Differentiated biological activity of Vero cytotoxins (VT) released by human and porcine Escherichia coli strains

Abstract We have investigated the biological activity in the filtered culture supernatants from 9 VT-producing Escherichia coli strains. The filtrates from 4 strains (3 of human and one of bovine origin), were cytotoxic on Vero and HeLa cells, and caused death in intraperitoneally injected adult mice. The 5 strains of porcine origin showed cytotoxic activity on Vero and Y-1 cells but not on HeLa cells. Filtrates of these latter strains were not lethal for adult mice. VT-cytotoxins produced by all strains were inactive in the infant mouse test and the filtrates from 7 of 8 VT-producing strains assayed in rabbit ileal loops caused fluid accumulation in at least one of the 3 rabbits employed.     

Enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ileum.

The enteropathogenicity of Vibrio parahaemolyticus was investigated by contrasting the effects of whole cells, cell fragments, cell-free preparations, and media constituents injected into rabbit ileal loops. Three of 20 cultures utilized were Kanagawa-negative strains from seawater and sea fish. The remaining 17 cultures included both Kanagawa-positive and -negative strains from Japanese victims of gastroenteritis. Broth culture filtrates concentrated 10-fold by dialysis against 30% Carbowax were unreactive, whereas lyophilized filtrates, regardless of Kanagawa type, as well as all sterile broth preparations containing greater than or equal to 5% NaCl gave positive reactions in the rabbit gut. In contrast, crude lysates derived from broth cultures of Kanagawa-positive strains caused loop dilatation; lysate supernatants were unreactive. Lysates of cells washed from brain heart infusion agar were more reactive than lysates from Trypticase soy agar-grown cells. When agar-grown cell lysates prepared by disruption in saline were dialyzed against distilled water, they were devoid of gut reactivity. Reactivity was restored in dialysands resuspended in saline and in dialysates concentrated 10-fold. The agar-grown cell lysates exhibited Kanagawa-type hemolysis. Our data support the conclusion that the rabbit loop reactivity observed with lyophilized, cell-free culture filtrates may result from excessively elevated NaCl concentrations, and that a toxic factor associated with large-cell particles may be dialyzable, depends on saline for expression, and resembles the Kanagawa hemolysin.     

Ileal Loop Fluid Accumulation and Production of Diarrhea in Rabbits by Cell-free Products of Clostridium perfringens

The ability of cell extracts and culture filtrates of various strains of C. perfringens to produce ileal loop fluid accumulation and overt diarrhea in rabbits was tested. Good correlation was obtained in the ability of whole cells and a toxic factor (present in cell extracts and concentrated culture filtrates) to produce both fluid accumulation in ileal loops and diarrhea when injected into the normal ileum of the rabbit. The toxic factor was present in cell-free preparations when cells were grown in a sporulation medium, but not when they were grown in an asporogenic medium. The factor was shown to be heat labile, nondialyzable, and was inactivated by Pronase but not by trypsin, lipase, or amylase. Loss of activity occurred at pH 1.0, 3.0, 5.0, and 12.0.     

Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139

Abstract The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.     

Assessment of in vivo revival, growth, and pathogenicity of Escherichia coli strains after copper- and chlorine-induced injury

Cells of one enteroinvasive and three enterotoxigenic strains of Escherichia coli were exposed to sublethal concentrations of copper and chlorine to produce 85 to 94% injury. Injured cells were intraluminally inoculated into ligated ileal loops of anesthetized mice, and injury was assessed at timed intervals. Substantial recovery (72-84%) of copper- and chlorine-injured cells was observed in the inoculated loops at 4 and 3 h, respectively. No appreciable increase in total numbers was observed during these time intervals. In vitro revival of copper-injured cells in phosphate-buffered saline alone after incubation at 35 degrees C for 4 h was not observed. However, a 60 to 70% revival occurred when 200 micrograms of protein per ml of mouse intestinal mucosal homogenate was incorporated into saline cell suspensions. The enterotoxigenic activity of copper-injured cells in rabbit ileal loops was somewhat reduced compared with that of chlorine-injured or uninjured cells. These results show that injured pathogenic E. coli cells can revive in the small intestine and appear to retain their enterotoxigenic activity.     

Assessment of in vivo revival, growth, and pathogenicity of Escherichia coli strains after copper- and chlorine-induced injury.

Cells of one enteroinvasive and three enterotoxigenic strains of Escherichia coli were exposed to sublethal concentrations of copper and chlorine to produce 85 to 94% injury. Injured cells were intraluminally inoculated into ligated ileal loops of anesthetized mice, and injury was assessed at timed intervals. Substantial recovery (72-84%) of copper- and chlorine-injured cells was observed in the inoculated loops at 4 and 3 h, respectively. No appreciable increase in total numbers was observed during these time intervals. In vitro revival of copper-injured cells in phosphate-buffered saline alone after incubation at 35 degrees C for 4 h was not observed. However, a 60 to 70% revival occurred when 200 micrograms of protein per ml of mouse intestinal mucosal homogenate was incorporated into saline cell suspensions. The enterotoxigenic activity of copper-injured cells in rabbit ileal loops was somewhat reduced compared with that of chlorine-injured or uninjured cells. These results show that injured pathogenic E. coli cells can revive in the small intestine and appear to retain their enterotoxigenic activity.     

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.     

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.     

Bacillus cereus-Induced Fluid Accumulation in Rabbit Ileal Loops

The usefulness of the ligated rabbit ileal loop as an experimental model of Bacillus cereus food poisoning was investigated. Positive responses, as measured by fluid accumulation in the loop, were obtained from 19 of 22 strains of B. cereus. Four of six strains of B. thuringiensis also elicited fluid accumulation, but eight strains of other Bacillus spp. failed to evoke a response. The growth medium employed markedly affected the ability of a given strain of B. cereus to provoke a response. Brain heart infusion broth (BHI) (Difco) proved to be best for this purpose. Loop fluid-inducing activity was produced by exponentially growing cells and was present in cell-free culture filtrates and associated with washed vegetative cells. Intraluminal growth of B. cereus did not elicit fluid accumulation. Cultures grown at temperatures in the range of 18 C to 43 C were loop active. When BHI cultures of selected loop positive strains were injected intraluminally into the normal ileum of rabbits, they failed to elicit diarrhea.     

Effects of crude and pure cholera toxin on prostaglandin.

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE2 equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE2/g tissue when incubated with crude toxin for 1-4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions. Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell

Rice culture filtrates of Bacillus cereus SA-50, an emetic-type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp-2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp-2 cells seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp-2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4-dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.     

Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin

Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared. Eleven B. cereus strains and one enterotoxigenic B. thuringiensis strain were evaluated. Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains. An emetic enterotoxin producing strain was negative with all three assays. Two B. cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay. The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative. Overall, the cell culture assay was the most sensitive. However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B. cereus diarrhoeal enterotoxin.     

Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin.

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re-examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 10(4)/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.     

Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Characterization of purified Shiga toxin from Shigella dysenteriae 1

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000-6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 microgram.     

Melanogenesis in murine B16 cells exposed to Aeromonas hydrophila cytotoxic enterotoxin

Specific markers (growth, melanogenesis) of B16 murine melanoma cells in culture were used as indicators of toxin production by Aeromonas hydrophila. Cytotonic enterotoxinlike activity (inhibited growth, raised tyrosinase activity, and melanin accumulation) occurred at cytotoxic end points of purified beta-hemolysin and several culture filtrates. Antihemolysin rabbit serum inhibited this activity. A hemolysin-neutralized culture filtrate concentrate (10X) failed to elevate tyrosinase relative to untreated and cholera toxin treated controls. Similar dilution profiles using Chinese hamster ovary cells showed limited cell extension only at cytotoxic end points with antihemolysin inhibiting this activity. Cytotoxicity of Chinese hamster ovary cells and B16 cells was proportional to hemolytic activity, with B16 cells showing about 100-fold greater sensitivity on a per cell basis. Cell culture cytotoxicity attributed to beta-hemolysin correlated with reactivity in rabbit ileal loop assays. The ADP-ribosyl transferase activity of concentrated (10X) A. hydrophila culture filtrates and fractions thereof was negative. Apparently sublethal doses of A. hydrophila beta-hemolysin can nonspecifically stimulate cyclic adenosine monophosphate mediated events in melanoma and Chinese hamster ovary cell assays, producing lower activities than cholera toxin with shorter lag times.     

Efficient expression of a 100-kilodalton mosquitocidal toxin in protease-deficient recombinant Bacillus sphaericus.

The expression of the 100-kDa mosquitocidal toxin (Mtx) during vegetative growth and sporulation in nine different mosquito-larvicidal strains of Bacillus sphaericus has been analyzed. In five out of the nine strains the 100-kDa toxin was found to be expressed predominantly in the vegetative phase of growth, and in all nine strains the level of the toxin in sporulated cells was very low or undetectable. Strains in four out of the six DNA homology groups of B. sphaericus produced intracellular and extracellular proteases, which degraded the 100-kDa toxin, during sporulation. The 100-kDa toxin gene was expressed by using its native promoter on a multicopy number plasmid in B. sphaericus 1693 (protease negative) and B. sphaericus 13052 (protease positive). High levels of the 100-kDa toxin were produced in vegetative cells of both strains as well as in sporulated cells of protease-negative strain 1693, which is in contrast to the low levels of the 100-kDa toxin produced in sporulated cells of protease-positive strain 13052. Thus, the small amount of the 100-kDa toxin in sporulated cells of the nine mosquito-larvicidal strains is probably due to degradation of the 100-kDa toxin synthesized during vegetative growth by a protease(s) produced during sporulation. B. sphaericus 1693 transformed with the 100-kDa toxin gene was as toxic to mosquito larvae during both vegetative growth and sporulation as the natural high-toxicity strains of sporulated B. sphaericus.(ABSTRACT TRUNCATED AT 250 WORDS)