Characterization of Clostridium sp. RKD producing botulinum-like neurotoxin

A Gram positive, motile, rod-shaped, strictly anaerobic bacterium isolated from intestine of decaying fish was identified as Clostridium sp. RKD and produced a botulinum type B-like neurotoxin as suggested by mouse bioassay and protection with anti botulinum antibodies. The neurotoxicity was functionally characterized by the phrenic nerve hemi-diaphragm assay. Phylogenetic analysis based on 16S rDNA sequence, placed it at a different position from the reported strains of Clostridium botulinum. The strain exhibited differences from both Clostridium botulinum and Clostridium tetani with respect to morphological, biochemical and chemotaxonomic characteristics. Botulinum group specific and serotype specific primers amplified the DNA fragments of 260 and 727 bp, respectively, indicating presence of botulinum type 'B' toxin gene. Sequence of nearly 700 bp amplified using primers specific for botulinum neurotoxin type B gene, did not show any significant match in the database when subjected to BLAST search.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

Recovery of a strain of Clostridium botulinum producing both neurotoxin A and neurotoxin B from canned macrobiotic food.

A rare strain of Clostridium botulinum subtype Ab was isolated from a canned macrobiotic food suspected of being linked to a fatal case of food-borne botulism. The strain was recovered and identified by conventional methods modified by the inclusion of a PCR assay (G. Franciosa, J.L. Ferreira, and C.L. Hatheway, J. Clin. Microbiol. 32:1911-1917, 1994). The titers of neurotoxins produced by the strain were evaluated by a mouse bioassay.     

Isolation of Clostridium acetobutylicum strains producing butanol and acetone

Summary A system is described for the isolation of bacteria (Clostridium acetobutylicum) from broad beans, potatoes or maize. The isolates were tested in molasses medium and solvent yields up to 18–20 g/litre of butanol plus acetone were obtained.     

Toxicity of Clostridium botulinum type E neurotoxin to Great Lakes fish: implications for avian botulism

Since 1999, large-scale mortalities of fish-eating birds have been observed on the Great Lakes, and more specifically on Lake Erie. Type E botulism has been established as the primary cause of death. The mechanism of type E botulism exposure in fish-eating birds is unclear. Given that these birds are thought to eat live fish exclusively, it seems likely that their prey play a key role in the process, but the role of fish as potential transport vectors of botulinum neurotoxin type E (BoNT/E) to birds has not been adequately investigated. Between June 2003 and April 2004 a methodological model for exposing fish to Clostridium botulinum was developed and used to compare the sensitivity of rainbow trout (Oncorhynchus mykiss), round goby (Neogobius melanostomas), walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) to four doses (0, 800, 1,500, and 4,000 Mouse Lethal Doses) of Clostridium botulinum type E neurotoxin. Each fish species expressed unique changes in both behavior and skin pigmentation prior to death. Yellow perch survived significantly longer (P < 0.05) than the three other species at all toxin treatments. Results of this study suggest that live fish can represent a significant vector for transfer of BoNT/E to birds.     

Metarhizium aciculare sp. nov. for euvesperins A and B producing Metarhizium strains

We have already found some Metarhizium isolates produce euvesperins A and B, which were circumventors of arbekacin resistance in MRSA. These isolates were obtained from soil samples collected from the Izu islands and Kannami town, Sizuoka, Japan. Using a combination of micro-morphological characteristics and multigene (SSU, LSU, TEF) phylogenics, the isolates were identified as a previously underscribed species of Metarhizium. Phylogenetically, the species is close to M. carneum but it is distinguished morphologically from M. carneum by size and the surface structure of conidia.     

Two cellulolytic Clostridium species: Clostridium cellulosi sp. nov. and Clostridium cellulofermentans sp. nov.

Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized. Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells. The optimum growth temperature is 55 to 60 degrees C, with a range of 40 to 65 degrees C. The deoxyribonucleic acid composition is 35 mol% G + C. The name Clostridium cellulosi sp. nov. is proposed. The type strain is AS 1.1777. Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells. The deoxyribonucleic acid composition is 34 mol% G + C. The name Clostridium cellulofermentans sp. nov. is proposed. The type strain is AS 1.1775. Both C. cellulosi AS 1.1777 and C. cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People's Republic of China.     

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.     

Phenotype and genotype of Candida albicans strains isolated from pregnant women with recurrent vaginitis

Fourteen out of 80 pregnant women receiving prenatal care presented signs and symptoms of recurrent vaginal candidiasis. Candida albicans strains were isolated from 12 patients (85.7%), and these were submitted to morphotyping (morphological characteristics of the colony), antifungal typing (pattern of sensitivity to amphotericin B, 5-fluorcytosine, myconazole, ketoconazole and fluconazole) and genotyping (electrophoretic migration of DNA fragments digested with EcoRI and HinfI). Alteration of morphotype and antifungal type was observed in 50% of the patients, but the genotype of the strains isolated from the same patients at different times was identical in all subjects. The predominant morphotypes presented continuous fringes and the basic changes observed among antifungal types was the emergence of strains resistant to myconazole, which was the drug used for the treatment of the first episode of vaginitis. We conclude that recurrent vaginal candidiasis is caused by the persistence of a single yeast genotype that undergoes morphological and behavioral changes in the presence of antifungal agents due to the selective pressure to which it is submitted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of selected Lactobacillus sp. on Clostridium difficile strains with different toxigenicity profile

This study was performed for determination of antagonistic activity of Lactobacillus spp. (L. plantarum 2017405, L. rhamnosus GG, L. acidophilus DSM 21007 and L. fernmentumn 353) on Clostridiunl difficile strains belonging to different toxigenicity profiles. Forty strains C. difficile isolated from patients suffering from antibiotic associated diarrhea (AAD) were used. Among C. difficile strains 13 produced toxin A and B (A+B+CDT-), 14 produced only toxin B (AB'CDT), 9 produced toxins A and B and possessing of binary toxin genes (A+B+CDT-) and 4 were non-toxigenic (A-B-CDT-). We did not observe relationship between degree of antagonistic activity Lactobacillus spp. and profile of toxigenicity of C. difficile strains.     

Clostridium barkeri sp. n

Clostridium barkeri sp. n. has been described, and its relationship to other clostridia is discussed.     

New solvent-producing Clostridium sp. strains, hydrolyzing a wide range of polysaccharides, are closely related to Clostridium butyricum

Thirteen new Clostridium strains, previously isolated from soil and found to produce high amounts of solvents from glucose, hydrolyzed a great variety of α- and β-glycans, including raw starch, xylan, pectin, inulin and cellulose. The sequences of the PCR-amplified DNA fragments containing the variable 3′ part of one of the 16S rRNA genes were 99.5% identical. The macrorestriction pattern of two endonucleolytic digests of chromosomal DNA in the pulsed-field gel electrophoresis (PFGE) confirmed their high homogeneity on the DNA level. The complete 16S rRNA gene sequence of three selected strains was 99.8% identical to the 16S rRNA gene sequence from Clostridium butyricum and separates them from C. acetobutylicum. To the closely related four species of solventogenic clostridia a new group of strains has to be added, which has a great potential for the direct fermentation of biomass. Journal of Industrial Microbiology & Biotechnology (2001) 27, 329–335. Received 12 September 2000/ Accepted in revised form 25 July 2001     

Urease-negative strains of Clostridium sordellii.

Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.     

Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani

Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.