D. C. A. Candy

Growth of Clostridium difficile and production of toxins A and B in complex and defined media.

The ability of several strains of Clostridium difficile to grow and to produce toxins A and B in complex and defined culture media has been studied with special reference to the amino-acid composition of the medium. The production of these toxins varied with the strain used and with the composition of the growth medium. Toxin A production was not inextricably linked to production of toxin B since conditions were found in which only one or other toxin was produced.

The Nature and Role of Mucosal Damage in Relation to Salmonella Typhimurium-Induced Fluid Secretion in the Rabbit Ileumx

Summary The time course and nature of mucosal damage induced in rabbit ileal loops by two strains of Salmonella typhimurium (TML and W118) isolated from human infections was assessed by immunofluorescence microscopy and by scanning and transmission electronmicroscopy. Salmonella-induced fluid secretion occurred in the presence or absence of gross mucosal architectural damage. Neither strain caused mucosal ulceration. When damage did occur, the villi were shortened by loss of their tip regions with concomitant reforming of an intact mucosal surface. Immediately preceding the onset of fluid secretion, marked infiltration of the mucosa with polymorphonuclear leukocytes and occasional macrophages was seen. This revives an earlier suggestion that interaction between invading salmonellae and acute inflammatory cells may be an important factor in initiation of fluid secretion. Brush-border invasion by salmonellae cannot per se be the immediate cause of fluid secretion, because the latter occurred several hours after initial invasion.

Enterotoxin production by Salmonella typhimurium strains of different virulence

Six strains of Salmonella typhimurium (TML, W118, LT7, SL1027, M206 and Thax-1) of known virulence and ability to induce fluid secretion when inoculated into the rabbit ileum were examined for enterotoxin production. Enterotoxic activity, assayed in the rabbit ileal-loop test, was detected in polymyxin-B extracts from all strains (with the possible exception of Thax-1) cultured for 6 h in casamino acid-yeast extract medium. The extracts were inactive in tissue-culture assays with CHO, Y-1 adrenal and Vero cells, and in the infant mouse assay for enterotoxin. There was no correlation between enterotoxigenicity in vitro and the ability of whole organisms to induce fluid secretion in vivo. The significance of these results in relation to salmonellosis is discussed.

The effects of Clostridium difficile crude toxins and toxin A on ileal and colonic loops in immune and nonimmune rabbits

Rabbits were solidly immunised by parenteral injection of purified Clostridium difficile toxin A such that they resisted an intravenous challenge with a normally lethal dose of toxin A. Ileal and colonic loops constructed in non-immune and immune animals received challenge injections of crude culture filtrate or purified toxin A of C. difficile. Protection of ileum was manifest after sufficient initial mucosal damage resulted in release of high levels of antitoxin A into the loop lumen of immune animals. There was less fluid accumulation in ligated ileal loops of immune than of non-immune rabbits. Less protection was observed when loops were challenged with crude culture filtrate containing toxins A and B than when challenged with purified toxin A. In-vitro studies with Ussing chambers yielded no evidence for tissue-localised immunity as judged by electrical responses and histology of toxin-treated tissue from non-immune and immune animals. No differences were found in the degree of epithelial damage, or volume or composition of fluid accumulating in colonic loops of non-immune and immune rabbits challenged with toxin A or crude culture filtrate. However, in colonic loops of immune rabbits there was no overt tissue-localised haemorrhage, whereas in those of non-immune rabbits tissue-localised haemorrhage was marked. In contrast to our findings with ileal loops, fluid accumulating in colonic loops was watery and contained substantially less total protein and (in immune animals) antitoxin A.

The role of leucocytes in the induction of fluid secretion by Salmonella typhimurium

Nitrogen mustard (N2M) treatment of rabbits induced neutropenia, and, in ligated ileal loops, it inhibited fluid secretion induced by salmonella or by cholera toxin (CT). Pretreatment of rabbits with indomethacin almost abolished salmonella-induced fluid secretion and significantly reduced that induced by CT. Similar effects of N2M and indomethacin on fluid secretion induced by salmonella, but not by CT, have been reported by other workers and used to implicate prostaglandins, from the salmonella-induced inflammation, as mediators of fluid secretion. In contrast, we show that N2M treatment, in addition to reducing CT-induced secretion, caused severe morphological alterations to ileal mucosa. Irradiation techniques were developed for inducing neutropenia, but they did not totally inhibit salmonella-induced leucocyte influx into ileal mucosa. We propose an alternative mechanism for the inhibitory effect of N2M on salmonella- and CT-induced secretion, based on the known anti-mitotic activity of N2M. Also, the anti-secretory effect of indomethacin cannot be attributed uniquely to its anti-inflammatory activity because it depressed CT-induced secretion as well as salmonella-induced secretion. These results support the concept of pathophysiological secretion in infectious diarrhoea, developed previously for rotavirus and extended to bacterial infections.

Biological mode of action of Clostridium difficile toxin A: a novel enterotoxin.

Antibody neutralisation and toxin A elution experiments showed that toxin A uptake from rabbit intestinal lumen was a continuous process. The kinetics of the ileal and colonic responses were significantly different; a much longer incubation (4 h) with toxin was required for colon, compared with 45 min for the ileum, to induce fluid accumulation at 12 h. Fluid secretion was induced only when toxin had gained access to deeper tissues, probably achieved by several toxin uptake-tissue damage cycles. Toxin A induced haemorrhage in both ileal and colonic tissues. In ileum, the villus architecture was severely damaged and this gave rise to protein-rich bloody luminal fluid. In the colon, although colonocytes were removed, the basement membrane remained intact; this resulted in a tissue-localised haemorrhage and a protein-low watery ultrafiltered luminal fluid. Toxin A is thus a novel type of histotoxic enterotoxin.

Production and Release of Toxins A and B by Clostridium Difficile

The production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated. Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells. Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay. Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle. The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed.

Expression of an antigen in strains of Salmonella typhimurium which reacts with antibodies to cholera toxin

Six strains of Salmonella typhimurium (W118, TML, SL1027, LT7, M206 and Thax 1) of different virulence were examined for the presence of antigens which react with antibodies to cholera toxin (anti-CT). A fluorescent-antibody-labelling technique employing anti-CT was used to analyse antigen expression. A rapid increase in the proportion of cells producing a CT-related antigen was demonstrated in cells in early log phase (1-4 h growth) followed by a rapid decline during mid-late log phase in each of the six strains. The nature of the CT-related antigen was analysed by immunoblotting using anti-CT. An antigen of mol. wt equivalent to a high-mol. wt species of CT B subunit was detected in polymyxin-B extracts of all strains but greater amounts were observed in the strains that we consider avirulent. Nothing equivalent to a CT A-related subunit was observed in any of the strains. The relatedness of the salmonella antigen to CT was limited. The high-mol. wt antigen was not disrupted in the denaturing conditions of SDS-PAGE; nothing was detected by enzyme-linked immunosorbent assays with either ganglioside or anti-CT as anchor.

Sporogenesis and toxin A production by Clostridium difficile

The kinetics of spore production by Clostridium difficile were not paralleled by release of C. difficile toxin A in vitro. Toxin A was not found to be associated with either purified whole spores or spore coats. Residual traces of toxin A detected in spore contents were almost certainly derived from contaminating vegetative cell debris. Thus, toxin A is unlikely to be a spore constituent or associated with sporogenesis.

The effects of Clostridium difficile crude toxins and purified toxin A on stripped rabbit ileal mucosa in Ussing chambers.

Clostridium difficile crude toxins and purified toxin A had similar effects on stripped rabbit ileal mucosa in Ussing chambers. Both toxin preparations caused secretion of sodium and chloride ions by increasing serosa to mucosa (s----m) fluxes. Transmural potential difference and resistance decreased after toxin treatment. Onset of changes in electrical measurements and ion fluxes coincided with onset of histological changes. The response to theophylline was greatly reduced in toxin-treated tissue compared with control tissue.