Thermostable direct haemolysin (TDH) produced by Vibrio parahaemolyticus is thought to play an important role in the severe diarrhoea caused by this organism. This study investigated the enterotoxicity of TDH for human intestinal cells. Addition of TDH to the mucosal side of human colonic tissue in Ussing chambers caused increased short circuit currents (Isc), a process that was inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), an inhibitor of Ca2+-activated chloride (Cl−) channels. With human colonic epithelial (Caco-2) cells, high Isc and intracellular Ca2+ concentrations ([Ca2+]in) were detected after the addition of TDH to the apical side of the cell monolayer. The Isc decreased with the addition of DIDS, but not with glybenclamide, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or gadolinium chloride. No Isc increase with TDH was observed when the Cl− in the medium was replaced by gluconate or when Ca2+ was depleted. Similarly, TDH did not raise [Ca2+]in after depletion of extracellular Ca2+. R7, a mutant form of TDH, reduced the effects of TDH on Isc and [Ca2+]in, as did protein kinase C (PKC) inhibitors. Thus, TDH increases Cl− secretion in human colonic epithelial cells, apparently through mechanisms involving cell binding and Ca2+ influx, followed by elevation of [Ca2+]in associated with PKC phosphorylation.
Variants of large colony β-haemolytic Lancefield group A, C and G streptococci that are non-haemolytic or α-haemolytic on sheep blood agar have been detected in clinical specimens due to their enhanced haemolytic activity when grown on a new selective and differential blood agar medium containing colistin, nalidixic acid and pH 7.5-adjusted PIPES buffer (CNA-P). The large colony Lancefield group C and G isolates were identified as Streptococcus dysgalactiae subsp. equisimilis by API 20 Strep classification and 16S rDNA profiling. The haemolytic activity of these variants on various blood agar media, including CNA-P, was closely similar to that of known streptolysin S-defective mutants of S. pyogenes and was blocked by addition of cholesterol, a specific inhibitor of the streptolysin O family of haemolysins. As haemolysin variants could be detected in large numbers in cultures from patients with clinical symptoms of pharyngitis it is suggested that they may function as primary pathogens in such infections. The high frequency with which haemolysin variants were isolated from clinical specimens during a 3-month trial (3%, 13% and 10%, respectively, of group A, C and G streptococcal isolates) indicated that a substantial proportion of streptococcal infections may go undetected if only conventional sheep blood agar media are used in clinical laboratories for the detection of β-haemolytic streptococci. As haemolysin variants have been implicated in the development of serious streptococcal sequelae, further investigation of the full extent of their contribution to streptococcal disease is indicated.
The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.