Laboratory diagnosis of Clostridium perfringens antibiotic-associated diarrhoea

Clostridium perfringens has been reported as the cause of up to 15% of cases of antibiotic-associated diarrhoea (AAD) and may be diagnosed by detection of enterotoxin (CPEnt) in faeces. The performance of a commercial ELISA method for CPEnt, with culture and PCR methods to confirm the presence of enterotoxigenic C. perfringens, was evaluated in 200 consecutive specimens from patients with clinical details suggestive of AAD: 8% of the specimens were positive for CPEnt, 16% were positive for C. difficile cytotoxin and 2% gave positive test results for both C. perfringens and C. difficile toxins. Culture and PCR results confirmed the majority of ELISA results, although 2 (12.5%) reactive specimens were only weakly positive. C. perfringens is a potentially important cause of infective AAD and can be detected with the C. perfringens enterotoxin ELISA kit, although weak positive results should be considered with caution.     

Epidemiology of diarrhoea caused by enterotoxigenic Clostridium perfringens

Enterotoxigenic strains of Clostridium perfringens have recently been implicated in some cases of antibiotic-associated diarrhoea. We present here the results of an epidemiological study of this disease. Five cases of diarrhoea caused by C. perfringens serotype 41 occurred during a 9-week period, and then during a 6-week period there were three cases due to serotype 27 and two due to serotype 24; in all but one case two geriatric wards were involved. In total there were 16 cases in 22 months. All cases were identified by the detection of C. perfringens enterotoxin in the faeces. The mean number of C. perfringens in these cases was 10(8.8) cfu/g of faeces. Of 37 patients who had negative test results for C. perfringens enterotoxin, 18 had positive cultures for C. perfringens, with mean faecal counts of 10(5.3) cfu/g, and nine of these patients had diarrhoea. Thirteen different serotypes were isolated from these 18 patients, including type 41 from seven patients and type 27 from one. Hand carriage of the offending serotype was demonstrated in three of four infected patients, none of four controls and two of 14 ward staff. C. perfringens of serotypes causing disease was isolated from 59% of environmental areas where there was active disease, 27% of areas where there had been disease which had since resolved and 9% of areas where there was no history of disease. The findings imply that cross infection may occur.     

Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin

Purpose: Clostridium perfringens is a significant cause of nosocomial AAD. The prevalence of C. perfringens enterotoxin (CPE)-positive stool specimens in hospitalised patients is very low in the Indian setting making the diagnostics very expensive. Therefore, a cost-effective diagnostic approach to screen faecal specimens for CPE was devised. Materials and Methods: Faecal specimens from 540 hospitalised patients with various ailments and from 340 healthy subjects were investigated for CPE. An aliquot of pooled faecal supernatants was made by mixing 100 μl each of 10 specimens to be tested. Each aliquot was investigated for the presence of CPE by an enzyme immunoassay. A repetition of the assay was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Results: Of the 540 patient specimens tested, 405 (75%) patients were on antibiotics, the predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides, etc. During the time of sampling, diarrhoea was present in 481 (89%), abdomen pain in 203 (37.6%) and fever in 242 (44.8%) patients. C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. Conclusion: Only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described herein will help to save institutional resources.     

Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhoea.

AIMS: To determine the incidence of sporadic and apparently non-food related diarrhoea associated with Clostridium perfringens enterotoxin. METHODS: Enzyme linked immunosorbent assay (ELISA) and reversed phase latex agglutination (RPLA) were used to detect C perfringens enterotoxin in faecal specimens from 818 sporadic cases of diarrhoea. RESULTS: C perfringens enterotoxin was identified as a cause of sporadic diarrhoea in 56 of 818 (6.8%) cases. Diarrhoea was prolonged (three days or more) in most cases. Ages ranged from 3 months to 89 years, although most patients were over 60 years of age. CONCLUSIONS: These results suggest that C perfringens may be a cause of sporadic cases of diarrhoea when causes such as food consumption or cross-infection are absent, particularly in the elderly.     

Clostridium perfringens enterotoxin

Current knowledge of CPE action is briefly summarized in Figure 1. After specific binding to a protein receptor(s), the entire CPE molecule rapidly inserts into membranes forming a complex of 150,000 Mr. Almost simultaneously with insertion, there is a sudden change in ion fluxes. The molecular events behind the induction of ion flux changes remain undefined, but might involve either direct formation of membrane pores by CPE or activation of pre-existing membrane pores. As intracellular ion levels change, cellular metabolism is affected and processes such as macromolecular syntheses are inhibited. One of the ion flux effects resulting from CPE treatment involves increased Ca2+ influx; as more Ca2+ enters the cell, morphologic damage and permeability alterations for larger molecules occur. It remains to be determined if both morphologic damage and larger permeability alterations are necessarily linked but, for example, it could be envisioned that CPE-induced Ca2+ influx causes a cytoskeletal collapse leading to altered membrane permeability. The cytoskeleton has been shown to be sensitive to intracellular Ca2+ levels and is important in normal membrane structure/function relationships. As the cumulative effects of CPE inhibit cellular metabolism, cell death occurs. The precise irreversible CPE lethal action still must be identified. As CPE-treated intestinal epithelial cells die in vivo, histopathologic damage appears. This damage results in loss of normal intestinal function causing secretion of fluids and electrolytes. This effect is clinically manifested as diarrhea. The strongly cytotoxic action of CPE clearly distinguished the action enterotoxin from STa or CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that Clostridium perfringens theta-toxin induces colloid-osmotic lysis of erythrocytes.

Clostridium perfringens theta-toxin was shown to lyse target erythrocytes by a colloid-osmotic mechanism. Analysis showed the onset of lysis of erythrocytes by theta-toxin could be temporarily stabilized with 0.3 M sucrose. Flow cytometry analysis of the size distribution of theta-toxin-treated erythrocytes showed swelling of the erythrocytes prior to lysis.     

Evidence that membrane rafts are not required for the action of Clostridium perfringens enterotoxin

The action of bacterial pore-forming toxins typically involves membrane rafts for binding, oligomerization, and/or cytotoxicity. Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with a unique, multistep mechanism of action that involves the formation of complexes containing tight junction proteins that include claudins and, sometimes, occludin. Using sucrose density gradient centrifugation, this study evaluated whether the CPE complexes reside in membrane rafts and what role raft microdomains play in complex formation and CPE-induced cytotoxicity. Western blot analysis revealed that the small CPE complex and the CPE hexamer 1 (CH-1) complex, which is sufficient for CPE-induced cytotoxicity, both localize outside of rafts. The CH-2 complex was also found mainly in nonraft fractions, although a small pool of raft-associated CH-2 complex that was sensitive to cholesterol depletion with methyl-β-cyclodextrin (MβCD) was detected. Pretreatment of Caco-2 cells with MβCD had no appreciable effect on CPE-induced cytotoxicity. Claudin-4 was localized to Triton X-100-soluble gradient fractions of control or CPE-treated Caco-2 cells, indicating a raft-independent association for this CPE receptor. In contrast, occludin was present in raft fractions of control Caco-2 cells. Treatment with either MβCD or CPE caused most occludin molecules to shift out of lipid rafts, possibly due (at least in part) to the association of occludin with the CH-2 complex. Collectively, these results suggest that CPE is a unique pore-forming toxin for which membrane rafts are not required for binding, oligomerization/pore formation, or cytotoxicity.     

Evidence for cross-infection in an outbreak of Clostridium difficile-associated diarrhoea in a surgical unit

Environmental studies were performed in a hospital outbreak of Clostridium difficile-associated diarrhoea. Transmission was associated with the sluice room and the storage room where medical equipment was found to be contaminated with C.difficile. Typing of isolates by antibiotic-susceptibility patterns and profiles of EDTA-extracted proteins showed the presence of an "epidemic" strain common to the majority of patients and environmental sites. Control of the outbreak was achieved by improvement of environmental hygiene and use of disposable equipment.     

Cholangiohepatitis in chickens induced by bile duct ligations and inoculation of Clostridium perfringens

The association of bile duct ligation with the development of cholangiohepatitis due to Clostridium perfringens was examined. Chickens with only the cystoenteric duct ligated had no significant liver lesions even if C. perfringens was inoculated into the cystoenteric duct. Chickens with both the cystoenteric and hepatoenteric ducts ligated had enlargement of the liver with an apparent acinar pattern. Histopathologically, proliferation of bile ductules was seen. Bile ductules proliferated extensively, and fibrosis formed bridges between triads. The gall bladder and extrahepatic bile ducts were thickened and distended with yellow inspissated material in chickens that had both ducts ligated and had C. perfringens inoculated into the cystoenteric duct. Multiple granulomas and intrahepatic cholangitis were frequently observed. Infiltration of lymphocytes and plasma cells with germinal centres, and heterophilic extramedullary haematopoiesis were seen in the portal areas. Thus, cholangiohepatitis similar to field cases can only be induced by both ligation of bile ducts and inoculation of C. perfringens.     

Evidence for coupling of Clostridium perfringens alpha-toxin-induced hemolysis to stimulated phosphatidic acid formation in rabbit erythrocytes.

When rabbit erythrocytes were exposed to low concentrations of Clostridium perfringens alpha-toxin, hot-cold hemolysis was observed. The toxin induced production of phosphatidic acid (PA) in a dose-dependent manner when incubated with erythrocytes at 37 degrees C. When erythrocyte membranes were incubated with the toxin and [gamma-32P]ATP in the presence or absence of ethanol, [32P]PA formation was maximal within 30 s, then sharply decreased, and began again after 5 min of incubation. Ethanol had no effect on the early appearance (at approximately 5 min) of PA formation induced by the toxin but significantly inhibited formation of PA over 10 min of incubation. Treatment of erythrocyte membranes with alpha-toxin resulted in the biphasic formation of 1,2-diacylglycerol and PA as well as an increase of inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-4,5-bisphosphate (PIP2) within 30 s. Neomycin inhibited the toxin-induced increase in turbidity of egg yolk suspensions but did not inhibit the toxin-induced hemolysis of intact erythrocytes. On the other hand, neomycin inhibited the toxin-induced hemolysis of saponin-treated erythrocytes. In addition, neomycin inhibited PA formation induced by the toxin in erythrocyte membranes. IP3 was released by incubation of PIP2 with erythrocyte membranes but not by incubation of PIP2 with the toxin. The toxin stimulated the membrane-induced release of IP3 from PIP2. These data suggest that the toxin-induced hemolysis is dependent on the action of phospholipase C in erythrocyte membranes.     

Clostridium perfringens septicemia and acute leukemia

Three cases of fatal clostridial septicemia in patients with acute leukemia are described. Predisposing factors and treatment are discussed. Clostridium septicemia should always be suspected when a patient with neutropenia suddenly develops diffuse abdominal pain, fever, and tachycardia over 120/min. The importance of early treatment with penicillin or another adequate antibiotic is discussed.     

Clostridium perfringens enterotoxin in antibiotic-associated diarrhea

Clostridium perfringens type A is associated with 5-20% cases of antibiotic-associated diarrhea (AAD) even though Clostridium difficile is implicated in the most severe cases. Fecal specimens from one hundred hospitalized patients, who developed diarrhea regardless of antibiotic intake and who were negative for C. difficile toxin assay, were investigated for C. perfringens enterotoxin (CPE). Simultaneously, cultures were set up for other possible aetiological factors. Ten healthy controls were also similarly investigated. CPE was positive in 2/100 (2%) of the patients and the samples were also positive for the organism in culture. Other organisms isolated were non-toxigenic C. difficile (4%), staphylococci (6%), Candida (18%) and Klebsiella pneumoniae (1%). Stool samples from healthy controls grew mixed growth of no significance and CPE was negative in all of them. Detection of CPE is not part of routine laboratory investigation due to resource implication. Criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens-associated AAD by further research.     

Lumbar Discitis Caused by Clostridium perfringens

We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of β-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern.     

Clostridium difficile-Associated diarrhoea in uremic patients

An outbreak of 94 episodes of Clostridium difficile-associated diarrhoea in 62 patients in a nephrology ward over a two-year period was investigated. Quantitative stool cultures were performed on ten uremic patients not on antibiotics and without diarrhoea and on ten healthy controls. All diarrhoeal episodes were associated with Clostridium difficile,and no other bacterial pathogens were isolated. Thirty-two relapses occurred in 16 patients, fourteen of the relapses without preceding antibiotic exposure.Clostridium difficile could not be isolated from the environment of the patients. Uremic patients, who had a significantly increased number of Clostridium spp. in their stools, are predisposed to Clostridium difficile infections.     

Role of Ca2+-binding motif in cytotoxicity induced by Clostridium perfringens iota-toxin

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). We investigated the role of the conserved Ca(2+)-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca(2+). A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca(2+). However, the addition of Ca(2+) had no effect on the binding of (125)I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca(2+)-binding motif of Ib with alanine. D8A, D10A, and D12A bound to the cell and formed an oligomer at about half of the wild-type Ib. The cytotoxicity of Ib variants in the presence of Ia was about 500-fold less than that of wild-type Ib. Immunofluorescence study showed that these variants were internalized in the early endosomes like wild-type Ib. However, wild-type Ib-induced internalization of Ia in the cells, but these variants did not. The result indicates that the conserved Ca(2+)-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca(2+).     

Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca(2+) concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes.