In order to develop a PCR assay for Escherichia coli O157:H7, a portion of the 60-MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then designed by employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxins I (SLT-I) and II (SLT-II), and the 60-MDa plasmid. PCR products of 1,087 bp (eaeA), 227 and/or 224 bp (SLT-I and/or SLT-II), and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC of serogroup O157.

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Acetanisole (4′‐methoxyacetophenone) (AC), benzaldehyde (BE), cinna‐maldehyde (CI), diacetyl (DI), phenylpropionaldehyde (PH), and pyruvaldehyde (PY) were tested against growth of S. aureus WRRC B124 in brain heart infusion broth. Activity was assessed in the presence and absence of oxygen at 12, 19 and 37C, and in combination with mild (20 min at 50 or 60C) heat treatments. The carbonyl compounds limited S. aureus growth at minimal inhibitory concentrations (MIC) of 0.5–8.0 mM. After 4 h at 37C, a 2 to 3‐log10 CFU/ml population reduction was observed with cultures containing 8.0 mM PH, PY, DI or CI. Activity was O2‐tension independent, with CI (0.5 mM), DI(2.0 mM) and PY(2.0 mM) being most active. The MIC for CI was temperature independent, while PY was most effective at 19C, and PH and DI at 12C. Mild heat treatment of carbonyl‐supplemented samples reduced previously observed MICs. At 60C, for example, the MICs for AC and BE, 4.0 and 8.0 mM respectively, were both reduced to 0.5 mM. The decimal reduction times for S. aureus exposed to both UV‐light and 8 mM flavor compounds were 3.3 and 4.3 s for CI and DI, respectively. However, the other compounds were not as effective in the presence of UV since the decimal reduction times ranged from 7.7 to 9.0s. The carbonyl compounds tested were effective antistaphylococcal agents and their use in combination with thermal processing may serve as a new approach to control S. aureus growth and other gram‐positive foodborne pathogens.

Plasmid DNA (pDNA) vaccines are effective at eliciting immune responses in a wide variety of animal model systems, however, pDNA vaccines have generally been incapable of inducing robust immune responses in clinical trials. Therefore, to identify means to improve pDNA vaccine performance, we compared various post-transcriptional and post-translational genetic modifications for their ability to improve antigen-specific CMI responses. Mice vaccinated using a sub-optimal 100 mcg dose of a pDNA encoding an unmodified primary isolate HIV-1 6101 env gp160 failed to demonstrate measurable env-specific CMI responses. In contrast, significant envspecific CMI responses were seen in mice immunized with pDNA expression vectors encoding env genes modified by RNA optimization or codon optimization. Further modification of the RNA optimized env gp160 gene by the addition of (i) a simian retrovirus type 1 constitutive RNA transport element; (ii) a murine intracisternal A-particle derived RNA transport element; (iii) a tissue plasminogen activator protein signal leader sequences; (iv) a beta-catenin derived ubiquitination target sequence; or (v) a monocyte chemotactic protein-3 derived signal sequence failed to further improve the induction of env-specific CMI responses. Therefore, modification of the env gp160 gene by RNA or codon optimization alone is necessary for high-level revindependent expression and results in robust env-specific CMI responses in immunized mice. Importantly, further modification(s) of the env gene to alter cellular localization or increase proteolytic processing failed to result in increased env-specific immune responses. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.