With continued development of novel molecular-based technologies for rapid, high-throughput detection of foodborne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. In fact, a number of unique approaches and variations on existing techniques are currently on the market or are being implemented that offer ease of use, reliability, and low cost compared with molecular tools. Approaches that enhance recovery of sublethally injured bacteria, differentiation among species using fluorogenics or chromogenics, dry plate culturing, differentiation among bacteria of interest using biochemical profiling, enumeration using impedence technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are summarized here and discussed in relation to their specific advantages or disadvantages when implemented in a food microbiology setting.

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis.

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid-based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR-based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

Thirty-nine Bacillus strains obtained from a variety of environmental and food sources were screened by PCR for the presence of five gene targets (hblC, hblD, hblA, nheA, and nheB) in two enterotoxin operons (HBL and NHE) traditionally harbored by Bacillus cereus. Seven isolates exhibited a positive signal for at least three of the five possible targets, including Bacillus amyloliquefaciens, B. cereus, Bacillus circulans, Bacillus lentimorbis, Bacillus pasteurii, and Bacillus thuringiensis subsp. kurstaki. PCR amplicons were confirmed by restriction enzyme digest patterns compared to a positive control strain. Enterotoxin gene expression of each strain grown in a model food system (skim milk) was monitored by gene-specific reverse transcription-PCR and confirmed with the Oxoid RPLA and Tecra BDE commercial kits. Lecithinase production was noted on egg yolkpolymyxin B agar for all strains except B. lentimorbis, whereas discontinuous beta hemolysis was exhibited by all seven isolates grown on 5% sheep blood agar plates. The results of this study confirm the presence of enterotoxin genes in natural isolates of Bacillus spp. outside the B. cereus group and the ability of these strains to produce toxins in a model food system under aerated conditions at 32°C.Bacillus cereus is traditionally considered the most problematic member of the genus Bacillus to the food industry due to the ability of many strains to produce enterotoxins, a topic which has been reviewed recently (9,11,14,24). B. cereus may express at least two distinct multiple-component enterotoxins, the genes for which have been cloned and sequenced (13,16,29). A tripartite hemolytic heat-labile enterotoxin designated HBL is the product of an operon that includes hblA, hblD, and hblC, which encode the binding subunit (B) and the L 1 and L 2 lytic components, respectively (17, 29). Additionally, a nonhemolytic enterotoxin (NHE) operon has recently been characterized (13). The subunits of the B. cereus NHE also include two apparent lytic components, NH 1 and NH 2 , and a third gene product that remains uncharacterized. In addition, a third enterotoxin has been described that is composed of a single 41-kDa subunit (2). The exact role of this toxin, BceT, is still unclear compared to what is known about HBL and NHE subunit enterotoxins.Consumption of enterotoxigenic Bacillus spp. at high cell densities results in symptoms of diarrhea, with possible vomiting from a separate heat-stable emetic toxin (3, 10). Symptoms may appear 10 to 14 h following ingestion of foodstuffs contaminated with enterotoxigenic strains. Foods most often implicated in the diarrheal syndrome include poultry, cooked meats, soups, desserts, and occasionally fluid and dry milk products (19,20). The infective dose is high (ca. Ͼ10 6 CFU/g) because symptoms rely on the ingestion of the viable cells or spores, not the preformed toxin, in affected foods (12). Such food may pose a threat to consumers if the product has been temperature abused during shipment or storage or when psychrotrophic str...

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens,Escherichia coli O157:H7 and enterotoxigenicStaphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

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Polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR using primers targeting 16S rRNA sequences in Escherichia coli O157:H7 were applied to monitor the stability of rDNA and rRNA in cells killed by mild heat treatment (60 degrees C) in skim milk. Serial dilutions of purified RNA and DNA from E. coli O157:H7 in skim milk were amplified by RT-PCR or PCR, respectively, before heat treatment and at time points 0, 6, 12, 24, and 48 h after heating. In general, DNA-PCR provided stronger amplification signals compared to RT-PCR at the corresponding time points with the same PCR primer set, indicating a lower efficiency of RNA amplification compared to that of DNA. Ribosomal RNA and rDNA could be amplified by RT-PCR or PCR from both viable and dead cells throughout the 48-h posttreatment holding period. For RT-PCR, amplification signals decreased in intensity with increased holding time, while the efficiency of amplification of DNA sequences from dead cells remained fairly stable throughout the study. DNA persistence was greater than that of rRNA following cell death by mild heat treatment in skim milk. Skim milk did not appear to accelerate nucleic acid degradation. While rRNA was less stable than DNA, its detection by RT-PCR may not be appropriate as an exclusive indicator of cell viability in minimally processed foods.