The convergence of molecular biology and miniaturized instrumentation has accelerated development of biosensors with the specifications necessary to support pathogen reduction and quality programs in the food supply. Advances in optoelectronics, thin layer deposition, and microfabrication have provided many options for achieving microbiological detection goals. Some promising technologies are reviewed.

Human disease caused by enterohemorrhagic E. coli O157:H7 and other serotypes (EHEC) has been associated with bovine fecal contamination of food and the environment. The range of serotypes, low infectious dose, and numerous transmission vehicles for EHEC render development of detection methods for this pathogen complex. In this study, the hemolysin gene (EHEC- hly A) was targeted with oligonucleotides, and probe-target hybrids were amplified using strand displacement amplification (SDA). Amplicons were resolved in the complete reaction mix through changes in the fluorescence polarization (FP) of a fluorescein-labeled detector probe hybridized to the amplicons during amplification. Results combining EHEC- hly A, SDA, and FP were obtained within 35 min of reaction initiation. The test specificity was determined on EHEC strains representing 13 serotypes (49 isolates); and control uropathogenic, commensal, and other organisms (10 isolates). Statistical analysis of results indicated a sensitivity in the reaction vessel to 4.3 bacteria (95% confidence interval), and a specificity for EHEC (n=59) at 100% (P=5.11E-17; i.e. P<<0.05). Detection based on combining EHEC- hly A, SDA, and FP was compatible with water sources directly associated with human infection (drinking and recreational supplies), and bovine drinking trough water representing an environmental matrix linked to the maintenance of an EHEC animal reservoir.

Vibrio cholerae strains with all known toxin genes deleted or inactivated still cause diarrhoea in some volunteers, suggesting the presence of an unknown virulence factor or factors. Lysozyme-EDTA treated cells of JBK70, a genetically manipulated cholera toxin negative strain of Vibrio cholerae O1, biotype El Tor, release a factor that causes elongation of Chinese hamster ovary (CHO) cells. CHO cell-elongating toxin (Cef) was purified by FPLC chromatography (anion exchange; Q Sepharose High Performance) followed by 2D electrophoresis (isoelectric focusing gel, IEF; pH 3–9 and SDS-PAGE, 8–25% gradient gel). Partly purified toxin (anion exchange or IEF-eluted concentrate) caused fluid accumulation in sealed infant mice suggesting that Cef shows some properties of an enterotoxin. On SDS-PAGE (8–25%) and IEF (pH 2.5–5.0) gels, CHO cell activity was associated with a single band at 85 kDa and a pI of 3.8, respectively. A unique amino terminal sequence, XGDETNSSGASTEVVYESYIQQ, was determined by automated Edman degradation of gel-purified protein. The unique molecular mass, N-terminal sequence and activity on CHO cells indicate that this factor is not zonula occludens toxin (Zot) or accessory cholera enterotoxin (Ace) or the Hly A haemolysin. Partly purified Cef did not increase cyclic AMP or prostaglandin E2levels in CHO cells which suggests that its mechanism of action differs from that of cholera toxin.

ABSTRACT Cholera vaccines developed by the deletion of CTX genes from Vibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467–470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with an M r of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V. cholerae.