA using guanidinium thiocyanate-phenol-chloroform (commercial TRIzol reagent), a reagent that inactivates cellular processes and allows separation of RNA from DNA and proteins (Chomczynski and Sacchi, 1987). Duquenne et al (2010) also used this type of extraction, after disruption of the cells by bead-beating. Bacterial cells may also be separated from cheese matrices using a Nycodenz gradient (Makhzami et al, 2008).…”