Toxigenic Clostridium Difficile Strains Research Articles

Comparative Effects of the Immediate and the Extended Release Formulations of Ciprofloxacin on Normal Human Intestinal Microflora

Ciprofloxacin is a well-known fluoroquinolone, active in vitro against most Gram-negative and Gram-positive bacteria. The purpose of the present study was to evaluate the ecological effects of an orally administered extendedrelease formulation of ciprofloxacin in comparison with an immediate-release formulation of ciprofloxacin on the normal human intestinal microflora. Thirty-six healthy female subjects were included in the study. The extended-release formulation of ciprofloxacin was given as 500 mg once daily and the immediate-release formulation as 250 mg twice daily. The treatment period was 3 days. The microbiological investigation was blinded with respect to treatment. Mean fecal concentration in the volunteers receiving the extended-release formulation ciprofloxacin was 453 mg/kg and in the immediate-release formulation ciprofloxacin volunteers, the mean fecal concentration was 392 mg/kg. The numbers of Escherichia coli were significantly suppressed while the enterococci decreased moderately in both treatment groups. No toxigenic Clostridium difficile strains were found. No major differences were observed between the two studied formulations on the normal human intestinal microflora.

Susceptibility of Clostridium difficile Toward Antimicrobial Agents Used as Feed Additives for Food Animals

A total of 65 toxigenic Clostridium difficile strains isolated from patients with antibiotic-associated diarrhea were tested for susceptibility to avilamycin, flavomycin, monensin, and salinomycin. Except for flavomycin the substances showed in vitro efficacy comparable to reports of the currently most commonly used drugs for treatment of C. difficile. This indicates that these old compounds may be useful for the treatment of C. difficile infections in man and perhaps for other bacterial causes of diarrhea.

The Attachment, Internalization, and Time-Dependent, Intracellular Distribution ofClostridium difficileToxin A in Porcine Intestinal Explants

Toxin A (TcdA), secreted by toxigenic strains of Clostridium difficile, produces lesions typical of C. difficile-associated disease (CDAD) in susceptible mammal species. Porcine colon explants maintained for 2 hours with TcdA developed severe lesions characterized by cell swelling, swelling of mitochondria and other organelles, distension of cytoplasmic vesicles, expansion of paracellular spaces, apoptosis, and necrosis. Severity of lesions was proportional to the dosage of toxin. No lesions were present in uninoculated control tissues after 2 hours. Receptor-mediated endocytosis is the keystone event in the pathogenesis of the toxin, and susceptibility of a given species is thought to depend on the presence of receptors in intestinal epithelial cells. The fate of TcdA applied to viable colon explants was determined by transmission electron microscopy in an anti-toxin-labeled gold assay. At 5 minutes postinoculation, the presence of TcdA was indicated at the membrane of microvilli or in the cytoplasm of epithelial cells. TcdA was also indirectly observed within endosomes or attached at their margin. A 30-minute inoculation period was associated with many more gold particles labeling structures inside the cell, although some were still attached to microvilli. Within the cell, most TcdA was associated with mitochondria of epithelial cells, but some gold particles decorated the nuclei. Endothelial cells of the lamina propria had evidence of TcdA at both their lumenal and basal aspects, as well as in the cytoplasm and, occasionally, nuclei. Gold particles also labeled the lumen of such vessels as well as leucocytes in blood vessels and the lamina propria.

Co-culture with potentially probiotic microorganisms antagonises virulence factors of Clostridium difficile in vitro

Toxigenic strains of Clostridium difficile were co-cultured with different strains of bifidobacteria and lactobacilli. Spent culture supernatants were tested for biological activity on cultured Vero cells. Co-culture of C. difficile with some potentially probiotic strains lead to a reduction of the biological activity of spent culture supernatants. The observed effects cannot be ascribed either to secreted factors from the probiotic strains or to toxin adsorption by bacterial cells. Immunological assays showed that there was significant diminution of both clostridial toxins (TcdA and TcdB) in spent culture supernatants of co-cultures as compared with pure clostridial cultures. Even though co-cultured clostridial cells showed a slight increase of intracellular toxins, this increase did not completely explains the reduction of toxin concentration in culture supernatants. The evidence suggests that the antagonism could be due to the diminution of the synthesis and/or secretion of both clostridial toxins. Our findings provide new insights into the possible mechanisms involved in the protective effect of probiotics in the context of C. difficile infection.

Diarrhoea caused by Clostridium difficile in patients with postoperative subhepatic abscess

Toxigenic strains of Clostridium difficile in the majority of cases cause disease of the intestinal tract of hospitalized patients. For a long time, Clostridium difficile was considered to produce both types of toxins (A+/B+ strain), however, the investigations conducted in the last ten years point to the existence of clinically significant isolates which produce only toxin B, i. e. toxin A negative/toxin B positive (A-/B+ strain) Clostridium difficile. We presented the case of a patient admitted to the Surgery Clinic, Clinical Center Nis due to the presence of calculus in the ductus choledochus. Twenty-four hours after the surgical intervention for calculus removal, the first signs of the operative wound infection began to appear. In the course of infection treatment, different antibiotics were administered (cefuroxine, ciprofloxacin, vancomycin, imipenem). After making etiological microbiological diagnosis and application of antibiotics according to antibiogram results, the signs of the operative wound infection began to withdraw, but the patient reported the abdominal pain and liquid stools with traces of blood (up to 17 stools per day). By microbiological examination, Clostridium diffidile was cultivated and the presence of toxin B was detected in the stool samples. The patient was sent to the Clinic for Infectious Diseases, where the causal therapy of mitronidazol was administered. Liquid and electrolytes were made up by substitution therapy. After the eight-day-treatment, the patient felt much better, and diarrheas stopped on the 10th day of the therapy application. Our results have shown that toxingen strains Clostridium difficile are present in our country so this bacterium sort have to be considered in differential causal diagnosis of diarrhoea syndrome. Considering that it can cause difficult form of the disease, it is an obligation to establish the presence of some toxins of Clostridium difficile in stool samples of patients and/or production of some toxins in liquid culturate of isolates to provide data for the presence of strains which produce only toxin B.

A New, More Virulent Strain of Clostridium Difficile

This paper examines the possibility of increasing virulent strains of toxigenic Clostridium difficile causing severe sepsis, shock and subsequent death within 36 hours at a community hospital. We have noted an increase in the incidence of new cases of Clostridium difficile associated diarrhea over the last 5 years. Moreover, we identified a subset of patients, from October 2004 to Febuary 2005, who based on positive toxigenic strains of Clostridium difficile met requirements for severe sepsis and had APACHE II scores of 25 or greater. We observed that the patients presented initially with anuric acute renal failure, acute respiratory failure and subsequent septic shock. They required multiple intravenous pressor agents and eventually died within 36 hours of admission. No other etiology of sepsis was identified and no colonic dilatation was noted. In these patients colectomy was felt not to be a viable option as the mortality is extremely high once patients develop hypotension requiring intravenous pressors agents. It is our opinion that a more virulent strain of Clostridium difficile triggered the inflammatory cascade causing severe sepsis and neither the use of antibiotics nor colectomy would be sufficient to supress the inflammatory response. It was hypothesized that activated protein C might have been a viable option to attempt to supress the inflammatory response, but the patients condition deteriorated much too rapidly. We believe that patients who develop Clostridium difficile associated diarrhea and severe sepsis should be initially treated with oral Vancomycin as a first line. It is our belief also that the management of suspected cases of fulminant colitis should be aimed at targeting the inflammatory cascade with more aggressive measures such as with the use of activated protein C. Colectomy should also be considered whenever feasible. With the possible increasing incidence of a new epidemic strain, the possibility of antibiotic resistance and the changing facets of the toxin itself, we believe that further research is needed to help in the management of this deadly disease.

Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile.

Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile, a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.

Infection of hamsters with epidemiologically important strains of Clostridium difficile.

Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters. Three strains-types B1, J9, and K14-have caused hospital outbreaks. Type Y2 is associated with a high rate of asymptomatic colonization in patients. The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C. difficile-associated diarrhea. Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C. difficile. Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals. Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003). The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains.

Rapid detection of toxigenic Clostridium difficile strains by a nested PCR of the toxin B gene.

Rapid detection of toxigenic Clostridium difficile strains by a nested PCR of the toxin B gene.

Rapid polymerase chain reaction method for specific detection of toxigenicClostridium difficile

A rapid polymerase chain reaction (PCR) method for detection of toxigenic Clostridium difficile directly from fecal samples by amplification of toxin A gene fragments was investigated. The technique was applied to monitor the spread of the microorganism in a long-term care ward with a relatively high incidence of overt episodes of diarrhea. The PCR approach has several advantages over traditional methods, rapidly allowing the specific detection of toxigenic Clostridium difficile strains from stool samples in both symptomatic and asymptomatic subjects with toxigenic strains. This PCR method allows early detection of toxigenic Clostridium difficile and could thus represent a powerful tool for the surveillance of epidemics.

Apparent outbreaks of Clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital.

Intestinal colonization with toxigenic strains of Clostridium difficile was documented in 9 of 10 horses with acute onset diarrhea in a veterinary medical teaching hospital, whereas a similar isolate was detected in only 1 of 23 other horses without diarrhea in the hospital. One horse with diarrhea was infected simultaneously with both C. difficile and Salmonella krefeld. Clostridium difficile was detected by fecal culture on selective medium, confirmed with a latex particle agglutination test, and identified as toxigenic by polymerase chain reaction amplification of toxin A and toxin B gene sequences. Using an arbitrarily-primed polymerase chain reaction, 6 distinct C. difficile isolates were detected in the feces of the 9 affected horses at the time of the outbreak of diarrhea.

Haemagglutination activity of toxigenic and non-toxigenic strains of Clostridium difficile

Haemagglutination activity of toxigenic and non-toxigenic strains of Clostridium difficile

Use of gene amplification to detect Clostridium difficile in clinical specimens

The combined use of an enrichment broth and gene amplification following simple DNA extraction to detect toxigenic strains of Clostridium difficile in feces was investigated by examining feces from 329 cases of suspected C. difficile infection. DNA was extracted by heating the washed centrifuged deposit from the broth in a microwave oven. For comparison, specimens were tested concurrently using standard methods for culture and cytotoxin testing. Amplified fragments were identified by molecular weight estimation, restriction enzyme digestion patterns and Southern blot hybridization. The combination of an enrichment broth followed by gene amplification was shown to be a sensitive, specific and rapid method for detecting toxigenic strains of C. difficile in feces. Use of the method in diagnostic laboratories may require the development of improved detection and verification systems for the amplified gene fragment.

Serogroup F strains of Clostridium difficile produce toxin B but not toxin A.

Most toxigenic strains of Clostridium difficile produce two toxins: an enterotoxin (toxin A) and a cytotoxin (toxin B). Only one strain (strain 8864) has been reported to produce toxin B but no toxin A. Serogroup F strains (44) of C. difficile, often isolated from asymptomatic infants, have been examined for toxin production. These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in vitro when examined in three distinct immunoassays. Nevertheless, all the strain produced a cytotoxin. Immunological differences between the cytotoxin of the serogroup F strains and that produced by C. difficile strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C. difficile strain VPI 10463 or C. sordellii lethal toxin (LT). Polymerase chain reaction amplification with primers derived from C. difficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene. When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces. However, cytotoxin was detected. Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C. difficile strain VPI 10463. The serogroup F strains appeared to be homogeneous and distinct from other C. difficile strains with regard to toxin production.

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.

Identification of toxigenic Clostridium difficile by the polymerase chain reaction

Toxigenic strains of Clostridium difficile are causative agents of pseudomembranous colitis and antimicrobial agent-associated diarrhea and colitis. The toxigenicity is routinely assayed by using highly sensitive cell cultures. We used a simple and rapid polymerase chain reaction (PCR) assay to differentiate toxigenic and nontoxigenic strains of C. difficile. Two sets of oligonucleotide primer pairs derived from nonrepeating sequences of the toxin A gene were used to amplify 546- and 252-bp DNA fragments. A primer pair derived from repeating sequences of the toxin A gene was used to amplify a 1,266-bp DNA product. Amplified products were visualized by polyacrylamide gel electrophoresis followed by ethidium bromide staining. All 35 cytotoxic strains of C. difficile tested generated the expected amplified DNA. In contrast, none of the 26 noncytotoxic strains tested gave positive results. Although the toxins of C. difficile have been demonstrated to cross-react serologically with the toxins of Clostridium sordellii, we did not detect any amplified DNA in two cytotoxic strains or seven noncytotoxic strains of C. sordellii. PCR was negative in all 30 strains of 20 other Clostridium species. Southern hybridization of HindIII-digested genomic DNA by use of subgenomic probes showed a single hybridization band in toxigenic strains but not in nontoxigenic strains. PCR appears to be a sensitive and specific assay for the rapid identification of toxigenic C. difficile. Nontoxigenic C. difficile appeared to lack the C. difficile toxin A gene.

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.

Effect of various diets on toxin production by two strains of Clostridium difficile in gnotobiotic mice.

When axenic mice fed a commercial diet were monoassociated with two toxigenic strains of Clostridium difficile, 100% of them died 3 days after inoculation and both enterotoxin and cytotoxin were produced in their intestinal tract. However, when axenic mice were fed various semisynthetic diets before C. difficile challenge, some of them survived and their fecal cytotoxin and enterotoxin productions were highly reduced, whereas the C. difficile population level did not decrease to a great extent. Thus, gnotobiotic mice associated with C. difficile were a good model for the study of modulation by the dietary regimen of intestinal cytotoxin and enterotoxin production.

Clostridium difficile--a spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis.

Each of nine different toxigenic strains of Clostridium difficile was administered orally to groups of hamsters pre-treated with clindamycin and housed individually in sterile isolator boxes. Faecal pellets and caecal contents from well, diarrhoeic, moribund and freshly dead animals were analysed for C. difficile and toxins A (enterotoxin) and B (cytotoxin), and tissue obtained when animals were killed was examined histologically. Not all strains were equally virulent in this model. Four strains of C. difficile killed all animals within 48 h and are designated as highly virulent for hamsters. These strains were clinical isolates from three cases of disease in man and one case in a hamster. Five strains caused death of some animals but only after 5 and upt to 13 days and are designated as less virulent for hamsters. These strains were isolated from asymptomatic infants (2) and household pets (2), and from the environment (1). The surviving test hamsters were killed after 14 days and, in most cases, were colonised by C. difficile, though levels of toxins A and B in caecal contents were low. None of the cultures used for challenge was capsulate or hydrophobic. There was no correlation between virulence and production of toxins A and B in vitro in tryptic-nitrate broth. With two strains examined, there was a correlation between virulence and toxin A (but not toxin B) production in caecal emulsions derived from clindamycin pre-treated hamsters. Caecal contents from the majority of moribund and freshly dead animals had quantities of toxin A sufficient to cause disease or death if given orogastrically. Toxin B was not produced in a fixed ratio with toxin A. The data support the view that high virulence of C. difficile is determined by efficient disease-inducing colonisation of the gut and the ability to generate, rapidly, high levels of toxin A in vivo.

Analysis of latex agglutination test for Clostridium difficile toxin A (D-1) and differentiation between C difficile toxins A and B and latex reactive protein.

Virulent toxigenic and avirulent non-toxigenic strains of Clostridium difficile gave a positive result in the latex agglutination test (LAT) for C difficile toxin A (D-1). Similar concentrations of latex agglutinating antigen were produced by these strains in vivo. Positive reactions were also given by C sporogenes, proteolytic C botulinum Types A, B, and A/F, and Bacteroides assaccharolyticus. The latex agglutinating antigen was denatured by boiling for 10 minutes, but not by heating at 56 degrees C for 30 minutes. The reaction was abolished by incubation of test material with crude C difficile antitoxin but not with other clostridial antitoxins or specific antitoxin to C difficile toxin A. The latex agglutinating antigen present in C difficile eluted between 0.39% and 0.47% M sodium chloride, and that produced by the other clostridia, between 0.35% and 0.43% M sodium chloride by fast protein liquid chromatography. The latex agglutinating antigen of C difficile was neither cytotoxic nor mouse lethal and was distinct from toxin A and toxin B. In the analysis of faecal specimens from patients with diarrhoea the latex agglutination test correlated better with the presence of C difficile than with toxin B and detected both toxigenic and non-toxigenic strains. The latex agglutination test should only be used in the laboratory as an alternative to culture for C difficile and not as a method for the detection of C difficile toxins.