Clostridium difficile-associated ileocecitis in clindamycin-treated infant hamsters

Infant hamsters, four days of age or older, developed aClostridium difficile-associated ileocecitis following clindamycin administration, whereas non-antibiotic-treated infant hamsters and hamsters less than four days old given clindamycin were asymptomatically colonized withC. difficile. The incidence of lethality among clindamycin-treated infant hamsters increased with age of the animals, such that all adult hamsters given clindamycin died within 72 h of treatment. Adult hamsters given clindamycin had significantly higher titers of cytotoxin throughout the intestinal tract compared with infant hamsters given clindamycin.     

Prevention of Clostridium difficile induced mortality in gnotobiotic mice by Saccharomyces boulardii

Oral preventive treatment of gnotobiotic mice by Saccharomyces boulardii significantly decreased mortality following Clostridium difficile infection. A single S. boulardii ingestion protected 16% of mice, whereas 56% were protected when S. boulardii was given continuously in the drinking water. No direct antagonistic effect of the yeast on C. difficile numbers was detected, whereas a modulation of fecal cytotoxin production was demonstrated.     

Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA

Techniques for the transformation of Clostridium acetobutylicum protoplasts with bacteriophage DNA are described. Transformation required regeneration of protoplasts and a 2-h eclipse period.     

Clostridium difficile electrophoretic typing

Abstract Two typing schemes for Clostridium difficile based on slide agglutinations and polyacrylamide gel electrophoresis (PAGE) have been described. We compared the reference strains of each typing system with a simplified PAGE method using whole cells and Coomassie blue staining. The method was also applied to clinical isolates and immunoblots were performed with a monospecific serum directed against a major band of low molecular weight. The results indicated the great heterogeneity of Clostridium difficile strains complicated by antigenic subdivision for strains belonging to the same electrophoretic type.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Antibiotics and Clostridium difficile

Clostridium difficile is now established as a major nosocomial pathogen. C. difficile infection is seen almost exclusively as a complication of antibiotic therapy, and is particularly associated with clindamycin and third-generation cephalosporins. Depletion of the indigenous gut microflora by antibiotic therapy has long been established as a major factor in the disease. However, the direct influence of antimicrobials upon virulence mechanisms such as toxin production and adhesion in the bowel, and the exact mechanisms by which the organism causes disease remain to be elucidated.     

Isolation of Bacteriophage for Clostridium tetani

The purpose of this report is to note the isolation of a Clostridium tetani phage from soil and to specify the procedure used to obtain suitable lawns for plaque formation.     

Thiosemicarbazones active against Clostridium difficile

A set of closely related furylidene thiosemicarbazones was prepared and screened against various clinically important Gram-positive bacteria. One compound containing an ethylene spacer and a 5-nitrofuryl group was found to have promising activity against Clostridium difficile.     

Clostridium difficile in association with sporadic diarrhoea.

A total of 154 patients admitted to an infectious diseases unit were included in a year''s prospective survey of sporadic diarrhoeal disease. Stools from 19 of them yielded Clostridium difficile, generally on more than one occasion. Twelve of these patients were assessed as having a severe or moderately severe gastrointestinal illness: Cl difficile was the only pathogen isolated from 10 of them, and two had an associated salmonella infection. Seven had had a recent course of antibiotics, but five had not taken antibiotics. Faeces from seven patients with moderate or mild gastrointestinal illness yielded Cl difficile, and two of these patients also had an associated salmonella infection. Two patients in this group had no antibiotic history. From these findings, the occurrence of C difficile in faeces could not be described as antibiotic-associated. Faecal Cl difficile cytotoxin was detected in only six patients, and generally at low levels. In such patients a more relevant pathogenic index might take account of the numbers of Cl difficile present and of their toxigenic potential.     

Clostridium difficile in emergency room

Clostridium difficile strains are known as etiological agents of pseudomembranous colitis (PMC), antibiotic-associated diarrhea (AAC) and colitis (AAC) and hospital-acquired infections. The aim of this study was to determine the frequency of C. difficile infection among patients in the emergency room and to compare isolated strains by phenotypic and genotypic characteristics. During a period of 11 months, 56 stool samples taken from diarrheic patients hospitalized in the emergency room of the Medical Center UC Davis and 14 environmental samples were cultured for isolation of C. difficile strains. Eighteen C. difficile strains were isolated from stool samples cultured on selective TCCCA plates and 5 strains from environmental samples using Rodac plates. Eleven toxigenic (TcdA+/TcdB+), 6 non-toxigenic (TcdA-/TcdB-) and unique toxin A-negative/toxin B-positive (TcdA-/TcdB+) C. difficile strains were detected among patients' isolates and 3 toxigenic and 2 non-toxigenic strains-among environmental samples. The majority of C. difficile-positive patients were treated previously by antibiotics. Four strains isolated from patients' fecal samples and one strain isolated from the environment demonstrated high-level resistance to erythromycin and clindamycin (MIC >256mug/mL). The results obtained by AP-PCR and PCR-ribotyping revealed genetic heterogeneity among the strains isolated from patients' fecal samples. However, similarity was observed among environmental strains and strains isolated from patients' fecal samples. Considering the importance of emergency room patients as a potential source of C. difficile strains, it appears to be important examine these patients for C. difficile before transfer to the other hospital units.     

Carbohydrate fermentation by Clostridium difficile

Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01% L-cysteine.HCl and 0.1% agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.     

Sporulation studies in Clostridium difficile

Clostridium difficile is a leading cause of healthcare-associated diarrhoea. In recent years, certain C. difficile types have become highly represented among clinical isolates and are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Endospores, produced during sporulation, play a pivotal role in infection and disease transmission and it has been suggested in the literature that these so-called ‘hypervirulent’ C. difficile types are more prolific in terms of sporulation in vitro. However, work in our laboratory has provided evidence to the contrary suggesting that although there is significant strain-to-strain variation in C. difficile sporulation characteristics this variation does not appear to be type-associated. On analysis of the literature, it is apparent that the methods used to quantify sporulation in previous studies have varied greatly and sample sizes have remained small. The conflicting data in the literature may, therefore, not necessarily be generally representative of C. difficile sporulation. Instead, these inconsistencies may reflect differences in the experimental design of each study. In this review, the need for further investigations of C. difficile sporulation rates is highlighted. Specifically, the advantages and disadvantages of the different experimental approaches previously used are discussed and a standard set of principles for measuring C. difficile sporulation in the future is proposed.     

Procalcitonin Levels Associate with Severity of Clostridium difficile Infection

Objective

Clostridium difficile infection (CDI) is a major cause of morbidity and biomarkers that predict severity of illness are needed. Procalcitonin (PCT), a serum biomarker with specificity for bacterial infections, has been little studied in CDI. We hypothesized that PCT associated with CDI severity.

Design

Serum PCT levels were measured for 69 cases of CDI. Chart review was performed to evaluate the presence of severity markers and concurrent acute bacterial infection (CABI). We defined the binary variables clinical score as having fever (T >38°C), acute organ dysfunction (AOD), and/or WBC >15,000 cells/mm³ and expanded score, which included the clinical score plus the following: ICU admission, no response to therapy, colectomy, and/or death.

Results

In univariate analysis log₁₀ PCT associated with clinical score (OR 3.13, 95% CI 1.69–5.81, P<.001) and expanded score (OR 3.33, 95% CI 1.77–6.23, P<.001). In a multivariable model including the covariates log₁₀ PCT, enzyme immunoassay for toxin A/B, ribotype 027, age, weighted Charlson-Deyo comorbidity index, CABI, and extended care facility residence, log₁₀ PCT associated with clinical score (OR 3.09, 95% CI 1.5–6.35, P = .002) and expanded score (OR 3.06, 95% CI 1.49–6.26, P = .002). PCT >0.2 ng/mL was 81% sensitive/73% specific for a positive clinical score and had a negative predictive value of 90%.

Conclusion

An elevated PCT level associated with the presence of CDI severity markers and CDI was unlikely to be severe with a serum PCT level below 0.2 ng/mL. The extent to which PCT changes during CDI therapy or predicts recurrent CDI remains to be quantified.     

Phospholipid profiles of Clostridium difficile.

Phospholipid molecular species present in 32 isolates of Clostridium difficile were examined by fast atom bombardment-mass spectrometry in negative-ion mode. This revealed major anions consistent with the expected presence of the following phosphatidylglycerol (PG) analogs: PG(31:2), PG(32:1), PG(33:2), PG(33:1), PG(34:2), and PG(34:1). The major phospholipid molecular species are distinct from those of other bacterial groups examined.     

Surface layers of Clostridium difficile endospores

Clostridium difficile is an important human pathogen and one where the primary cause of disease is due to the transmission of spores. We have investigated the proteins found in the outer coat layers of C. difficile spores of pathogenic strain 630 (CD630). Five coat proteins, CotA, CotB, CotCB, CotD, and CotE, were shown to be expressed on the outer coat layers of the spore. We demonstrate that purified spores carry catalase, peroxiredoxin, and chitinase activity and that this activity correlates with the predicted functions of three spore coat proteins identified here, CotCB, CotD, and CotE. CotCB and CotD are putative manganese catalases, and CotE is a novel bifunctional protein with peroxiredoxin activity at its amino terminus and chitinase activity at its carboxy terminus. These enzymes could play an important role in coat assembly by polymerizing protein monomers in the coat. CotE, in addition to a role in macromolecular degradation, could play an important role in inflammation, and this may be of direct relevance to the development of the gastrointestinal symptoms that accompany C. difficile infection. Although specific enzyme activity has not yet been assigned to the proteins identified here, this work provides the first detailed study of the C. difficile spore coat.     

Secretome analysis of Clostridium difficile strains

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.