Effect of the cytotoxin of Clostridium difficile on cultured hepatoma cells

Clostridium difficile is the major etiologic agent of human pseudomembranous colitis. It produces two toxins: an enterotoxin and a cytotoxin. In cultured hepatoma cells, at very low doses, the cytotoxin inhibits the incorporation of precursors into biological macromolecules. Protein synthesis is more affected than RNA and DNA synthesis. The toxin also induces severe alterations of the cell morphology consisting in damages to the cytoskeleton and to the cell shape.     

Effect of environmental stress on Clostridium difficile toxin levels during continuous cultivation.

A method for the continuous culture of Clostridium difficile has been described. It has been shown that subjecting continuous cultures of this microorganism to environmental stress results in increased levels of toxin in culture medium. Factors found to cause this release include alteration of the Eh from --360 to +100 mV or increasing the temperature from 37 to 45 degrees C. The increased toxin levels were not associated with a change in viable cell density or the numbers of spores present. Additional studies have shown that subinhibitory concentrations of vancomycin and penicillin, but not clindamycin, also cause an increase in toxin levels during continuous culture. The increase in supernatant toxin levels occurs concomitant with a decrease in sonicated cell extract toxin levels. The data suggest that a number of factors can cause a release of toxin from C. difficile into the surrounding medium.     

Effect of environmental stress on Clostridium difficile toxin levels during continuous cultivation.

A method for the continuous culture of Clostridium difficile has been described. It has been shown that subjecting continuous cultures of this microorganism to environmental stress results in increased levels of toxin in culture medium. Factors found to cause this release include alteration of the Eh from --360 to +100 mV or increasing the temperature from 37 to 45 degrees C. The increased toxin levels were not associated with a change in viable cell density or the numbers of spores present. Additional studies have shown that subinhibitory concentrations of vancomycin and penicillin, but not clindamycin, also cause an increase in toxin levels during continuous culture. The increase in supernatant toxin levels occurs concomitant with a decrease in sonicated cell extract toxin levels. The data suggest that a number of factors can cause a release of toxin from C. difficile into the surrounding medium.     

Purification and properties of Clostridium difficile cytotoxin B

Toxin B, a potent cytotoxin produced by Clostridium difficile, was purified to homogeneity from 6-day broth cultures of a toxigenic isolate. Cytotoxin was purified approximately 4000-fold by sequential ammonium sulfate precipitation, DEAE-Sepharose chromatography, and high performance liquid chromatography on a Mono Q anion-exchange column. The molecular weight of reduced purified toxin was 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared to 150,000 for unreduced toxin. Dose-response studies indicated that subpicogram concentrations of purified toxin caused rounding of approximately 20,000 IMR-90 fibroblasts. The phenomenon of cell rounding caused by toxin B was correlated with the ratio of globular to filamentous actin in fibroblasts as measured by two techniques. The toxin caused a significant increase in the ratio of globular to filamentous actin which was nearly completed prior to the onset of rounding. We conclude that cell rounding of fibroblasts exposed to toxin B is related to an increase in the ratio of globular to filamentous actin which is produced by small numbers of toxin molecules/cell.     

Enhanced fermentation of mannitol and release of cytotoxin by Clostridium difficile in alkaline culture media.

Clostridium difficile ATCC 43255 fermented less than 10% of the mannitol in a medium at pH 7; however, when the initial pH of the medium was adjusted to 8.5 or 9, about 80% of the mannitol was fermented. Cell extracts of C. difficile phosphorylated mannitol with phosphoenolpyruvate, not ATP, indicating a phosphoenolpyruvate phosphotransferase system transport phosphorylation of mannitol. The phosphorylation product was dehydrogenated by D-mannitol-1-phosphate:NAD oxidoreductase. Growth at an initial pH of 8.5 yielded cytotoxin titers of 10(7) to 10(8) in Trypticase-yeast extract-mannitol medium, wit a titer of 10(8) as early as 13 h.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B

Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA-1470 and toxB-1470 . A polymerase chain reaction (PCR)-based approach to the sequencing of the total toxB-1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VPI10463, the toxB-1470 gene has 16 additional nucleotides, 13 within the 5'-untranslated region and three within the coding region. The M _r of ToxB-1470 is 269 262, with an isoelectric point (IP) of 4.16. The equivalent values for ToxB are M _r 269 709 and IP 4.13. In comparison with ToxB, ToxB-1470 differs primarily in the N-terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C-terminal region between residues 869–2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB-1470 with ToxA and ToxB reveals the highest homology between ToxB-1470 and ToxB. Thus ToxB-1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB-1470 is less potent than ToxB. The cytopathic effects of ToxB-1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii , but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or α-toxin (Tcnα) of Clostridium novyi . The LT-like action of ToxB-1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells. Since the differences between ToxB and ToxB-1470 are clustered within the N-terminal third of the respective proteins, we conclude that these domains carry the toxic determinants. A three-domain structure is proposed for the family of large clostridal cytotoxins.     

Clostridium difficile and its cytotoxin in infants admitted to hospital with infectious gastroenteritis.

During a prospective study of infectious gastroenteritis in children under 2 years, 19 out of 390 patients (4.9%) were found to have Clostridium difficile cytotoxin in the faeces. In several there was no history of use of antibiotics. The symptoms of many infants with toxin settled spontaneously, but one child became acutely and severely ill and developed a toxic megacolon and five others required, and responded to, vancomycin. Cl difficile was cultured from the stools in 191 (49%) of the children. The highly significant increased prevalence of past use of antibiotics in 118 control patients was not associated with an increased incidence of either isolation of Cl difficile or presence of faecal cytotoxin. Cl difficile should not be overlooked as a cause of acute diarrhoea and vomiting in children under 2 years.     

Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.     

Effect of Clindamycin on Cytotoxin Production by Clostridium difficile

A total of 80 strains of Clostridium difficile, 33 toxigenic and 11 nontoxigenic clindamycin (CLDM)-sensitive (MIC less than 12.5 μg/ml), and 23 toxigenic and 13 nontoxigenic CLDM-resistant (MIC 200 to 6,400 μg/ml) were tested for cytotoxin production in the presence of CLDM. None of the 24 nontoxigenic strains produced cytotoxin regardless of the presence of CLDM and only six out of the 56 toxigenic strains showed 16- to 64-fold higher levels of cytotoxic activity in the presence of CLDM at the concentrations of 1/2 to 1/32 of the MIC than in the absence of CLDM; all of the six strains were CLDM sensitive. Further studies revealed that addition of CLDM to the culture caused enhanced cytotoxin synthesis, and that the maximum production of cytotoxin was obtained when CLDM was added to the medium at the time of inoculation or of the ensuing early logarithmic phase. Also, the influence of other antibiotics on the effect of CLDM was examined. Addition of metronidazole, vancomycin, chloramphenicol, cephaloridine, or penicillin, which induced cytotoxin to medium containing CLDM did not increase the effect of CLDM any further. Addition of CLDM to medium containing tetracycline, which inhibited cytotoxin production, induced cytotoxin production but not fully.     

Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture.

Batch cultures (pH 6.7) of Streptococcus bovis JB1 were severely inhibited by 1.25 and 5 microM lasalocid and monensin, respectively, even though large amounts of glucose remained in the medium. However, continuous cultures tolerated as much as 10 and 20 microM, respectively, and used virtually all of the glucose. Although continuous cultures grew with high concentrations of ionophore, the yield of bacterial protein decreased approximately 10-fold. When pH was decreased from 6.7 to 5.7, the potency of both ionophores increased, but lasalocid always caused a larger decrease in yield. The increased activity of lasalocid at pH 5.7 could largely be explained by an increased binding of the ionophore to the cell membrane. Because monensin did not show an increased binding at low pH, some other factor (e.g., ion turnover) must have been influencing its activity. There was a linear increase in lasalocid binding as the concentration increased, but monensin binding increased markedly at high concentrations. Based on the observations that (i) S. bovis cells bound significant amounts of ionophore (the ratio of ionophore to cell material was more important than the absolute concentration), (ii) batch cultures responded differently from continuous cultures, and (iii) pH can have a marked effect on ionophore activity, it appears that the term "minimum inhibitory concentration" may not provide an accurate assessment of microbial growth inhibition in vivo.     

Interaction of Clostridium difficile toxin A with L cells in culture

Toxin A of Clostridium difficile was purified by column chromatography and acetic acid precipitation. Cells exposed to toxin A showed polarization of nuclei towards one pole of the cells. Toxin A was conjugated to ferritin and applied to L cells to localize binding sites of this toxin to the cell surface. It was found that toxin A conjugate attached to the cell membrane in aggregated form. Antibody specific to toxin A was prepared and used for localization of intracellular toxins in intoxicated cells. Toxin A was found inside the cytoplasm 6 h after cell treatment, mainly in the form of aggregates inside the cytoplasmic vacuoles. At 24 h after exposure, toxin A could be detected within the cytoplasm. Tunicamycin treatment of cells reduced the cell-binding efficiency of toxin A to 50%, but neuraminidase did not effect toxin binding significantly.     

Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes.

Three different dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested.     

Improved Bacterial Mutagenesis by High-Frequency Allele Exchange,Demonstrated in Clostridium difficile and Streptococcus suis

Here we show that the frequency of mutant isolation by two-step allele exchange can be improved by increasing the length of homologous DNA and the opportunity for recombination, obviating the need for counterselection markers. These principles are demonstrated in Clostridium difficile and Streptococcus suis but are likely to be generally applicable.     

Production of butanol by Clostridium puniceum in batch and continuous culture

Summary The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l^-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.     

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h^-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article no. 95-07-064.     

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.