The distinction between viable and dead cells is a major issue in many aspects of biological research. We have developed a novel concept for quantification of viable and dead cells in complex samples. The viable/dead stain ethidium monoazide (EMA) is used in combination with realtime PCR to inhibit amplification of DNA from dead cells that have taken up EMA (Fig. 1). Viable/dead determinations are key issues in many aspects of biological research. The current technologies addressing this important issue have severely limited application ranges (4,5,14,18,19). There are for instance no approaches enabling accurate viable/dead quantifications in mixed cell populations (2, 13).Real-time PCR is the most widely applied technology for direct quantification of cells in mixed samples. Real-time PCR is increasingly being used for direct detection and quantification of pathogens in foods and environmental or clinical samples. Still, a major obstacle with PCR diagnostics is how to distinguish between DNA from viable and dead cells. Intact DNA can be present although the organisms are dead. This is particularly relevant for pathogens subjected to killing treatments such as disinfections or antibiotics. Even greater challenges are encountered with organisms such as Campylobacter jejuni that have specific growth requirements and may enter a state where it is viable and infectious but not cultivable. The lack of viable/dead differentiation has been a serious limitation for the implementation of DNA diagnostics in routine applications (15,19,22).We have recently used ethidium monoazide (EMA)-PCR for qualitative DNA-based viable/dead differentiation of bacteria in pure monoculture models (21). Viable/dead analyses of pure monocultures are not new or novel. A wide range of different approaches exist (2,5,10,12,25,26). Methods for direct quantitative analyses of complex samples, however, are still lacking. Solving these analytical problems would be a major technological breakthrough. We discovered during the work with the monoculture models that EMA-PCR has this potential. Thus, the aim of the present work was to use EMA-PCR to show that it is possible to develop quantitative assays for specific viable and dead bacteria in complex samples with mixed bacterial populations. We developed the assay for the major food-borne pathogenic bacterium C. jejuni due to the apparent need for new viable/dead diagnostics of this bacterium. The conditions analyzed include detection in mixed and natural samples, survival in foods, and after disinfection and antibiotic treatments. This knowledge is crucial both for diagnostics and in the control of C. jejuni.A dynamic range of more than 4 log 10 was obtained for the EMA-PCR viable/dead assay. We were able to reliably quantify the fraction of viable C. jejuni under all conditions tested, including complex samples with mixed populations. This is to our knowledge the first time that quantitative viable/dead information has been obtained from specific bacteria in mixed populations. C. jejuni was used as...

No abstract

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobicinteraction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated Mc of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.

No abstract