Purification and characterisation of toxin B from a strain of Clostridium difficile that does not produce toxin A

Most toxigenic strains of Clostridium difficile produce both toxin A and toxin B. The toxin produced by C. difficile strain 8864 was characterised and compared with those produced by C. difficile strain 10463. Toxin A was not detected by immunoassay in cultures from strain 8864 and all the cytotoxic activity produced by this strain was neutralised by antiserum to toxin B. Toxin B from strain 8864 was purified and compared with toxin B from strain 10463. The size of the purified subunits of toxin B from strain 8864 differed slightly from those of strain 10463 and there were small immunological differences. The effect on fibroblast cells was more like that of C. sordellii cytotoxin than of toxin B from strain 10463. These results suggest that C. difficile strain 8864 produces a modified toxin B and does not produce toxin A.     

The large clostridial toxins from Clostridium sordellii and C. difficile repress glucocorticoid receptor activity

We have previously shown that Bacillus anthracis lethal toxin represses glucocorticoid receptor (GR) transactivation. We now report that repression of GR activity also occurs with the large clostridial toxins produced by Clostridium sordellii and C. difficile. This was demonstrated using a transient transfection assay system for GR transactivation. We also report that C. sordellii lethal toxin inhibited GR function in an ex vivo assay, where toxin reduced the dexamethasone suppression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Furthermore, the glucocorticoid antagonist RU-486 in combination with C. sordellii lethal toxin additively prevented glucocorticoid suppression of TNF-alpha. These findings corroborate the fact that GR is a target for the toxin and suggest a physiological role for toxin-associated GR repression in inflammation. Finally, we show that this repression is associated with toxins that inactivate p38 mitogen-activated protein kinase (MAPK).     

New types of toxin A-negative,toxin B-positive strains among Clostridium difficile isolates from Asia

A total of 56 C. difficile strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of tcdA and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A(-)B(+) strain, C. difficile 8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A(+)B(+) strains. Toxinotypes XVI and XVII represent two new groups of A(-)B(+) strains. Strains of the well-known A(-)B(+) group from toxinotype VIII have a nonsense mutation at the beginning of tcdA gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5'-end sequence of tcdA in two newly described A(-)B(+) strains does not contain an identical mutation. The prevalence of variant C. difficile strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.     

Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B.

A total of 39 toxigenic and 20 nontoxigenic strains of Clostridium difficile were tested for the presence of either toxin A or toxin B by the polymerase chain reaction (PCR). All toxigenic strains produced cytotoxin as assayed by using highly sensitive fetal lung fibroblasts and were positive for toxin A as well as toxin B in the PCR assay. All nontoxigenic strains failed to produce toxin and were negative in the PCR assay. This study shows that nontoxigenic strains of Clostridium difficile lack the toxin A as well as the toxin B gene.     

Clonal spread of a Clostridium difficile strain with a complete set of toxin A, toxin B, and binary toxin genes among Polish patients with Clostridium difficile-associated diarrhea

Clinically relevant Clostridium difficile strains usually produce toxins A and B. Some C. difficile strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with C. difficile-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented.     

Macrofragment localization of the toxin A and toxin B genes of Clostridium difficile.

We report the physical mapping of the toxin A and B genes to the bacterial chromosome of Clostridium difficile ATCC 43594 by pulsed-field gel electrophoresis. Single and double digestions with restriction endonucleases NruI and SacII allowed localization of the toxin genes to a specific 577-kb fragment and estimation of genome size to be approximately 3.8 megabases. This effort represents the initial step in the construction of a physical map of the whole genome.     

Positive regulation of Clostridium difficile toxins.

The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli. To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters. The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay. When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold. Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans. Our results suggest that TxeR is a positive regulator that activates expression of the C. difficile toxins.     

Commercial latex test for Clostridium difficile toxin A does not detect toxin A.

The rapid latex test recently marketed by Marion Scientific (Div. Marion Laboratories, Inc., Kansas City, Mo.) for the detection of Clostridium difficile toxin A does not react with the toxin, based on the following findings: culture filtrates from nontoxigenic strains of C. difficile gave positive reactions in the test, culture filtrate in which toxin A had been removed gave positive reactions, purified toxin A did not react in the test, and the latex reagent bound an antigen which is distinct from toxin A and which is produced in various amounts by both toxigenic and nontoxigenic strains of C. difficile.     

Immunization with Bacillus spores expressing toxin A peptide repeats protects against infection with Clostridium difficile strains producing toxins A and B

Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A(+)B(+)). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A.     

Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.     

Community-acquired Clostridium difficile diarrhea caused by binary toxin, toxin A, and toxin B gene-positive isolates in Hungary

The aim of this work was to study the toxin types of Clostridium difficile isolates originating from different parts of Hungary. A PCR method was used for amplification of the two major toxin genes and the binary toxin gene and to detect the deletion or insertion in the 3' end of the toxin A gene. The findings were compared with the results of cytotoxicity assays on the HeLa cell line. One hundred twelve isolates were tested; the toxin A and toxin B genes were detected in 79 strains by the PCR method. All of the isolates that were positive by the PCR method were also positive by the cytotoxicity assay. All of the other strains (n = 33) were negative for the toxin A and toxin B genes; in these cases, cytopathic effects on the cell line were not observed. No tcdA-negative and tcdB-positive isolates were found by the PCR method. In two cases, the presence of a binary toxin gene was observed by PCR; both isolates that were isolated from diarrheal feces carried the tcdA and tcdB genes. No prior hospitalization had occurred in either case.     

Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.     

Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxins.

Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxin was studied by cytotoxicity assay in tissue culture. The sources of toxin were stools from two patients with pseudomembranous colitis and a culture filtrate of C. difficile isolated from one of the patients. C. sordellii antitoxin was available either in monovalent form or as gas gangrene polyvalent antitoxin. The potency of antitoxins against C. difficile determined by cytotoxicity assay did not correlate with the established values reported for mouse protection tests against C. sordellii toxin. An equivalent zone of optimal neutralization was demonstrated for stool toxin, and a slightly different one for culture toxin. The rate of neutralization appeared to be instantaneous, either at 24 or at 37 degrees C. The efficacy of antitoxin in preventing cytotoxicity in cultured cells preexposed to toxin decreased rapidly with preexposure time. The union between toxin and antitoxin could be readily dissociated by simple dilution or by ammonium sulfate precipitation followed by dissociated by simple dilution or by ammonium sulfate precipitation followed by dilution. Continued incubation of toxin-antitoxin mixture did not increase the firmness of the union; on the contrary, more dissociation occurred. The unusual looseness of the toxin-antitoxin union is probably relatd to lack of serological specificity or affinity. Based on these observations, a practical diagnostic method for antibiotic-induced colitis is outlined.     

Comparison of two toxins produced by Clostridium difficile.

Clostridium difficile was shown to produce a toxin which could be biochemically separated from the previously described cytotoxin of the same organism. The two proteins differ in biological activity and physical properties. Antiserum prepared to the second toxin does not neutralize the biological activity of the cytotoxin, and immunological cross-reactivity could not be demonstrated. However, some relationship may exist between the two toxins, since the newly described toxin degrades on polyacrylamide electrophoresis into two molecules, one of which appears to migrate with the band of purified cytotoxin. We suggest that this newly described toxin be designated toxin A until its primary biological activity and physical relationship to cytotoxin is determined. This toxin is active in biological assays of enteric disease and may play an important role in C. difficile-induced colitis.     

Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of Clostridium difficile have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (tcdA) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with C. difficile-associated diarrhea in France. The repeating segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol. 36:2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire tcdA gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11, C. difficile variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other tcdA variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of C. difficile-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion of C. difficile-associated diarrhea but a negative immunoassay for toxin A.     

Molecular epidemiology of Clostridium difficile strains in children compared with that of strains circulating in adults with Clostridium difficile-associated infection

Molecular analysis of Clostridium difficile (28 isolates) from children (n = 128) in Oxfordshire, United Kingdom, identified eight toxigenic genotypes. Six of these were isolated from 27% of concurrent adult C. difficile-associated infections studied (n = 83). No children carried hypervirulent PCR ribotype 027. Children could participate in the transmission of some adult disease-causing genotypes.     

艰难梭菌分离株快速鉴定和毒素检测的多重PCR方法的建立

目的 建立可同时进行艰难梭菌分离株菌种鉴定和毒素检测的多重PCR方法。方法 用于多重PCR中的3对引物分别为艰难梭菌的种特异性的磷酸内糖异构酶(triose phosphate isomerase,tpi)基因、毒素A基因部分序列、毒素B基因部分序列。艰难梭菌ATCC 9689等21株标准菌株和47株临床分离艰难梭菌分别被应用于多重PCR最低检出限、特异性评估试验和验证试验。同时,应用ELISA对47株分离株进行毒素A/B检测。结果 该多重PCR方法可检测到最低DNA浓度为0.5 pg/μ l,特异性为100％。47株艰难梭菌分离株中tpi基因均为阳性,其中毒素基因A(+)/B(+)为37株,毒素基因A（-）/B（-）为10株,未检出毒素基因A（-）/B(+)菌株。47株毒素A/B检测结果为20株阳性、27株阴性。毒素A/B阳性的20株菌均为多重PCR检测毒素基因A(+)/B(+)。结论 成功建立用于艰难梭菌的菌种鉴定和毒素分析结合为一体的多重PCR方法,对临床诊断艰难梭菌感染有着重要应用价值。     

Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin

A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.