Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Purification and cloning of piscicolin 61, a bacteriocin fromCarnobacterium piscicola LV61

Piscicolin 61, a bacteriocin produced byCarnobacterium piscicola LV61, inhibits the growth of strains ofCarnobacterium, Lactobacillus, andEnterococcus. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential hydrophobic interaction and reversed-phase chromatography. The N-terminal amino acid sequence of piscicolin 61 was determined by Edman degradation. The plasmid-located structural gene encoding piscicolin (psc61) was cloned and sequenced. It encoded a primary translation product of 71 amino acid residues, which is cleaved between amino acid residues 18 and 19 to yield the active bacteriocin. The calculatedM _r from the deduced protein sequence, 5052.6, agreed with that obtained by mass spectrometry. Piscicolin 61 did not show any sequence similarities to other known bacteriocins. However, the leader sequence resembled those of the pediocin-like bacteriocins. Piscicolin 61 may be able to form amphiphilic helices and may thus act on the membrane of sensitive cells.     

Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake Lb674

Abstract Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes , was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 ( sakR ) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.     

Biofilm Formation and the Presence of the Intercellular Adhesion Locus ica among Staphylococci from Food and Food Processing Environments

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.     

A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5′ ‘HEAD’ region and a 3′ region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1–2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5′ nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.     

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750

Abstract Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus . Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated ifM_r from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 ( dvn750 ) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M _rmr = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.     

Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

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.