Search

Background: The incidence of Clostridioides difficile infection (CDI) has increased over the past 2 decades and is considered an urgent threat by the Centers for Disease Control and Prevention. Hypervirulent strains such as ribotype 027, which possess genes for the additional toxin C. difficile binary toxin (CDT), are contributing to increased morbidity and mortality., Methods: We retrospectively tested stool from 215 CDI patients for CDT by enzyme-linked immunosorbent assay (ELISA). Stratifying patients by CDT status, we assessed if disease severity and clinical outcomes correlated with CDT positivity. Additionally, we completed quantitative PCR (PCR) DNA extracted from patient stool to detect cdtB gene. Lastly, we performed 16 S rRNA gene sequencing to examine if CDT-positive samples had an altered fecal microbiota., Results: We found that patients with CdtB, the pore-forming component of CDT, detected in their stool by ELISA, were more likely to have severe disease with higher 90-day mortality. CDT-positive patients also had higher C. difficile bacterial burden and white blood cell counts. There was no significant difference in gut microbiome diversity between CDT-positive and -negative patients., Conclusions: Patients with fecal samples that were positive for CDT had increased disease severity and worse clinical outcomes. Utilization of PCR and testing for C. difficile toxins A and B may not reveal the entire picture when diagnosing CDI; detection of CDT-expressing strains is valuable in identifying patients at risk of more severe disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Clostridium difficile is the most common known cause of antibiotic-associated diarrhea. Upon the disturbance of gut microbiota by antibiotics, C. difficile establishes growth and releases toxins A and B, which cause tissue damage in the host. The symptoms of C. difficile infection disease range from mild diarrhea to pseudomembranous colitis and toxic megacolon. Interestingly, 10-50 % of infants are asymptomatic carriers of C. difficile. This longitudinal study of the C. difficile colonization in an infant revealed the dynamics of C. difficile presence in gut microbiota., Methods: Fifty fecal samples, collected weekly between 5.5 and 17 months of age from a female infant who was an asymptomatic carrier of C. difficile, were analyzed by 16S rRNA gene sequencing., Results: Colonization switching between toxigenic and non-toxigenic C. difficile strains as well as more than 100,000-fold fluctuations of C. difficile counts were observed. C. difficile toxins were detected during the testing period in some infant stool samples, but the infant never had diarrhea. Although fecal microbiota was stable during breast feeding, a dramatic and permanent change of microbiota composition was observed within 5 days of the transition from human milk to cow milk. A rapid decline and eventual disappearance of C. difficile coincided with weaning at 12.5 months. An increase in the relative abundance of Bacteroides spp., Blautia spp., Parabacteroides spp., Coprococcus spp., Ruminococcus spp., and Oscillospira spp. and a decrease of Bifidobacterium spp., Lactobacillus spp., Escherichia spp., and Clostridium spp. were observed during weaning. The change in microbiome composition was accompanied by a gradual increase of fecal pH from 5.5 to 7., Conclusions: The bacterial groups that are less abundant in early infancy, and that increase in relative abundance after weaning, likely are responsible for the expulsion of C. difficile.

Clostridium difficile infection (CDI) is a gastrointestinal disease caused by C. difficile, a spore-forming bacterium that in its spore form is tolerant to standard antimicrobials. Ramoplanin is a glycolipodepsipeptide antibiotic that is active against C. difficile with MICs ranging from 0.25 to 0.50 μg/ml. The activity of ramoplanin against the spores of C. difficile has not been well characterized; such activity, however, may hold promise, since posttreatment residual intraluminal spores are likely elements of disease relapse, which can impact more than 20% of patients who are successfully treated. C. difficile spores were found to be stable in deionized water for 6 days. In vitro spore counts were consistently below the level of detection for 28 days after even brief (30-min) exposure to ramoplanin at concentrations found in feces (300 μg/ml). In contrast, suppression of spore counts was not observed for metronidazole or vancomycin at human fecal concentrations during treatment (10 μg/ml and 500 μg/ml, respectively). Removal of the C. difficile exosporium resulted in an increase in spore counts after exposure to 300 μg/ml of ramoplanin. Therefore, we propose that rather than being directly sporicidal, ramoplanin adheres to the exosporium for a prolonged period, during which time it is available to attack germinating cells. This action, in conjunction with its already established bactericidal activity against vegetative C. difficile forms, supports further evaluation of ramoplanin for the prevention of relapse after C. difficile infection in patients., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We report the selection of Clostridium difficile resistant to the rifamycin class of antibiotics in a patient within 32 h of receiving rifaximin for the treatment of recurrent C. difficile diarrhea. Resistance was associated with single nucleotide substitutions within rpoB.

gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

Clostridium difficile is an emerging nosocomial pathogen and one of the major causes of antibiotic-associated diarrhoea. Cases of Clostridium difficile-associated disease (CDAD) are likely initiated by the ingestion of dormant C. difficile spores, which then germinate, outgrow and rapidly proliferate to cause gastrointestinal (GI) infections. To understand the initial stages of CDAD pathogenesis, we have characterized the germination of spores from a collection of C. difficile strains, including some clinical isolates obtained from a CDAD outbreak (CDAD isolates). Spores of one laboratory strain and five CDAD isolates did not germinate with amino acids, but did germinate on a nutrient-rich medium. However, bile salts had little effect on spore germination, either alone or in a nutrient-rich medium. These spores also germinated with KCl, as well as the non-nutrient germinants dodecylamine and a 1 : 1 chelate of Ca(2+) and dipicolinic acid. An unexpected finding was that spores of most of the C. difficile strains also germinated with inorganic phosphate (P(i)) with a pH optimum of 6. The in vitro germination of spores of CDAD strains with KCl and P(i), two molecules present at significant levels in the GI tract, suggests that C. difficile spores germinate in the human body by sensing P(i) in the early segments of the duodenum and KCl in the colon.

Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains. Unlike other clostridial binary toxins, binary toxin CDT required exogenous trypsin for activation. Supernatants from all A-B-CDT+ strains caused marked fluid accumulation in the rabbit ileal loop assay after concentration and trypsinization. In addition, the ileal loop response was neutralized by antisera raised against other binary toxin-producing clostridia. Challenge of clindamycin-treated hamsters with these strains resulted in colonization but not diarrhea or death. Binary toxin CDT may play an adjunctive role to toxins A and B in the pathogenesis of C. difficile-associated disease but by itself may not be sufficient to cause disease.

Introduction: The binary toxin genes cdt and cdtB have been detected in approximately 5% of Clostridium difficile strains. Severe C. difficile disease (CDD) may be associated with strains that carry the binary toxin genes., Methods: From April 2001 through March 2002, 8 severe and 41 nonsevere cases of nosocomial CDD were studied. Severe cases of CDD were defined by the presence of >or=2 of the following criteria: (1) abdominal pain, (2) a white blood cell count of >20,000 or <1500 cells/mm(3), and (3) ileus or bowel wall thickening with ascites. Underlying disease was assessed by 2 methods: a modified Horn score and the presence of comorbid conditions. The presence of cdtA, cdtB, and the toxin A and toxin B genes was determined, and molecular subtyping was performed., Results: All strains were positive for the toxin A and B genes, and 65.3% of the strains carried the cdtA and cdtB genes. Strains that carried the binary toxin genes accounted for 87.5% of the cases of severe CDD and 61.0% of the nonsevere cases (P=.23). Severity of CDD was not associated with either severe underlying disease or comorbid conditions. The strains that caused severe CDD belonged to 4 protein profile groups and >or=3 restriction endonuclease analysis (REA) groups. All (i.e., 5 of 5) strains in REA group BI, compared with none (i.e., 0 of 7) of the strains in REA group J carried the binary toxin genes (P=.001). Strains that belonged to REA groups BK and BR also carried the binary toxin genes., Conclusions: The binary toxin genes were present in nearly two-thirds of the C. difficile strains, and they were correlated with the REA group. Severity of CDD was not closely associated with a specific clone or underlying disease, but it may be associated with the presence of the binary toxin genes. Larger studies are needed to discern whether a true association exists and whether the binary toxin alters the pathogenicity of the C. difficile strain.

Clostridium difficile causes approximately 25% of nosocomial antibiotic-associated diarrheas and most cases of pseudomembranous colitis. We evaluated C. DIFF CHEK, a new screening test that detects glutamate dehydrogenase of C. difficile. Our results showed that this test was comparable to PCR in sensitivity and specificity and outperformed bacterial culture.

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest that C. perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal cpe gene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpe isolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive, C. perfringens fecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpe gene, but the North American food poisoning isolates all carry a chromosomal cpe gene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpe isolates cause most CPE-associated AAD cases and chromosomal cpe isolates cause most C. perfringens type A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms.

The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.

Ten healthy ponies from a single herd were found by repeated fecal culture to be free of Salmonella species and Clostridium cadaveris. In a preliminary study, four ponies administered a single oral dose of 10 mg/kg lincomycin did not develop idiopathic colitis when the drug was administered alone. Four other ponies were administered 10 mg/kg lincomycin by stomach tube together with 0.45 L of colonic content from a horse with idiopathic colitis induced earlier by lincomycin alone. Two of the four ponies were treated with 25 g oral zinc bacitracin premix (110 g/kg active ingredient) 24 h later. Forty-two hours after inoculation the two untreated ponies had severe signs of idiopathic colitis and were euthanized. Postmortem findings were typical of idiopathic colitis. The two treated ponies had milder illness but the more severely affected was also euthanized; the other was retreated at 42 h with bacitracin pre-mix and again 12 h later. Its illness and diarrhea resolved over the next 24 h. Clostridium cadaveris was isolated in large numbers from the cecum of the euthanized ponies and their cecal content contained mouse lethal and guinea pig dermonecrotic, but not cytotoxic, activity. Enterotoxins of Clostridium perfringens and Clostridium difficile could not be demonstrated. No toxin could be demonstrated in culture supernatants of C. cadaveris or in supernatants of cecal contents treated with ethanol prior to culturing in anaerobically incubated broth. No Salmonella spp. were isolated. A further two ponies were administered 10 mg/kg lincomycin orally with 0.45 L colonic content from a horse with idiopathic colitis, as described.(ABSTRACT TRUNCATED AT 250 WORDS)

Washed cells and Sarkosyl-insoluble outer membrane preparations of the black-pigmented bacteroides Porphyromonas gingivalis W50 bound hemin. The amount of hemin removed from a buffered solution by both cells and outer membranes was significantly larger if bacteria had been grown in broths supplemented with 5 mg of hemin per liter rather than none. Conversely, cells grown without supplemental hemin bound relatively little. However, all preparations bound some hemin. In addition, hemin regulated the production of significantly higher levels of trypsinlike protease by P. gingivalis W50. The nonpigmented variant, W50 BE1, showed no such responses to the levels of hemin in the growth medium.

Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.

A PTFE cannula was introduced into the rabbit abdomen through the right flank. A purse string suture was used to fix a small plastic flange within the caecum. A larger flange was sutured under the skin to fix the cannula. Caecal contents could be collected from the chronic fistula via a threaded stainless steel cap on the cannula. Animals tolerated the fistulation well and sepsis was never seen.

A helically coiled, anaerobic, gram-positive sporeforming bacillus, identified as Clostridium spiroforme, was isolated from the cecal contents of all of 27 rabbits with spontaneous diarrhea, at a mean concentration of 10(6.0) spores per g of material. All of these rabbits also had a toxin present in their cecal contents that was neutralized by anti-Clostridium perfringens type E iota toxin, but not by other clostridial antitoxins. In addition, four rabbits with clindamycin-associated colitis were positive for C. spiroforme at a mean concentration of 10(4.5). All of these animals also had iota-like toxin present. Iota-like toxin was not detected in the cecal contents of 72 healthy animals, although C. spiroforme was found in two of these animals at a mean concentration of 10(6.0). C. spiroforme was shown to produce a toxin in vitro that was lethal to mice and caused dermonecrosis in guinea pigs. In all cases, this toxin was neutralized by anti-C. perfringens type E iota toxin.