Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolyticClostridium botulinum type F

For investigation of the genes of proteins associated in vivo with botulinum neurotoxin (BoNT), polymerase chain reaction (PCR) experiments were carried out with oligonucleotide primers designed to regions of the nontoxic-nonhemagglutinin (NTNH) gene ofClostridium botulinum type C. The primers were used to amplify a DNA fragment from genomic DNA ofC. botulinum types A, B, E, F, G and toxigenic strains ofClostridium barati andClostridium butyricum. The amplified product from all of these strains hybridized with an internal oligonucleotide probe, whereas all nontoxigenic clostridia tested gave no PCR product and showed no reaction with the probe. TheNTNH gene was shown to be located upstream of the gene encoding BoNT, thereby revealing a conserved structure for genes encoding the proteins of the M complex of the progenitor botulinum toxin in these organisms. The sequence of theNTNH gene of nonproteolyticC. botulinum type F was determined by PCR amplification and sequencing of overlapping cloned fragments. NTNH/F showed 71% and 61% identity with NTNH ofC. botulinum type E and type C respectively.     

Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C. difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty-four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 10^4.1/g in young adults and 10^4.6/g in elderly adults, with a range of 10^2.0 to 10^6.9/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.     

Clostridium difficile in vegetables, Canada

Background: Clostridium difficile is an important gastrointestinal pathogen of humans and animals. It has been isolated from various foods, including meat and ready‐to‐eat salads, and concern has been expressed regarding food as a possible source of human C. difficile infection (CDI). Aims: We sought to isolate C. difficile from a variety of vegetables obtained from local grocery stores and to characterize these isolates. Materials and Methods: Vegetables were purchased from 11 different grocery stores in Guelph, Ontario, Canada between May and August 2009. Enrichment culture was performed and isolates were characterized by ribotyping, PFGE, toxinotyping and PCR detection of toxin genes. Results: Clostridium difficile was isolated from 4.5% (5/111) of retail vegetables. Two different ribotypes and two different toxinotypes were identified. Three isolates were ribotype 078/NAP 7/toxinotype V, possessing all three toxin genes. The other two isolates shared a ribotype with a toxigenic strain previously found in humans with CDI in this region. Discussion: Contamination of vegetables was found at relatively low levels, however, all isolates were toxigenic and belonging to ribotypes previously associated with CDI. Conclusions: Contamination of vegetables with CDI‐associated isolates can occur and although the implications for food safety practices remain elusive, the presence of toxigenic isolates suggests vegetables could be a source of C. difficile in humans.     

Toxigenicity and antimicrobial susceptibility ofClostridium difficile,a cause of antimicrobial agent-associated colitis

Fourteen of 16 strains ofClostridium difficile, a recently recognized cause of antimicrobial agent-associated ileocolitis in laboratory animals and colitis in man, were found to be toxigenic. The susceptibility of these isolates to a variety of antimicrobial agents provides information that may be of value in assessing the means by whichC. difficile may produce colitis, in developing selective bacteriologic media for diagnosis, and in selecting appropriate antimicrobial therapy for colitis.     

Analysis of genome-specific sequences inPhleum species: Identification and use for study of genomic relationships

Sau3AI shot gun cloning and colony hybridization with total genomic probes were used to isolate genome-specific sequences inPhleum species. The total DNA isolated from diploid speciesP. alpinum andP. bertolonii was partially digested withSau3AI and cloned using pUC19 as a vector to anE. coli strain DH5mcr. A partial genomic DNA library consisting of 3030 colonies for the genome ofP. alpinum and one consisting of 3240 colonies for the genome ofP. bertolonii were constructed. Twelve hundred and thirty colonies from the DNA library ofP. alpinum and 1320 from that ofP. bertolonii were respectively blotted to membrane filters and hybridized to the total genomic probes from these two species. Eight clones specific toP. alpinum and 13 specific toP. bertolonii were isolated through colony hybridization and further dot-blot hybridization. Most of these clones may carry highly or moderately repetitive sequences. Three sequences specific toP. alpinum and 3 specific toP. bertolonii were used as probes to hybridize theEcoRI-digested DNA samples from four species,P. alpinum,P. bertolonii,P. pratense andP. montanum, on Southern blot. The results from these hybridization experiments showed that all 3P. bertolonii-specific probes and 2 of the 3P. alpinum-specific probes hybridized to the DNA ofP. pratense, thus confirming the conclusion of the close relationships between the cultivated timothy and its two wild relatives that was drawn in our previous study using the C-banding technique.     

EhRho1, a RhoA-Like GTPase of Entamoeba histolytica, Is Modified by Clostridial Glucosylating Cytotoxins

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[¹⁴C]glucose.     

Characterization of 10 mesophilic cellulolytic clostridia isolated from a municipal solid waste digestor

By hybridization experiments with three cloned fragments carrying cellulase genes ofClostridium cellulolyticum, we tried to differentiate 10 cellulolytic mesophilic clostridia, isolated from a municipal solid waste digestor. On the basis of hybridization experiments, three major groups were found among the 10 isolates. The two endoglucanase genes,cel CCA andcel CCB ofC. cellulolyticum, hybridized with nine strains of our isolates, suggesting homology and widespread distribution of these genes. Withcel CCA the strain A31 exhibited a different pattern. In contrast to these nine strains, the strain A11 was found to share no or very weak homology with these two probes, which indicated that this strain of cellulolytic clostridia possesses nonidentical cellulase complex. None of these new strains hybridized withnif genes, indicating that these clostridia did not appear to be nitrogen-fixing bacteria. With other biochemical characteristics, we found that these bacteria appeared to be different from the presently known mesophilic cellulolytic clostridia.     

Vaccination against lethalClostridium difficile enterocolitis with a nontoxic recombinant peptide of toxin A

Toxin A ofClostridium difficile has a complex series of repeating units, each 20 or 30 amino acids in length, located at the COOH-terminus of the molecule. In the following study, we found that antiserum against a nontoxic recombinant peptide comprising 33 of the 38 repeating units neutralized the enterotoxic and cytotoxic activity of the toxin and that hamsters vaccinated with the recombinant peptide were partially protected againstC. difficile disease.     

A simple procedure for identification ofClostridium botulinum colonies

For direct identification of toxigenic colonies ofClostridium botulinum type E, suspected colonies are uniformly suspended in a phosphate buffer containing 0.5% (w/v) gelatin and 0.05% (w/v) Tween 20. After centrifuging, the supernatant is tested for botulinal toxin by an enzyme-linked immunosorbent assay (ELISA). The assay is specific for this type as it did not react with culture filtrates of otherClostridium species, including non-toxigenic E-like organisms.     

Homology of Balbiani rings among chironomid species and localization of a new mobile element on the polytene chromosomes

The results ofin situ cross-hybridization of the cloned DNA fragments of BRa, BRb and BRc fromChironomus thummi to the polytene chromosomes of 14Chironomus species andCamptochironomus tentans are presented. BRs of the species studied were demonstrated to contain the homologus DNA sequences. The cloned fragment from the BRa ofC. thummi hybridized with the BRa ofC. piger and with a region on the arm G ofC. tentans andC. plumosus, the latter species had no extra BR. The clone 16 from the BRb ofC. thummi hybridized only with the developed BR on the arm G of all species studied. The sequence from the BRc ofC. thummi was located in the BRc ofC. piger and in the developed BR ofC. plumosus andC. nuditarsis, which were located at the most distal end of arm G. These clones can be used as markers of homologous BRs. The new mobile elements C6.10 fromC. thummi genome were localized on the polytene chromosomes of 10Chironomus species andCamptochironomus tentans. The species of the generaLipiniella, Cryptochironomus andGlyptotendipes did not contain the sequences homologous to ME C6.10.     

Characterisation and carriage ratio of Clostridium difficile strains isolated from a community-dwelling elderly population in the United Kingdom

Background

Community-associated Clostridium difficile infection (CDI) appears to be an increasing problem. Reported carriage rates by C.difficile are debatable with suggestions that primary asymptomatic carriage is associated with decreased risk of subsequent diarrhoea. However, knowledge of potential reservoirs and intestinal carriage rates in the community, particularly in the elderly, the most susceptible group, is limited. We have determined the presence of C.difficile in the faeces of a healthy elderly cohort living outside of long-term care facilities (LCFs) in the United Kingdom.

Methods

Faecal samples from 149 community-based healthy elderly volunteers (median age 81 years) were screened for C.difficile using direct (Brazier''s CCEY) and enrichment (Cooked Meat broth) culture methods and a glutamate dehydrogenase (GDH) immunoassay. Isolates were PCR-ribotyped and analysed for toxin production and the presence of toxin genes.

Results

Of 149 faecal samples submitted, six (4%) were found to contain C.difficile. One particular sample was positive by both the GDH immunoassay and direct culture, and concurrently produced two distinct strain types: one toxigenic and the other non-toxigenic. The other five samples were only positive by enrichment culture method. Overall, four C.difficile isolates were non-toxigenic (PCR-ribotypes 009, 026 (n = 2) and 039), while three were toxigenic (PCR-ribotypes 003, 005 and 106). All individuals who had a positive culture were symptom-free and none of them had a history of CDI and/or antibiotics use in the 3 month period preceding recruitment.

Conclusions

To our knowledge, this is the first study of the presence of C.difficile in healthy elderly community-dwelling individuals residing outside of LCFs. The observed carriage rate is lower than that reported for individuals in LCFs and interestingly no individual carried the common epidemic strain PCR-ribotype 027 (NAP1/BI). Further follow-up of asymptomatic carriers in the community, is required to evaluate host susceptibility to CDI and identify dynamic changes in the host and microbial environment that are associated with pathogenicity.     

Assessment of changes in the epidemiology of Clostridium difficile isolated from diarrheal patients in Hungary

150 Clostridium difficile strains isolated from diarrheal feces were collected from three parts of Hungary and the presence of genes responsible for toxin A and B, and binary toxin production were examined. MIC distribution against clindamycin, erythromycin, metronidazole, moxifloxacin and rifampin of 80 toxigenic strains selected from the above-mentioned strains and 20 large clostridial toxins (LCTs)-positive strains chosen from our earlier strain collection were determined. 80% of the examined 150 strains were positive for both tcdA and tcdB, and no toxin A-negative, toxin B-positive isolates were found during the study period. 5.3% of toxigenic strains proved to be positive for binary toxin too. Among binary toxin-positive strains, one strain showed the same pattern characteristic of PCR ribotype 027. Comparison of recent findings and our earlier results, the prevalence of toxin-producing and binary toxin-positive strains among C. difficile isolated from diarrheal specimens increased. No metronidazole resistant isolate was detected among strains isolated in 2002–2003 and 2006–2007. The rates of resistance to erythromycin, clindamycin, moxifloxacin and rifampin among strains isolated between 2006 and 2007 were 25%, 27.5%, 25% and 6.3%, respectively. Erythromycin resistance was frequently associated with clindamycin and moxifloxacin resistance, however this resistant phenotype was not found among strains isolated in 2002–2003.     

Clostridium difficile testing algorithms: What is practical and feasible?

There has been renewed interest in the laboratory diagnosis of Clostridium difficile infections due in large measure to the increase in both numbers and severity of cases of this disease. For the past two decades, enzyme-immunoassays (EIAs) for the detection of first C. difficile toxin A and then toxins A and B have been the most widely used diagnostic test for diagnosis of C. difficile infections. Recently this diagnostic approach has been called into question by the recognition that a screening test which detects glutamate dehydrogenase, a cell wall antigen of C. difficile, was significantly more sensitive than toxins A and B EIAs making it an effective screening test for C. difficile infection. Although sensitive, GDH lacks specificity and so if this test was utilized, a confirmatory test to differentiate false positives from true positives was needed. Studies to date have used cytotoxin neutralization or toxigenic culture as confirmatory tests but both of these have their limitations. A testing algorithm using rapid immunochromatographic devices for detection of GDH and toxins A and B as screening tests will give an accurate test result in approximately 90% of specimens within one hour when using cytotoxin neutralization as a reference method. For the other 10% of specimens, a third test would be needed in the algorithm. This test could be cytotoxin neutralization, toxigenic culture, or PCR for toxin or toxin operon genes.     

Are Rapid Immunoassays for in vivo Detection of Toxin A Sufficient for Diagnostic Purposes of Clostridium difficile -Associated Diseases?

One hundred and fifty-five stool specimens of patients suspected for Clostridium difficile -associated diarrhoea, colitis or pseudomembranous colitis (PMC) were investigated. All patients were pre-treated with antibiotics, suffered from watery diarrhoea and abdominal pain and were hospitalized in different hospital units. Units varied from departments of surgery, internal medicine, intensive care, paediatry, dermatology, orthopaedy to gastroenterology. Fifty C. difficile strains were isolated from the faecal samples. Clostridium difficile toxin detection was done directly in the stool samples, and also in cultured C. difficile strains (in vivo and in vitro, respectively). We observed clear differences between in vivo and in vitro toxin A detection by using commercial rapid immuno-enzymatic tests: from 25 in vivo toxin A-negative samples, 17 were positive in vitro. This observation suggests that culturing of C. difficile on selective medium is mandatory for adequate toxin detection and necessary for confirming the presence of toxin-producing C. difficile. This is especially important among patients with clinical symptoms and history of pretreatment with antibiotics and when in vivo toxin A detection is negative. It was established that toxin gene detection by PCR is optimal and PCR results were concordant with results of other in vitro assays. Genotyping by using AP-PCR and PCR ribotyping showed heterogeneity among the toxigenic C. difficile strains cultured from in vivo toxin A-negative stool samples.     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of membrane functions in intact HeLa cells byClostridium difficile cytotoxic culture filtrates

The basis for cytotoxicity to intact HeLa cells by culture filtrates ofClostridium difficile has been investigated. Decrease in intracellular K⁺ levels and inhibition of -aminoisobutyric acid uptake were detected first after exposure to filtrates, followed by inhibition of macromolecular synthesis. Twenty-five percent of the K⁺ remained associated with the cell monolayer, and amino acid uptake and macromolecular synthesis were not totally abolished. These results indicate thatC. difficile culture filtrates preferentially inhibited membrane functions, either by exhausting ATP supplies or by disrupting the permeability barrier of the cell.     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.