An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-AspTyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.Vibrio tubiashii is a marine organism that causes bacillary necrosis in larval and juvenile bivalve mollusks (24,25). The disease is characterized by a rapid onset of symptoms, such as a generalized reduction in larval motility, an increase in larval quiescence, and extensive soft tissue necrosis. The pathogen has been isolated from hard clam larvae, juvenile hard clams, and Eastern oyster spat and larvae (7,12,25). V. tubiashii has been isolated from diseased mollusks in the United States and United Kingdom and has been associated with red tides caused by Mesodinum rubrum along the northwest coast of Spain (12,23,24,25). In spite of the economic importance of V. tubiashii in the cultivation of bivalve mollusks, nothing is known about the virulence mechanisms of this pathogen. Romalde et al. (23) reported that culture supernatants of the pathogen exhibited cytotoxicity towards fathead minnow peduncle cells and mouse lung fibroblasts in tissue culture. We describe the purification and properties of a cytolysin that lyses various types of erythrocytes and Chinese hamster ovary (CHO) cells and is cytotoxic to human intestinal (Caco-2) cells and fish (Atlantic menhaden liver [AML]) cells in tissue culture.Cytolysin production and purification. Two V. tubiashii strains (ATCC 19105 and ATCC 19109) were obtained from the American Type Culture Collection (Manassas, Va). Both strains were confirmed to be V. tubiashii using biochemical tests and were stored at Ϫ70°C. The ATCC 19105 frozen culture was rapidly thawed and inoculated onto two plates containing Trypticase soy agar (BBL, Cockeysville, Md.) supplemented with 1% NaCl. The plates were incubated at 30°C for 16 to 18 h, and the bacteria were harvested in 5 ml of Casamino Acids-yeast extract broth (3% Casamino Acids, 0.4% yeast extract, 0.05% K 2 HPO 4 [pH 7.4] supplemented with 1% NaCl). A 2-liter flask containing 500 ml of Casamino Acids-yeast extract broth was inoculated with the seed culture suspension (25 optical density units at 650 nm; ca. 10 10 CFU), and the culture was incubated for 7 h at 37°C on a rotary shaker at 100 rpm. Culture supernatant fluids (stage 1) were recovered by centrifugation at 16,000 ϫ g (20 min). Disodium hydrogen phosphate and sodium chloride were dissolved in the sta...

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 omega10c and 17:1 anteiso, which are unique in these species. Iso 17:1 omega10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, analysis of FAMEs from bacteria and spores can provide a sensitive procedure for the identification of food-borne pathogens.

Campylobacter jejuni-mediated pathogenesis involves gut adherence and translocation across intestinal cells. The current study was undertaken to examine the C. jejuni interaction with and translocation across differentiated Caco-2 cells to better understand Campylobacter's pathogenesis. The efficiency of C. jejuni 81-176 invasion of Caco-2 cells was two-to threefold less than the efficiency of invasion of INT407 cells. Adherenceinvasion analyses indicated that C. jejuni 81-176 adhered to most INT407 cells but invaded only about two-thirds of the host cells over 2 h (two bacteria/cell). In contrast, only 11 to 17% of differentiated Caco-2 cells were observed to bind and internalize either C. jejuni strain 81-176 or NCTC 11168, and a small percentage of infected Caco-2 cells contained 5 to 20 internalized bacteria per cell after 2 h. Electron microscopy revealed that individual C. jejuni cells adhered to the tips of host cell microvilli via intimate flagellar contacts and by lateral bacterial binding to the sides of microvilli. Next, bacteria were observed to bind at the apical host membrane surface via presumed interactions at one pole of the bacterium and with host membrane protrusions located near intercellular junctions. The latter contacts apparently resulted in coordinated, localized plasma membrane invagination, causing simultaneous internalization of bacteria into an endosome. Passage of this Campylobacter endosome intracellularly from the apical surface to the basolateral surface occurred over time, and bacterial release apparently resulted from endosome-basolateral membrane fusion (i.e., exocytosis). Bacteria were found intercellularly below tight junctions at 60 min postinfection, but not at earlier times. This study revealed unique host cell adherence contacts, early endocytosis-specific structures, and a presumptive exocytosis component of the transcellular transcytosis route.

a b s t r a c tThe mechanism of Cronobacter pathogenesis in neonatal meningitis and potential virulence factors (aside from host cell invasion ability) remain largely unknown. To ascertain whether Cronobacter can invade and transcytose across intestinal epithelial cells, enter into the blood stream and then transcytose across the blood-brain-barrier, we have utilized human intestinal INT407 and Caco-2 cells and brain microvascular endothelial cell (HBMEC) monolayers on Transwell filters as experimental model systems. Our data indicate a wide range of heterogeneity with respect to invasion efficiency among twenty-three Cronobacter isolates screened. For selected isolates, we observed significant levels of transcytosis for Cronobacter sakazakii across tight monolayers of both Caco-2 and HBMEC, mimicking in vivo ability to cross the intestine as well as the blood brain barrier, and at a frequency equivalent to that of a control meningitis-causing Escherichia coli K1 strain. Finally, EM analysis demonstrated intracellular Cronobacter bacteria within host vacuoles in HBMEC, as well as transcytosed bacteria at the basolateral surface. These data reveal that certain Cronobacter isolates can invade and translocate across both cultured human intestinal epithelial cells and HBMEC, thus demonstrating a potential path for neonatal infections of the central nervous system (CNS) following oral ingestion.Published by Elsevier Ltd.