Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus.

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.

Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products.

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.

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review.

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.

A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR.

Rapid nucleic acid-based methods to detect human pathogens in foods are dependent on the reliability of the DNA or RNA extraction method used. Skim milk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powder were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food product using a solvent-based procedure and (ii) using a guanidinium isothiocyanate (GITC) procedure after previous bacterial concentration. Both the efficiency of DNA extraction and the overall PCR detection limits were evaluated. In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method. However, the purity of the DNA obtained after bacterial concentration was significantly better than that obtained after organic extraction alone. PCR detection limits after each DNA recovery method varied with the specific food, ranging from 10(1) to 10(4) cfu ml(-1) for all products except whey powder. DNA yields and subsequent PCR detection limits for reconstituted whey powder were extremely poor, and neither procedural changes nor the addition of PCR enhancement agents were able to improve recovery and/or detection. It is concluded that the efficiency of DNA extraction is an extremely important and frequently overlooked variable impacting the overall detection limits of PCR-based detection strategies.

Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk.

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.

Survival of Escherichia coli O157:H7 in buttermilk as affected by contamination point and storage temperature.

The effects of contamination point (during fermentation versus postfermentation) and storage temperature (5 and 12 degrees C) were determined for survival of Escherichia coli O157:H7 in fermented buttermilk. E. coli O157:H7 was recovered from buttermilk inoculated during fermentation for 22 days and in buttermilk inoculated postfermentation for 32 days. For storage temperatures of 5 and 12 degrees C, D-values were lower for E. coli O157:H7 inoculated during fermentation (2.5, 2.2 days) than postfermentation (5.6, 4.8 days) (P < 0.05). Developed acidity in inoculated buttermilks was not different from controls (P > 0.05). The extended recovery of viable enterohemorrhagic E. coli O157:H7 from both processing scenarios indicates that the presence of E. coli O157:H7 in buttermilk is not limited to postprocessing contamination.

Nucleic acid persistence in heat-killed Escherichia coli O157:H7 from contaminated skim milk.

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.

rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.

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 enterotoxigenic Staphylococcus 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 degrees 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 degrees 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 degrees 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.

Biochemical characterization, developmental expression, and induction of the immune protein scolexin from Manduca sexta.

The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M(r) approximately 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M(r) approximately 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis.