Clostridia

Int J Syst Bacteriol, 1996 Oct, 46(4), 1174 - 6
Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences; Kuhnert P et al.; The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined . After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers . A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C . chauvoei and C . septicum in Clostridium cluster I (M . D . Collins, P . A . Lawson, A . Willems, J . J . Cordoba, J . Fernandez-Garayzabal, P . Garcia, J . Cai, H . Hippe, and J . A . E . Farrow, Int . J . Syst . Bacteriol . 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani . We found that 99.3% of the nucleotides in the genes of C . chauvoei and C . septicum are identical.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1145 - 52
Clostridium ultunense sp . nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium; Schnurer A et al.; A syntrophic acetate-oxidizing bacterium, strain BST (T = type strain), was isolated from a previously described mesophilic triculture that was able to syntrophically oxidize acetate and form methane in stoichiometric amounts . Strain BST was isolated with substrates typically utilized by homoacetogenic bacteria . Strain BST was a spore-forming, gram-positive, rod-shaped organism which utilized formate, glucose, ethylene glycol, cysteine, betaine, and pyruvate . Acetate and sometimes formate were the main fermentation products . Small amounts of alanine were also produced from glucose, betaine, and cysteine . Strain BST grew optimally at 37 degrees C and pH 7 . The G+C content of the DNA of strain BST was 32 mol% . A 16S rRNA sequence analysis revealed that strain BST was a member of a new species of the genus Clostridium . We propose the name Clostridium ultunense for this organism; strain BS is the type strain of C . ultunense.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1105 - 12
Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component; East AK et al.; The cluster of genes encoding components of the botulinum neurotoxin (BoNT) complex was mapped in proteolytic (group I) Clostridium botulinum strains encoding BoNT types A, B, and F . Two different arrangements of genes were found: type A strain 62A and type B strain NCTC 7273 have similar organizations of genes encoding BoNT, the nontoxic nonhemagglutinin component (NTNH), hemagglutinin components, and P-21; type F strain Langeland has genes encoding BoNT, NTNH, and P-21, and a previously unidentified open reading frame encoding a protein of 416 amino acids . A group of type A strains typified by infant strain Kyoto-F, which is unlike type A strain 62A, lacks genes for hemagglutinin components and exhibits an organization similar to that of type F . Sequencing and pairwise analysis revealed the presence of possible chimeric sequences in some NTNH genes of proteolytic C . botulinum . Discordance in genealogical trees derived from different regions of the NTNH genes was observed which could be symptomatic of recombination and which may indicate that the NTNH gene represents a hot spot for such events within the cluster of genes encoding the BoNT complex . It is also evident that the phylogenetics of the NTNH gene, which is linked to the gene encoding BoNT, does not mirror the evolutionary history of the BoNT, upon which the C . botulinum species complex is defined and subdivided.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1083 - 7
Phylogenetic relationships of the genera Acetobacterium and Eubacterium sensu stricto and reclassification of Eubacterium alactolyticum as Pseudoramibacter alactolyticus gen . nov., comb . nov; Willems A et al.; 16S rRNA gene sequences of the type strains of the seven previously described Acetobacterium species were determined . The Acetobacterium species were found to form a tight phylogenetic cluster within the Clostridium subphylum of the gram-positive bacteria . Within this subphylum these organisms belong to cluster XV as defined by Collins et al . (M.D . Collins, P.A . Lawson, A . Willems, J.J . Cordoba, J . Fernandez-Garayzabal, P . Garcia, J . Cai, H . Hippe, and J . A . E . Farrow, Int . J . Syst . Bacteriol . 44:812-826, 1994) together with Eubacterium alactolyticum barkeri, Eubacterium callanderi, and Eubacterium limosum . Our data indicate that Clostridium cluster XV consists of at least the following three genera: the genus Acetobacterium, the genus Eubacterium sensu stricto (comprising E . limosum, E . barkeri, and E . callanderi), and the genus Pseudoramibacter gen . nov., which is created for E . alactolyticum, which we reclassify as Pseudoramibacter alactolyticus comb . nov.

J Nucl Med, 1996 Oct, 37(10), 1683 - 5
Abnormal colonic accumulation of fluorine-18-FDG in pseudomembranous colitis; Hannah A et al.; A 51-yr-old man with a history of pancreatic carcinoma was studied with {18F}fluorodeoxyglucose ({18F}FDG) and PET as part of staging for residual disease after chemotherapy . The PET study was performed during a clostridium difficile-associated diarrheal illness . Striking {18F}FDG uptake was demonstrated in the wall of the colon over its entire length . Clostridium difficile associated diarrhea and mechanisms of {18F}FDG uptake in normal and abnormal tissues are briefly reviewed and a mechanism for FDG uptake in this patient is postulated.

Biochemistry, 1996 Oct 1, 35(39), 12842 - 8
Coordination of the {2Fe-2S} cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy; Shergill JK et al.; The {2Fe-2S} ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60 . This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the {2Fe-2S} cluster . The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates . Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine . The mutated ferredoxins have been purified and were all found to contain {2Fe-2S} clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein . The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein . The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the {2Fe-2S} center . The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone . 2H2O ESEEM spectra showed that the {2Fe-2S} cluster is accessible for exchange with solvent deuterons . ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein . There was no evidence for coordination of the {2Fe-2S} cluster by {14N}histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin . By these criteria, the environment of the {2Fe-2S} center is not distinguishable from those in plant-type ferredoxins . Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues . The data shown here indicate that the fourth ligand of the {2Fe-2S} cluster is neither a histidine residue nor another nitrogenous ligand . The possibility of oxygenic coordination for this molecular variant is discussed.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 115 - 20
Cloning of a gene encoding cinnamoyl ester hydrolase from the ruminal bacterium Butyrivibrio fibrisolvens E14 by a novel method; Dalrymple BP et al.; A gene (cinI) encoding a cinnamoyl ester hydrolase (CEH) has been isolated from the ruminal bacterium, Butyrivibrio fibrisolvens E14, using a model substrate, MUTMAC {4-methylumbelliferoyl (p-trimethylammonium cinnamate chloride)} . CinI has significant amino-acid similarities with members of a large and diverse family of hydrolases with a serine/aspartic acid/histidine catalytic triad . Our analyses identified two previously unclassified amino acid sequences, the amino-terminal domain of unknown function in XynZ from Clostridium thermocellum and XynC, an acetylxylan esterase from Caldicellulosiruptor saccharolyticus, as members of the same family of hydrolases . A previously described esterase with CEH activity, XylD from Pseudomonas fluorescens ssp . cellulosa, is not similar to CinI . CinI was expressed at high levels in the periplasmic fraction of E . coli TOPP2 and released ferulic acid from Fara {5-O-(trans-feruloyl)-arabinofuranose} prepared from wheat bran.

Dis Colon Rectum, 1996 Oct, 39(10), 1107 - 11
Initial North American experience with botulinum toxin type A for treatment of anismus; Joo JS et al.; PURPOSE: Botulinum toxin type A (BTX-A), produced by Clostridium botulinum, is a potent neurotoxin . The purpose of this study was to evaluate the efficacy of BTX-A for treatment of anismus . MATERIALS AND METHODS: All patients treated with BTX-A for anismus were evaluated . Eligibility criteria included a history of chronic assisted evacuation (laxatives, enemas, or suppositories), demonstration of anismus by cinedefecogram and electromyography, and failure of a minimum of three sessions of supervised biofeedback therapy (BF) . Contingent on body mass, 6 to 15 units of BTX-A was injected bilaterally under electromyography guidance into the external sphincter or the puborectalis muscle . Treatment was repeated as necessary for a maximum of three sessions during a three-month period . Success was considered as discontinuation of evacuatory assistance and was evaluated between one and three months and again at up to one year . RESULTS: Between July 1994 and May 1995, four patients ranging from 29 to 82 years in age (2 females, 2 males) had anismus that failed to respond to between 3 and 15 biofeedback sessions . All patients improved between one and three months after BTX-A injection, and two had sustained improvement for a range of three months to one year . There was no morbidity or mortality associated with BTX-A injection . CONCLUSIONS: BTX-A is extremely successful for temporary treatment of anismus that is refractory to BF management . However, because the mechanism of action is short, longer term results are only 50 percent successful . Hopefully, modifications in the strain of BTX-A and dose administered will allow longer periods of success or a repeat trial of BF . Nonetheless, this preliminary report is very encouraging in offering a method of managing this recalcitrant condition.

J Bacteriol, 1996 Oct, 178(19), 5844 - 6
Phospholipid profiles of Clostridium difficile; Drucker DB et al.; 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.

J Bacteriol, 1996 Oct, 178(19), 5732 - 40
Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome; Ahsan MM et al.; The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055 . The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative . CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily E1 endoglucanase domain, a family J endoglucanase domain, a docking domain, and another domain of unknown function (UD-2) . UD-1 has no significant similarity to UD-2 . CelJ hydrolyzed carboxymethylcellulose and xylan, and xylanase activity was ascribed to the family J domain . Antiserum raised against the truncated CelJ cross-reacted with proteins contained in the cellulosome of C . thermocellum F1 . These results strongly suggest that CelJ is equivalent to S2, which was identified as the largest catalytic component in the cellulosome of C . thermocellum YS . A second but incomplete ORF encoding an enzyme classified in subfamily E2 endoglucanase, was located downstream of celJ.

Curr Microbiol, 1996 Oct, 33(4), 220 - 3
Relapses or reinfections: analysis of a case of Clostridium difficile-associated colitis by two typing systems; Kato H et al.; Immunoblotting and pulsed-field gel electrophoresis of Clostridium difficile isolates were employed to differentiate reinfection by a newly acquired strain from relapse by an original strain in a 10-year-old patient with four episodes of C . difficile-associated colitis . Immunoblot typing demonstrated subserogroup K-1 of serogroup K for the first and second organisms, subserogroup A-1 of serogroup A for the third organism, and subserogroup G-4 of serogroup G for the fourth organism . PFGE analysis revealed consistent results with immunoblot analysis except that the strains from the fourth episode, whose DNA constantly degraded, were nontypable by this method . Five separate isolates of C . difficile from a specimen of each episode showed identical PFGE patterns, indicating that infections of multiple strains probably did not occur in this patient . These typing results suggested that the second episode after a 17-day course of vancomycin therapy represented a relapse by the strain causing the first episode, and that the third and fourth episodes after tapering vancomycin therapy were reinfections by other strains . Both immunoblot and PFGE typing systems are promising tools for analyzing recurrence of C . difficile infection.

Ann Intern Med, 1996 Oct 1, 125(7), 558 - 63
An outbreak of type A botulism associated with a commercial cheese sauce; Townes JM et al.; BACKGROUND: Although botulism is rare, recognition of a possible case of this illness represents a public health emergency . To prevent more cases, prompt investigation must be done to determine whether illness is linked to commercial product or restaurant . Botulism can masquerade as other illnesses, and seemingly unlikely foods can harbor botulinum toxin . OBJECTIVE: To confirm the diagnosis and determine the cause and extent of an outbreak of botulism associated with food served at a delicatessen . DESIGN: Retrospective cohort study of patrons of the delicatessen; laboratory analysis of food, serum samples, and stool samples; and traceback of implicated food . SETTING: Community in Georgia . PARTICIPANTS: Patrons of the delicatessen . MAIN OUTCOME MEASURES: Botulinum toxin in food, serum, or stool and Clostridium botulinum in food and stools . RESULTS: 8 of 52 patrons (15%) met the case definition for botulism . In 4 of the 8 patrons, and illness other than botulism was initially diagnosed . Five of the 8 were hospitalized, and 1 died . Stool cultures from 4 patrons yielded type AC . botulinum, and two serum samples contained botulinum toxin . All ill persons ate food from the delicatessen on 1 October 1993 . Of the 22 persons who ate at the delicatessen that day, all 8 ill persons but none of the 14 well persons ate a potato stuffed with meat and cheese sauce . An open can of cheese sauce contained type A botulinum toxin and yielded C botulinum on culture . Cheese sauce experimentally inoculated with C botulinum spores became toxic after 8 days at a temperature of 22 degrees C (room temperature) . CONCLUSIONS: A commercial, canned cheese caused a botulism outbreak . This product readily becomes toxic when contaminated by C botulinum spores and left at room temperature . Mild botulism caused by unusual vehicles may be misdiagnosed . Botulism should be included in the differential diagnosis of persons with signs or symptoms of acute cranial nerve dysfunction.

Gene, 1996 Sep 26, 174(1), 145 - 50
A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile; Mullany P et al.; We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile . Physical analysis of this transposon demonstrated that it contained a group II intron . This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria . The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916 . DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C . difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.

Biochem Biophys Res Commun, 1996 Sep 24, 226(3), 735 - 40
Molecular cloning of Clostridium perfringens epsilon-toxin gene and its high level expression in E . coli; Goswami PP et al.; A gene coding for epsilon-toxin was isolated from a field isolate of Clostridium perfringens type D by PCR amplification and was cloned under the control of T5 promoter fused with six-histidine tag at the amino terminal end . Escherichia coli cells harbouring this construct expressed high levels of the recombinant protein in the form of inclusion bodies . The protein was purified using single step affinity chromatography on a Ni(2+)-nitrilotriacetic acid (NTA) agarose column . Upon immunization of rabbit with the purified recombinant protein, high antibody titre was detected . The antibodies raised against the recombinant protein were able to recognize the recombinant as well as the native toxin . Anti epsilon-toxin monoclonal antibody was able to detect the recombinant protein in a Western blot . N-terminal sequence of the recombinant protein matched with the known sequence of the toxin . At the shake flask level, up to 20 mg of pure epsilon-prototoxin was produced per litre of culture.

Biochemistry, 1996 Sep 17, 35(37), 12119 - 25
Evidence that carbon monoxide is an obligatory intermediate in anaerobic acetyl-CoA synthesis; Menon S et al.; Carbon monoxide is produced by several biological reactions . It is proposed to act as an intracellular signaling molecule and can serve as the carbon and electon source for certain bacteria . Direct evidence for a new biological role for CO is presented here . The results strongly indicate that CO is produced as an obligatory intermediate during growth of the acetogenic bacterium Clostridium thermoaceticum on glucose, H2/CO2, or aromatic carboxylic acids . Our results are consistent with earlier hypotheses of the intermediacy of CO during growth of acetogenic bacteria on CO2 and hexoses {Diekert, G., & Ritter, M . (1983) FEMS Microbiol . Lett . 17, 299-302} and methanogenic Archaea on CO2 {Stupperich, E., Hammel, K . E., Fuchs, G., & Thauer, R . K . (1983) FEBS Lett . 152, 21-23} . Therefore, CO production is a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis . The carbonyl group of acetyl-CoA is shown to be formed from the carboxyl group of pyruvate by the following steps . (i) Pyruvate undergoes decarboxylation by pyruvate:ferredoxin oxidoreductase to form acetyl-CoA and CO2 . (ii) CO2 is reduced to CO by the CODH site of the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) . (iii) CO generated in situ combines with the ACS active site to form a paramagnetic adduct that has been called the NiFeC species, and (iv) the bound carbonyl group combines with a bound methyl group and CoA to generate acetyl-CoA . To our knowledge, this paper represents the first demonstration of a pathway in which CO is produced and then used as a metabolic intermediate.

Eur J Biochem, 1996 Sep 15, 240(3), 707 - 12
Restoration of Clostridium difficile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate; Schmidt M et al.; Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins . Since phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P2} has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridium difficile toxin B, which inactivates Rho proteins . Addition of MgATP to permeabilized HEK cells increased basal PLD activity and potentiated PLD stimulation by the stable GTP analogue, guanosine 5'-{gamma-thio}triphosphate (GTP{S}), concomitant with a large increase in PtdIns(4,5)P2 . On the other hand, neomycin, which binds to PtdIns(4,5)P2, inhibited basal and GTP{S}-stimulated PLD activities . Addition of PtdIns(4,5)P2 increased PLD activity in HEK cell membranes by 2-3-fold, whereas various other phospholipids were ineffective . Prior treatment of HEK cells with toxin B reduced the level of PtdIns(4,5)P2, measured either in intact cells or in membrane preparations, by about 40% . In membranes of toxin-B-treated cells, basal and GTP{S}-stimulated PLD activities were reduced, when measured with exogenous phosphatidylcholine as enzyme substrate . Inclusion of PtdIns(4,5)P2 with phosphatidylcholine in the substrate vesicles or addition of PtdIns(4,5)P2 fully restored basal and GTP{S}-stimulated PLD activities in membranes of toxin-B-treated cells . In conclusion, the data indicate that PtdIns(4,5)P2 is an essential cofactor for PLD activity in HEK cells and that inhibition of PLD activity by the Rho-inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P2.

Biochemistry, 1996 Sep 10, 35(36), 11710 - 8
4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction; Muh U et al.; 4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond . The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA . Addition of crotonyl-CoA to the dehydratase, which contains FAD as well as non-heme iron and acid labile sulfur, led to a decrease of the flavin absorbance at 438 nm and an increase in the region from 500 to 800 nm . The protein-bound FAD was easily reduced to the semiquinone (redox equilibration within seconds) and only slowly to the hydroquinone (redox equilibration minutes to hours): the redox potentials were not unusual for flavoproteins (Eox/sq = -140 +/- 15 mV and Esq/red = -240 +/- 15 mV; pH 7.0, 25 degrees C) . There was no equilibration of electrons between the flavin and the Fe-S cluster, which was difficult to reduce . After extensive photoreduction, an EPR signal indicative of a {4Fe-4S}+ cluster was detected (g-values: 2.037, 1.895, 1.844) . Upon exposure to air at 0 degrees C, the enzyme lost dehydration activity completely within 40 min, but isomerase activity dropped to about 40% of the initial value and persisted for more than a day . The properties of the protein-bound FAD are consistent with a mechanism involving transient one-electron oxidation of the substrate to activate the the beta-C-H bond . The putative {4Fe-4S}2+ cluster could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group.

Arch Intern Med, 1996 Sep 9, 156(16), 1883 - 8
Preventable disease in correctional facilities . Desmoteric foodborne outbreaks in the United States, 1974-1991; Cieslak PR et al.; BACKGROUND: Various disease outbreaks have been reported among prisoners . Recent foodborne outbreaks in correctional facilities in Georgia and Delaware prompted us to review the epidemiological characteristics of such outbreaks reported in the United States . METHODS: Foodborne outbreaks reported to the Centers for Disease Control and Prevention as part of routine surveillance from 1974 to 1991 were examined to identify outbreaks in jails, prisons, correctional facilities, and juvenile detention centers . Outbreak sizes, temporal trends, food vehicles, pathogens, and hygienic transgressions were analyzed . RESULTS: Eighty-eight desmoteric foodborne outbreaks involving 14307 cases of illness were reported from 31 states and territories . The mean outbreak size was 163 cases, compared with a mean of 31 cases for the 9107 reported outbreaks not involving prisoners . No fatalities among prisoners were reported . No pathogen was identified in 47 (53%) of the 88 outbreaks Salmonella species accounted for 15 (37%) of 41 outbreaks of known cause from 1974 to 1991, Clostridium perfringens for 14 (34%), and Staphylococcus aureus for 9 (22%) . Fourteen of 15 Salmonella outbreaks occurred from 1984 to 1991 . Food vehicles were reported for 63 (72%) of the outbreaks . Beef and poultry each were implicated in 9 (14%) of these, followed by fish or poultry salads and Mexican food, which accounted for 6 outbreaks (10%) . Food-handling errors were reported for 69 (78%) of the 88 outbreaks . Improper food storage was reported in 62 (90%) of these . CONCLUSIONS: Foodborne outbreaks are reported regularly from correctional facilities in the United States . Outbreaks caused by Salmonella species, a special threat to prisoners with human immunodeficiency virus infection, seem to be increasing . Food production in correctional facilities should meet minimum safety standards, including sufficient refrigeration facilities, training of food handlers, and exemption of ill food handlers from work.

Southeast Asian J Trop Med Public Health, 1996 Sep, 27(3), 606 - 9
Antibacterial activity of teicoplanin against Clostridium difficile; Wongwanich S et al.; The in vitro inhibitory action of teicoplanin, vancomycin, metronidazole and clindamycin against clinical isolates of Clostridium difficile was investigated . Minimum inhibitory concentrations (MICs) were determined using E test . Teicoplanin (MIC range 0.023-0.75 microgram/ml), vancomycin (MIC range 0.5-3 micrograms/ml) and metronidazole (MIC range 0.19-1 microgram/ml) were all very active against the isolates examined . No resistant strains of C . difficile to those three antimicrobial agents were observed, whereas resistance to clindamycin was found in 39.5% of the tested strains . Teicoplanin was about 4-times more potent than vancomycin . It appears to be a more promising antimicrobial for treatment of C . difficile enteric disease.

Enzyme Microb Technol, 1996 Sep, 19(4), 267 - 76
Site-directed mutations of the catalytic and conserved amino acids of the neuraminidase gene, nanH, of Clostridium perfringens ATCC 10543; Chien CH et al.; The small nanH gene encoding the neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector . Sequence analysis revealed an ORF encoding 382 amino acids without a signal peptide sequence . Four regions of amino-acid sequence, 71-82, 140-151, 208-219, and 255-266 constituted four repeated and conserved sequence motifs-Ser-X-Asp-X-Gly-X-Thr-Trp-, the "Asp boxes." When compared, the nanH polypeptides of C . perfringens ATCC 10543 and Salmonella typhimurium LT12 shared 33% sequence identity and 60% similarity if conservative replacements were included . The homology-modeled structure of C . perfringens NanH showed the same folding topology as the x-ray three-dimensional structure of NanH in S . typhimurium LT12 . Amino acid residues Arg37, Arg56, Asp62, His63, Asp100, Glu230, Asp247, Tyr347, and Glu362 located around the pocket of modeled C . perfringens small nanH were superimposed with the active-site pocket of S . typhimurium LT12, nanH . The catalytic amino-acid residues as well as the role of the "Asp boxes" have not been characterized for C . perfringens and S . typhimurium . In this study, Asp100, Glu230, and Asp62 were found to be involved in the catalytic activity of C . perfringens small nanH with immunoreactive properties and site-directed mutagenesis analysis . Four "Asp-box" motifs were found remote from the active-site pocket . Mutational and immunoreactive analysis of the highly conserved amino acids located in the "Asp boxes" suggest that these highly conserved residues are important in maintaining the tertiary structure of NanH . The results of this study provide some knowledge for the design of new inhibitors of small neuraminidase.

Diagn Microbiol Infect Dis, 1996 Sep, 26(1), 47 - 51
Evaluation of two commercial microtiter cytotoxin assays for the detection of Clostridium difficile toxin B in stool specimens; Fedorko DP et al.; Two commercial microtiter cytotoxin assays using a fibroblast cell line (Bartels, Baxter Diagnostics, Inc., Deerfield, IL) and an epithelial cell line (Cytotoxi Test, Advanced Clinical Diagnostics, Toledo, OH) were evaluated for their ability to detect Clostridium difficile toxin B in stool specimens . After 48 hours, the assays had comparable sensitivity (90 versus 92%) and specificity (99 versus 98%) . Although not statistically significant, the Bartels assay detected more toxin-positive specimens at 24 hours (84 versus 72%, P = 0.089, Fisher's Exact Test) and the Cytotoxi assay had fewer nonspecific reactions requiring repeat testing (2.2 versus 1.1%, P = 0.186, Fisher's Exact Test) . Both cytotoxin assays had comparable analytic performance.

Glycobiology, 1996 Sep, 6(6), 599 - 609
Molecular mimicry in the recognition of glycosphingolipids by Gal alpha 3 Gal beta 4 GlcNAc beta-binding Clostridium difficile toxin A, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor; Teneberg S et al.; Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13 . However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase . To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay . The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed . A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes . This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source . Two additional binding-active glycosphingolipids were found . One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation . The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis . A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands . Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.

Intensive Care Med, 1996 Sep, 22(9), 990 - 4
Clostridium difficile infection as a cause of severe sepsis; Lowenkron SE et al.; Although colitis is often seen in critically all patients who have received multiple broad-spectrum antibiotics, there are no reports describing severe sepsis as a result of Clostridium difficile infection . We describe three cases of severe sepsis with local intestinal Clostridium difficile infection as the only identifiable etiology . The mechanism of severe sepsis may be a derangement of the gastrointestinal barrier function . This could result in absorption of microbes or endotoxin or activation of inflammatory cascades in the submucosa of the intestine or liver.

Mol Microbiol, 1996 Sep, 21(6), 1219 - 25
Conversion of an extracellular cytolysin into a phagosome-specific lysin which supports the growth of an intracellular pathogen; Jones S et al.; Listeria monocytogenes is a facultative intracellular pathogen which secretes a pore-forming cytolysin, listeriolysin O (LLO), necessary for intracellular growth . Clostridium perfringens is an extracellular pathogen which secretes a related cytolysin, perfringolysin O (PFO) . When PFO is secreted by intracellular L . monocytogenes, it is toxic to the infected host cell . PFO-mediated toxicity renders the infected host cell permeable to gentamicin and leads to the death of the intracellular bacteria . In this study, we selected for L . monocytogenes mutants in which PFO supported the intracellular growth of L . monocytogenes . Six independent mutants were isolated, each containing a single amino acid change within the PFO protein . Three classes of PFO mutations were identified, all capable of mediating lysis of the vacuole but without a toxic effect upon the infected host cell . The first class had a severe defect in haemolytic activity . The second class had a change in the pH optimum of PFO . The third class had nearly wild-type levels of haemolytic activity, but had a decrease in protein half-life in the host-cell cytosol . Acquisition of single amino acid changes in PFO were sufficient to convert an extracellular cytolysin into a vacuole-specific lysin which mediated growth of L . monocytogenes in cultured cells.

Toxicon, 1996 Sep, 34(9), 975 - 85
Therapeutic botulinum type A toxin: factors affecting potency; Mclellan K et al.; The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurons . This property has resulted in the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms . At present, the only recognised assay to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the endpoint . Such bioassay is inherently variable and large interlaboratory variability has been reported, highlighting problems for standardisation of activity in the absence of any commonly used reference preparation . In the present study, we have confirmed that many different assay conditions can affect potency estimates of clinical formulations of type A botulinum toxin . Further, our studies indicate that different preparations, because of their unique formulation and stability, are differentially affected by some of these assay conditions and that these differences might well contribute to the differences observed in their clinical use.

Cell Transplant, 1996 Sep-Oct, 5(5), 543 - 51
Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas; Klock G et al.; Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus . The isolation of islets from pancreata requires the specific dissociation of the tissue . Commercial collagenases from Clostridium histolyticum are widely used for this purpose . Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot . This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants . Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities . Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease . These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas . To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C . histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite . Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease . The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.

Mol Biol Cell, 1996 Sep, 7(9), 1419 - 27
Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton; Tilly BC et al.; Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume . Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles . In addition, an increase in total cellular F-actin was observed . Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho . In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux . Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity . Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux . Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

J Hosp Infect, 1996 Sep, 34(1), 23 - 30
Financial burden of hospital-acquired Clostridium difficile infection; Wilcox MH et al.; Clostridium difficile infection has become endemic in many hospitals and yet few data on the associated costs of such cases are available . We prospectively followed 50 consecutive cases of C . difficile infection and 92 control patients, who were admitted to the same geriatric wards within 72 h of the cases . Cases and controls had similar age, sex and major diagnosis distributions . Cases stayed significantly longer (mean 21.3 days, median 20.5 days; P < 0.001) in hospital than controls, including an average 14 days in a side room . Diarrhoea developed in cases on average 10.8 days after admission, which, when compared with a mean duration of stay for controls of 25.2 days, implies that C . difficile infection caused an increased duration of stay, as opposed to infection occurring because of longer residence . There was a significantly higher death rate in cases compared with controls (P < 0.01) . Antibiotic treatment of C . difficile infection cost an average of Pounds 47 per case . The average number of laboratory investigations per day was similar for cases and controls, but the increased length of stay meant an extra cost for tests of approximately Pounds 210 per case . Assuming hotel costs of Pounds 150 (Pounds 200) per day stay (in a side room), 94% of the additional costs associated with C . difficile infection were due to increased duration of stay (Pounds 3850) . The total identifiable increased cost of C . difficile infection was, therefore, in excess of Pounds 4000 per case . Such high costs can be used to justify expenditure on personnel and/or other control measures to reduce the incidence of this hospital-acquired infection.

Int J Food Microbiol, 1996 Sep, 32(1-2), 225 - 33
Mathematical model for the combined effect of temperature and pH on the thermal resistance of Bacillus stearothermophilus and Clostridium sporogenes spores; Fernandez PS et al.; Two mathematical models have been studied to establish the relationship between the pH, treatment temperature and thermal destruction constant (k) of Bacillus stearothermophilus and Clostridium sporogenes spores . The study was carried out by heating the spores in mushroom extract acidified with two different acidulants (citric acid and glucono-delta-lactone) . Among the models studied, the one that best described the inactivation was a second order polynomial equation, the precision of which depended on the microorganism studied.

Int J Food Microbiol, 1996 Sep, 32(1-2), 145 - 58
Chemiluminescent enzyme immunoassay for detection of PCR-amplified enterotoxin A from Clostridium perfringens; Baez LA et al.; A PCR protocol was developed for the rapid and specific detection of Clostridium perfringens strains harboring the enterotoxin A gene in artificially contaminated ground beef . A biotinylated primer pair was designed for amplification of a 750 bp fragment of the C . perfringens enterotoxin A gene . A combination of 4 h enrichment incubation and nucleic acid extraction, followed by 2 h of PCR amplification allowed detection at levels below 10 CFU of freshly grown cells in raw and cooked beef samples . PCR amplified products were confirmed by a Southern hybridization assay using a digoxigenin-labeled internal probe, and two hybridization ELISA protocols (PCR-ELISA) applying a streptavidin capture step for the hybridized PCR products . Both enzyme immunoassays utilized chemiluminescent detection with Lumiphos 530TM as substrate, after hybridization to an internal digoxigenin-labeled probe or a 5' conjugated alkaline phosphatase-labeled probe . The PCR-ELISA resulted in faster confirmation of the PCR products while providing a level of sensitivity comparable to Southern hybridization, and has potential for development into an automated method.

Int J Food Microbiol, 1996 Sep, 32(1-2), 115 - 23
Growth of Clostridium perfringens from spore inocula in sous-vide turkey products; Juneja VK et al.; Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag ground turkey (pH 6) that included 0.3% (w/w) sodium pyrophosphate, and sodium chloride at 0, 1, 2, or 3% (w/w) . The packages were processed to an internal temperature of 71.1 degrees C, ice chilled and stored at various temperatures . The total C . perfringens population was determined by plating diluted samples on tryptose-sulfite-cycloserine agar followed by anaerobic incubation at 37 degrees C for 48 h . At 28 degrees C, the addition of 3% salt in turkey was effective in delaying growth for 12 h . At 15 degrees C, growth occurred at a relatively slow rate in the presence of 1-2% salt . Vegetative cells were not observed even after 28 days of storage in the presence of 3% salt . C . perfringens growth was not observed at 4 degrees C regardless of salt levels . The D-values ranged from 23.2 min (no salt) to 17.7 min (3% salt) . Cyclic and static temperature abuse of refrigerated products for 8 h did not lead to growth by C . perfringens from a spore inoculum.

Clin Diagn Lab Immunol, 1996 Sep, 3(5), 605 - 7
Effect of Clostridium difficile toxin A on CD11/CD18 expression in vitro; Warny M et al.; Clostridium difficile toxin A is chemotactic for neutrophils and induces their emigration into the colonic mucosae of rodents . We found that toxin A did not upregulate neutrophil beta 2 integrins on isolated human neutrophils . These data support the hypothesis that in C . difficile colitis, these adhesion molecules are upregulated by endogenous mediators.

Lett Appl Microbiol, 1996 Sep, 23(3), 199 - 202
The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi; Kopecny J et al.; The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp . strain ChK5, which shows strong phenotypic similarity to Clostridium tertium . The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus . Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp . strain G-1 and Caecomyces sp . strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis . Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O . joyonii strain A4 . Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin . Mixtures of culture supernates from this bacterium and from O . joyonii strain A4 showed cellulase activity that was less than that of the component cultures . It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.

Lett Appl Microbiol, 1996 Sep, 23(3), 195 - 8
The isolation and characterization of a rumen chitinolytic bacterium; Kopecny J et al.; Chitinolytic bacteria were detected in faeces and digesta of wild and domesticated herbivores . The presence of chitinolytic bacteria in two cows was verified following enrichment culture of rumen fluid on colloidal chitin . In three other cows, direct counts on chitin agar showed that the numbers of these bacteria in the rumen fluid ranged from 5 x 10(4) to 2 x 10(8) ml-1 . Most of these bacteria were Clostridium-like spore producers . The most typical strain, Clostridium sp . ChK5, was characterized further . This bacterium degraded colloidal chitin and produced mainly acetate, butyrate and lactate . Endochitinase and chitobiase were produced when chitin was the growth substrate . Endochitinase was also detected in cultures grown on N-acetylglucosamine and glucose . Optimal conditions for endochitinase activity were 37 degrees C and pH 4.5-6.1 . The Michaelis constant (Km) for this enzyme was 19.3 mg ml-1 . Strain ChK5 shows strong phenotypic similarity to Clostridium tertium.

Microbiology, 1996 Sep, 142 ( Pt 9), 2561 - 6
An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter; Matsushita C et al.; The plc gene, which encodes phospholipase C (alpha-toxin) of Clostridium perfringens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter . The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNA-containing sequence, depending on its proper linear and rotational orientation . The in vitro transcriptional activity of the plc gene was also stimulated by the upstream sequence . In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively . A similar temperature effect was also observed with the chromosomal plc gene . These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.

J Neuropathol Exp Neurol, 1996 Sep, 55(9), 999 - 1008
Ependymal denudation, aqueductal obliteration and hydrocephalus after a single injection of neuraminidase into the lateral ventricle of adult rats; Grondona JM et al.; To investigate the role of sialic acid in the ependyma of the rat brain, we injected neuraminidase from Clostridium perfingens into the lateral ventricle of 86 adult rats that were sacrificed at various time intervals . After administration of 10 micrograms neuraminidase, ciliated cuboidal ependymal cells of the lateral ventricles, third ventricle, cerebral aqueduct, and the rostral half of the fourth ventricle died and detached . The ependymal regions sealed by tight junctions such as the choroid plexus and the subcommissural organ were not affected . Debris was removed by infiltrating neutrophils and macrophagic cells . At the same time, after ependymal disappearance, the aqueduct was obliterated . In this region, mitoses were evident and cystic ependymal cells were frequent . Hydrocephalus of the lateral and third ventricles was evident 4 days after neuraminidase injection . Gliosis was restricted to the dorsal telencephalic wall of the injected lateral ventricle . It is thought that cleavage of sialic acid from ependymal surface glycoproteins or glycolipids, likely involved in cell adhesion, led to the detaching and death of the ependymal cells . Thereafter, ependymal loss, together with edema, led to fusion of the lateral walls of the cerebral aqueduct and this in turn provoked hydrocephalus of the third and lateral ventricles . This model of experimental hydrocephalus is compared with other models, in particular those of hydrocephalus after viral invasion of the cerebral ventricles.

Infect Immun, 1996 Sep, 64(9), 3930 - 3
Phospholipid metabolism induced by Clostridium perfringens alpha-toxin elicits a hot-cold type of hemolysis in rabbit erythrocytes; Ochi S et al.; GTP and AIF4- significantly stimulated the late phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin in rabbit erythrocyte lysates . Pertussis toxin blocked the PA production . AIF4- markedly enhanced phosphatidylethanol production induced by alpha-toxin in the presence of ethanol . GTP{gamma S} stimulated the PA formation and hemolysis induced by alpha-toxin, and GDP{beta S} inhibited them . An H-to-G mutation at position 126 (H126G) induced the PA formation and hemolysis in a Co2+ concentration-dependent manner . H148G induced neither the PA formation nor hemolysis . These results suggest that the toxin-induced hemolysis is due to activation of phospholipid metabolism systems through GTP-binding protein.

Arch Microbiol, 1996 Sep, 166(3), 176 - 83
Ruminococcus hydrogenotrophicus sp . nov., a new H2/CO2-utilizing acetogenic bacterium isolated from human feces; Bernalier A et al.; A new H2/CO2-utilizing acetogenic bacterium was isolated from the feces of a non-methane-excreting human subject . The two strains S5a33 and S5a36 were strictly anaerobic, gram-positive, non-sporulating coccobacilli . The isolates grew autotrophically by metabolizing H2/CO2 to form acetate as sole metabolite and were also able to grow heterotrophically on a variety of organic compounds . The major end product of glucose and fructose fermentation was acetate; the strains also formed ethanol, lactate and, to a lesser extent, isobutyrate and isovalerate . The G+C content of DNA of strain S5a33 was 45.2 mol% . 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa . Based on phenotypic and phylogenetic considerations, a new species, Ruminococcus hydrogenotrophicus, is proposed . The type strain of R . hydrogenotrophicus is S5a33 (DSM 10507) . Furthermore, H2/CO2 acetogenesis appeared to be a common property of most of the species phylogenetically closely related to strain S5a33 (Clostridium coccoides, Ruminococcus hansenii, and Ruminococcus productus).

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8836 - 40
Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein; Dauter Z et al.; The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles . The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A . The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins . Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum . The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar . The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A) . An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed . Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain . Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site . The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein . The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

Biochim Biophys Acta, 1996 Aug 15, 1296(1), 112 - 20
Site-directed mutagenesis of putative catalytic and nucleotide binding sites in N10-formyltetrahydrofolate synthetase; Kounga K et al.; To determine the importance of specific amino-acid residues in catalysis and substrate binding by N10-formylH4 folate synthetase, one lysine and three histidine residues in the enzyme from Clostridium cylindrosporum were mutated to glutamine and serine residues, respectively . These residues, Lys-71, His-125, His-131, and His-268, are conserved in four bacterial and five eukaryotic proteins for which the amino-acid sequences are known . Previous evidence indicated that a histidine residue may play a role in catalysis and it has been proposed that Lys-71 could be a member of a putative nucleotide binding consenus sequence . The histidine mutations, H125S, H131S, and H268S, produced proteins that were unstable and were proteolytically degraded to different extents . No activity of purified H268S could be detected and the 240 kDa native tetramer was also absent . Activities of the H125S and H131S mutants could be measured and the Km values of the substrates were similar to those for the wild-type enzyme . It is concluded that the mutations resulted in monomers that do not fold properly and/or do not associate to the active tetramer and, as a consequence, are susceptible to intracellular proteolytic digestion . On the other hand, the K71Q mutation did not produce proteolyzed material . The resulting protein had a kcat value which was reduced by a factor of 3.3 x 10(-4) . Km values of the substrates were not affected, nor were the affinty constants for MgATP and H4PteG3 . CD and fluorescence spectra demonstrated that little change in the tertiary structure of the protein had occurred as a result of the mutation . The monomer form of K71Q was less stable than the monomer of the wild-type enzyme and reassociated less efficiently than the wild-type . From these results it is suggested that Lys-71 plays a critical role in catalysis by N10-formylH4 folate synthetase and that this residue may reside at an intersubunit interface.

J Biol Chem, 1996 Aug 9, 271(32), 19219 - 24
Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora; Chou MY et al.; Sialidase L is a NeuAcalpha2-->3Gal linkage-specific sialidase that releases 2,7-anhydro-NeuAc instead of NeuAc from sialoglycoconjugates (Chou, M.-Y., Li, S.-C., Kiso, M., Hasegawa, A., and Li, Y.-T.(1994) J . Biol . Chem . 269, 18821-18826) . A 2 . 5-kilobase cDNA of sialidase L was cloned by a combination of methods based on polymerase chain reactions . The composite cDNA sequence reveals an open reading frame coding for 762 amino acids, including a putative 28-residue signal peptide at the N terminus that is similar to the signal sequence of the Clostridium septicum sialidase . The result suggests that sialidase L is a secretory enzyme . The coding sequence excluding the putative signal peptide of sialidase L was overexpressed in Escherichia coli . The purified recombinant enzyme was characterized to be as active as the enzyme isolated from the leech . It also possessed the strict NeuAcalpha2-->3Gal linkage specificity and released the unique cleavage product, 2,7-anhydro-NeuAc from sialoglycoconjugates . The deduced amino acid sequence of sialidase L exhibits little similarity with other reported sialidases . However, sialidase L contains a conserved "FRIP region" and four repeating "Asp box" motifs that align well with the corresponding positions of bacterial sialidases . The predicted beta-strand structures near the conserved motifs of sialidase L are similar to those of Salmonella typhimurium sialidase . Several conserved single amino acid residues of bacterial sialidases, including those known to be involved in the active site of Salmonella enzyme, are conserved in the deduced amino acid sequence of sialidase L . This observation suggests that part of the catalytic mechanism of sialidase L may be similar to the ordinary sialidase.

J Biol Chem, 1996 Aug 2, 271(31), 18743 - 8
EPR and Mössbauer spectroscopic studies on enoate reductase; Caldeira J et al.; Enoate reductase (EC 1.3.1.31) is a protein isolated from Clostridium tyrobutyricum that contains iron, labile sulfide, FAD, and FMN . The enzyme reduces the alpha,beta carbon-carbon double bond of nonactivated 2-enoates and in a reversible way that of 2-enals at the expense of NADH or reduced methyl viologen . UV-visible and EPR potentiometric titrations detect a semiquinone species in redox intermediate states characterized by an isotropic EPR signal at g = 2.0 without contribution at 580 nm . EPR redox titration shows two widely spread mid-point redox potentials (-190 and -350 mV at pH 7 . 0), and a nearly stoichiometric amount of this species is detected . The data suggest the semiquinone radical has an anionic nature . In the reduced form, the {Fe-S} moiety is characterized by a single rhombic EPR spectrum, observed in a wide range of temperatures (4 . 2-60 K) with g values at 2.013, 1.943, and 1.860 (-180 mV at pH 7.0) . The gmax value is low when compared with what has been reported for other iron-sulfur clusters . Mossbauer studies reveal the presence of a {4Fe-4S}+2/+1 center . One of the subcomponents of the spectrum shows an unusually large value of quadrupole splitting (ferrous character) in both the oxidized and reduced states . Substrate binding to the reduced enzyme induces subtle changes in the spectroscopic Mossbauer parameters . The Mossbauer data together with known kinetic information suggest the involvement of this iron-sulfur center in the enzyme mechanism.

Gastroenterol Hepatol, 1996 Aug-Sep, 19(7), 363 - 5
{Severe massive pseudomembranous colitis with a fulminant course}; Sebastian JJ et al.; Pseudomembranous colitis is an inflammatory disease of rectal and colonic mucosa caused by Clostridium difficile produced toxin . The inflammation is produced as the consequence of a non-specific response to several agents . It usually presents with abdominal pain and mild watery diarrhea which used to decrease when removing the antibiotic or when starting the therapy with metronidazole or vancomycin . In aged patients, with severe concomitant diseases, may appear complications such as dehydration, electrolyte imbalance, hypotension and toxic megacolon, which occasionally may lead to fatal outcome . We report the case of a severe pseudomembranous colitis, with a fulminant clinical course, in a patient without a recent antibiotic therapy.

Eur J Clin Microbiol Infect Dis, 1996 Aug, 15(8), 625 - 34
Imipenem or cefoperazone-sulbactam combined with vancomycin for therapy of presumed or proven infection in neutropenic cancer patients; Bodey G et al.; The purpose of this prospective randomized study was to compare the efficacy and safety of imipenem and cefoperazone-sulbactam combined with vancomycin for the treatment of fever in neutropenic cancer patients . Patients were assigned to either imipenem 500 mg/m2 (500 mg for bone marrow transplant recipients) every 6 h or cefoperazone (2 g)-sulbactam (1 g) every 8 h All patients received vancomycin 1 g every 12 h . A total of 457 febrile or infectious episodes occurring in 407 patients were entered in the study . The response rate was 73% for imipenem plus vancomycin and 74% for cefoperazone-sulbactam plus vancomycin among the 369 episodes that could be evaluated . Response rates were comparable for the two regimens with regard to infecting organism, administration of antimicrobial prophylaxis, and neutrophil count and trend . The frequency of side-effects was significantly higher for imipenem plus vancomycin (11% vs.5%, p = 0.02), due to therapy-associated nausea and vomiting (5.3% vs . 0%, p = 0.0004) . The overall frequency of superinfections was similar with both regimens, but Clostridium difficile colitis occurred significantly more often in patients receiving imipenem plus vancomycin (5 vs . 0, p = 0.02) . In this study cefoperazone-sulbactam plus vancomycin was an effective alternative to imipenem plus vancomycin for initial therapy of fever in neutropenic patients.

Eur J Epidemiol, 1996 Aug, 12(4), 391 - 4
Clostridium difficile acquisition rate and its role in nosocomial diarrhoea at a university hospital in Turkey; Soyletir G et al.; Infection with Clostridium difficile can present with various clinical pictures ranging from an asymptomatic carrier state to pseudomembranous colitis and plays an important part in the etiology of nosocomial diarrhoea . To identify risk factors for C . difficile colonization and diarrhoea in hospitalized subjects, patients admitted to a general medicine ward at Marmara University hospital during a one year period were entered into the study . Of the 202 patients, nosocomial diarrhoea developed in 45 (22.3%) . Fourteen patients (6.9%) were colonized with C . difficile during their hospitalization period . Ten of the colonized patients (71.4%) developed diarrhoea and were found to be positive by toxin assay . Pseudomembranous colitis was confirmed endoscopically in 3 of the patients with diarrhoea . Administration of beta lactam agents such as ampicillin and cephalosporins; gastrointestinal manipulations and admission to the intensive care unit were found as major risk factors for C . difficile colonization.

J Dairy Sci, 1996 Aug, 79(8), 1467 - 75
How many ruminal bacteria are there?
Krause DO, Russell JB.
With the development of strictly anaerobic techniques and habitat-simulating media, a variety of bacteria were isolated from the rumen in the 1940s and 1950s . Based on standard morphological and physiological characteristics, the microbial ecosystem of the rumen contains a very complex population of bacteria . In recent years, ruminal bacteria have been re-evaluated with newer, more objective, and genetically valid methods of classification . Ribosomes are complicated structures, and their DNA-encoding sequences are relatively free from selective pressure . Because ribosomes have evolved slowly, they provide a long-term natural history of evolution . The invariable and hypervariable regions of rRNA genes can be used to group bacteria into kingdoms, genera, and species . The 16S rRNA sequences have provided a basis for renaming some ruminal species (Bacteroides amylophilus is now Ruminobacter amylophilus and Bacteroides succinogenes is now Fibrobacter succinogenes) and for classifying at least one recently isolated ruminal bacterium (e.g., Clostridium aminophilum) . The DNA:DNA hybridization is a more sensitive method of assessing bacterial relatedness than is 16S rRNA . Bacterial strains within a species should have a high degree of DNA:DNA homology, but some species of ruminal bacteria (e.g., Prevotella ruminicola and Butyrivibrio fibrisolvens) had highly unrelated strains . Studies of 16S rRNA and DNA:DNA hybridization indicate that the diversity of ruminal bacteria has been greatly underestimated . Traditional studies of phylogeny of ruminal bacteria were stymied by the fastidious growth requirements of many ruminal bacteria, and enumeration was tedious and inaccurate . Modern methods of bacterial classification do not require in vitro culture and have the potential of detecting even a single cell.

Int J Food Microbiol, 1996 Aug, 31(1-3), 357 - 65
Detection of Clostridium botulinum in fish and environmental samples using polymerase chain reaction; Hielm S et al.; A test protocol for the detection and enumeration of Clostridium botulinum in fish and sediment samples with specific identification of neurotoxin types A, B, E and F was developed . Specific amplification products generated by polymerase chain reaction (PCR) formed the basis of identification of the toxin-producing organism, whereas quantification of the results was achieved with the most probable number (MPN) method . Twenty-six C . botulinum strains studied with PCR assays after enrichment in trypticase-peptone-glucose-yeast extract (TPGY) broth gave identical results as with the mouse bioassay . The suitability of the detection method for food and environmental surveys was assessed by running it on 32 samples of rainbow trout inoculated with spore loads ranging from 10(2) to 10(6) C . botulinum type E spores per kg . The organism was detected in all samples, and MPN estimates corresponded well to inoculum levels . In order to assess possible natural contamination, 16 fish and 16 visceral samples of rainbow trout, as well as ten aquatic sediment samples were tested . Of these, eight (80%) of the sediment samples were positive, with estimated spore counts of C . botulinum type E ranging from 95-2710 per kg sample.

Int J Food Microbiol, 1996 Aug, 31(1-3), 69 - 85
Predictive model of the effect of temperature, pH and sodium chloride on growth from spores of non-proteolytic Clostridium botulinum; Graham AF et al.; Non-proteolytic strains of Clostridium botulinum are capable of growth at chill temperatures and thus pose a potential hazard in minimally-processed chilled foods . The combined effect of pH (5.0-7.3), NaCl concentration (0.1-5.0%) and temperature (4-30 degrees C) on growth of non-proteolytic C . botulinum in laboratory media was studied . Growth curves at various combinations of pH, NaCl concentration and temperature were fitted by the Gompertz and Baranyi models, and parameters derived from the curve-fit were modelled . Predictions of growth from the models were compared with data in the literature and this showed them to be suitable for use with fish, meat and poultry products . This model should contribute to ensuring the safety of minimally-processed foods with respect to non-proteolytic C . botulinum.

Biochem Mol Biol Int, 1996 Aug, 39(6), 1141 - 6
Organization and nucleotide sequence of genes for hemagglutinin components of Clostridium botulinum type B progenitor toxin; Yang GH et al.; The genes for hemagglutinin components (33 kD, 17 kD, and 21.5 kD) of Clostridium botulinum type B progenitor toxin were cloned and sequenced . Analysis of the nucleotide sequence showed that the 33 kD, 17 kD, and 21.5 kD hemagglutinin genes were organized into an operon in the 5'upstream region of the toxin gene and their ORF orientation were opposite to that of the toxin gene . A comparison of amino acid sequences between the hemagglutinin components in type B and type C progenitor toxin showed significant homology . Northern blot analysis also revealed that all of the genes for the hemagglutinin components were transcribed as a polycistronic RNA.

Nebr Med J, 1996 Aug, 81(8), 279 - 80; discussion 281
Clostridium perfringens septicemia . A case report; van Geem T; A rare case of cervical pregnancy complicated by Clostridium perfringens septicemia is presented . The relationship of sepsis in association with cervical pregnancy is reviewed.

Eur J Biochem, 1996 Aug 1, 239(3), 686 - 91
Purification and partial characterisation of a reversible artificial mediator accepting NADH oxidoreductase from Clostridium thermoaceticum; Bayer M et al.; An NAD(H)-dependent artificial mediator accepting pyridine nucleotide oxidoreductase present in Clostridium thermoaceticum has been purified 50-fold by three chromatographic steps to apparent electrophoretical homogeneity with a yield of 25% . By PAGE and gel filtration the molecular mass of the native enzyme was estimated to be 200 kDa and 210 kDa, respectively . By SDS/gel electrophoresis, a single band was found at 17000 Da, suggesting a homododecamer . Reducing carbamoylmethylviologen or hexacyanoferrate(III) with NADH, the enzyme was most active at pH 10 and the specific activities were 100 mumol min-1 mg-1 protein and 800 mumol min-1 mg-1 protein, respectively . The K(m) values for hexacyanoferrate(III), carbamoylmethylviologen and NADH at pH 8.5 were determined to be 0.40, 0.55 and 1.1 mM, respectively . Other electron acceptors for the dehydrogenation of NADH were 2,6-dichlorophenolindophenol, anthraquinone-2,6-disulphonate, ubiquinone 0 and FAD . In the reduction of NAD+ with reduced methyl viologen (MV+), the specific activity was about 225 mumol min-1 mg-1 protein at the pH maximum of 5.0 . The K(m) values for reduced methylviologen, NADH and NAD+ were 1.0, 1.1 and 0.25 mM, respectively . The enzyme had 10.6 atoms iron and 12.7 atoms sulphur per dodecamer . A significant content of flavin or molybdopterin cofactor could not be detected . The first 45 amino acids of the oxidoreductase show a surprisingly high degree of identity or similarity with the ribosomal L12 protein of various eubacteria, the acyl carrier proteins of microorganisms, but also with bovine heart mitochondria and a 3-phosphoglycerate dehydrogenase as well as a gyceraldehyde-3-phosphate dehydrogenase from bacteria and pea chloroplasts, respectively.

J R Coll Surg Edinb, 1996 Aug, 41(4), 235 - 8
The management of chronic fissure in-ano with botulinum toxin; Mason PF et al.; Five patients with a chronic fissure in-ano each received an injection of Clostridium botulinum type A toxin into the lower internal anal sphincter . A mean lowering of maximum resting anal pressure by 23.3 (SEM 5.6) cm H2O was achieved within seven days . Maximum voluntary squeeze pressures were not significantly altered . Anal compliance increased in all cases . Healing of the fissure with an apparent reduction in anal sensation occurred in three of the patients and partial resolution of symptoms in the other two . No adverse effects resulted from injections of the toxin . A controlled trial to compare the relative efficacies of botulinum toxin and lateral sphincterotomy is required.

Epidemiol Infect, 1996 Aug, 117(1), 203 - 11
Role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in Spain; Munoz M et al.; Faeces samples from diarrhoeic and non-diarrhoeic lambs and goat kids aged 1-45 days were examined for enteric pathogens . Cryptosporidium parvum was detected in both diarrhoeic lambs (45%) and goat kids (42%) but not in non-diarrhoeic animals . F5+ (K99+) and/or F41+ Escherichia coli strains were isolated from 26% and 22% of the diarrhoeic lambs and goat kids, respectively, although these strains, which did not produce enterotoxins ST I or LT I, were found with similar frequencies in non-diarrhoeic animals . A F5-F41-ST I+ E . coli strain was isolated from a diarrhoeic lamb (0.6%) . Verotoxigenic E . coli was isolated from both diarrhoeic and non-diarrhoeic lambs (4.1% and 8.2%, respectively) and there was no association between infection and diarrhoea . The prevalence of group A rotavirus infection in diarrhoeic lambs was very low (2.1%) . Groups A and B rotaviruses were detected in three (8.1%) and five (13.5%) diarrhoeic goat kids from two single outbreaks . Group C rotaviruses were detected in four non-diarrhoeic goat kids . An association of diarrhoea and infection was demonstrated only for group B rotavirus . Clostridium perfringens was isolated from 10.8% of the diarrhoeic goat kids but not from non-diarrhoeic goat kids or lambs . Salmonella arizonae was isolated from a diarrhoeic goat kid (2.7%) and the clinical characteristics of the outbreaks where these two latter enteropathogens were found different from the rest . Picobirnaviruses were detected in a diarrhoeic lamb . No coronaviruses were detected using a bovine coronavirus ELISA . No evidence was found of synergistic effect between the agents studied . Enteric pathogens were not found in four (8.7%) and three (20%) outbreaks of diarrhoea in lambs and goat kids, respectively.

Microbiology, 1996 Aug, 142 ( Pt 8), 2087 - 95
Clostridium paradoxum DSM 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences; Rainey FA et al.; Sequence analysis of the cloned 16S rRNA genes of Clostridium paradoxum DSM 7308T revealed the presence of 15 different sequences in variable region I (Escherichia coli position 73-97) of the 16S rRNA . The majority of the cloned genes contained intervening sequences (IVSs), which varied in length from 120-131 nt, and were present in the DNA obtained from single colonies of C . paradoxum . The absence of IVSs in the mature rRNA was demonstrated by Northern hybridization and sequence analysis of the 16S rRNA reverse transcriptase (RT)-PCR product . This finding was supported by the failure of oligonucleotide probes specific for certain IVSs to hybridize to the RT-PCR product obtained from C . paradoxum . Alterations in culture conditions (temperature, pH, salt) or culture age did not lead to expression of RNA containing IVSs, as indicated by the size of RT-PCR products . Hybridization of the restriction-enzyme-digested genomic DNA of C . paradoxum with probes derived from the IVSs demonstrated that the 16S rRNA genes containing different IVSs are located at different sites on the chromosome.

Microbiology, 1996 Aug, 142 ( Pt 8), 2079 - 86
Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824; Green EM et al.; Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824 . Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome . Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk . Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production . Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

J Bacteriol, 1996 Aug, 178(16), 4854 - 60
Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli; Berry AM et al.; Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene . Only one stable pneumococcal neuraminidase gene (designated nanA) has been described . In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream) . nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames . NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide . There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum . NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa . The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.

Infect Immun, 1996 Aug, 64(8), 3301 - 9
Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C . perfringens; Czeczulin JR et al.; Clostridium perfringens enterotoxin (CPE), the virulence factor responsible for symptoms associated with C . perfringens type A food poisoning, is produced by enterotoxigenic C . perfringens type A isolates when these bacteria sporulate in the gastrointestinal tract . Less than 5% of the global C . perfringens population apparently carries the cpe gene . To assess the distribution of cpe-regulatory factors, we investigated whether the cpe gene of a C . perfringens food poisoning isolate can be expressed and properly regulated (i.e., expressed in a sporulation-associated manner) when transformed into naturally cpe-negative C . perfringens isolates . Sporulation-associated CPE expression was observed when low-copy-number plasmids carrying either a 5.7-kb DNA insert, containing the cpe open reading frame plus >1 kb each of upstream and downstream flanking sequences from C . perfringens food poisoning isolate NCTC 8239, or a 1.6-kb insert, containing only the cpe open reading frame of NCTC 8239, were electroporated into cpe-negative C . perfringens type A, B, and C isolates . Northern (RNA) blot analysis demonstrated that the sizes of the cpe message in the transformants and the naturally enterotoxigenic C . perfringens NCTC 8239 were similar and that this message was detectable only in sporulating cultures of the transformants or NCTC 8239 . These studies strongly suggest that many, if not all, cpe-negative C . perfringens isolates (including type B isolates, which are not known to naturally express CPE) produce a factor(s) involved in normal (i.e., sporulation-associated) transcriptional regulation of CPE expression by C . perfringens food poisoning isolates . These findings are consistent with this CPE-regulatory factor(s) also regulating the expression of other genes in C . perfringens.

Infect Immun, 1996 Aug, 64(8), 3252 - 8
Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes; Konig B et al.; Previously, we have shown that Pseudomonas aeruginosa lipase and phospholipase C (PLC), two extracellular lipolytic enzymes, interact with each other during 12-hydroxyeicosatetraenoic acid (HETE) generation from human platelets . In this regard . the addition of purified P . aeruginosa lipase to PLC-containing crude P . aeruginosa culture supernatants enhances the generation of the chemotactically active 12-HETE from human platelets . Therefore, we analyzed the interaction of purified P . aeruginosa lipase and purified hemolytic P . aeruginosa PLC with regard to inflammatory mediator release from human platelets, neutrophilic and basophilic granulocytes, and monocytes . Purified P . aeruginosa PLC, but not purified lipase by itself, induced 12-HETE generation from human platelets, the generation of leukotriene B4 (LTB4) and oxygen metabolites, enzyme release from human neutrophils, and histamine release from basophils but diminished interleukin-8 (IL-8) release from human monocytes in a dose-dependent manner . The addition of purified lipase enhanced PLC-induced 12-HETE and LTB4 generation, did not influence enzyme, histamine, or IL-8 release, but diminished the PLC-induced chemiluminescent response . Similar results were obtained when the hemolytic PLC from Clostridium perfringens was used instead of P . aeruginosa PLC . For further comparison, we used the well-defined calcium ionophore A23187 and phorbol-12-myristate-13-acetate (PMA) as stimuli . Lipase enhanced calcium ionophore-induced LTB4 generation and beta-glucuronidase release but reduced calcium ionophore-induced and PMA-induced chemiluminescence . In parallel, we analyzed the role of lipase in a crude P . aeruginosa culture supernatant containing PLC and lipase . Lipase activity in the P . aeruginosa culture supernatant was inhibited by treatment with the lipase-specific inhibitor hexadecylsulfonyl fluoride, leaving the activity of PLC unaffected . The capacity of "lipase-inactivated culture supernatant" to induce 12-HETE and LTB4 generation was diminished by 50 to 100% . Our results suggest that the simultaneous secretion of lipase and PLC by P . aeruginosa residing in an infected host may result in severe pathological effects which cannot be explained by the sole action of the individual virulence factor on inflammatory effector cells.

Infect Immun, 1996 Aug, 64(8), 3168 - 73
Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin; Anderson R et al.; Tetanus toxin is a potent neurotoxin synthesized by Clostridium tetani . Immunization with fragment C protein, the nontoxic C-terminal domain of tetanus toxin, will protect mice against lethal challenge with tetanus toxin . A synthetic gene encoding fragment C (tetC) had previously been shown to express high levels of fragment C in Saccharomyces cerevisiae . A plasmid, pcDNA3/tetC, which encodes the synthetic tetC gene expressed under the control of the human cytomegalovirus major intermediate-early promoter/enhancer region, was constructed . Expression of fragment C was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/tetC . The immune response induced by intramuscular immunization with pure pcDNA3/tetC DNA was evaluated in a murine model . Anti-fragment C serum immunoglobulin and proliferative responses in splenocytes were observed in BALB/c mice following two immunizations with pcDNA3/tetC . The major immunoglobulin G subclass that recognized fragment C was immunoglobulin G2a, and the stimulated splenocytes secreted high levels of gamma interferon . Immunity to tetanus is dependent on the presence of neutralizing serum antibodies against tetanus toxin . Sufficient anti-fragment C serum immunoglobulins were induced by DNA-mediated immunization to protect mice against lethal challenge with tetanus toxin.

J Bacteriol, 1996 Aug, 178(15), 4597 - 603
Effect of nitrate on the autotrophic metabolism of the acetogens Clostridium thermoautotrophicum and Clostridium thermoaceticum; Frostl JM et al.; Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H2-CO2-dependent growth . Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources . This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H2-CO2 did not replace CO . These results indicated that nitrate blocked the reduction of CO2 to the methyl and carbonyl levels . On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive . Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway . However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy . Collectively, these findings indicated that (i) C . thermoautotrophicum and C . thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.

Appl Environ Microbiol, 1996 Aug, 62(8), 3069 - 72
Growth of and toxin production by nonproteolytic Clostridium botulinum in cooked puréed vegetables at refrigeration temperatures; Carlin F et al.; Seven strains of nonproteolytic Clostridium botulinum (types B, E, and F) were each inoculated into a range of anaerobic cooked pureed vegetables . After incubation at 10 degrees C for 15 to 60 days, all seven strains formed toxin in mushrooms, five did so in broccoli, four did so in cauliflower, three did so in asparagus, and one did so in kale . Growth kinetics of nonproteolytic C . botulinum type B in cooked mushrooms, cauliflower, and potatoes were determined at 16, 10, 8, and 5 degrees C . Growth and toxin production occurred in cooked cauliflower and mushrooms at all temperatures and in potatoes at 16 and 8 degrees C . The C . botulinum neurotoxin was detected within 3 to 5 days at 16 degrees C, 11 to 13 days at 10 degrees C, 10 to 34 days at 8 degrees C, and 17 to 20 days at 5 degrees C.

Appl Environ Microbiol, 1996 Aug, 62(8), 2758 - 66
Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824; Boynton ZL et al.; The enzymes phosphotransacetylase (PTA) and acetate kinase (AK) catalyze the conversion of acetyl coenzyme A to acetate in the fermentation of Clostridium acetobutylicum . The acetate-producing step is an important element in the acidogenic fermentation stage and generates ATP for clostridial cell growth . The genes pta and ack, encoding PTA and AK, respectively, were cloned and sequenced . Enzyme activity assays were performed on cell extracts from Escherichia coli and C . acetobutylicum harboring the subclone, and both AK and PTA activities were shown to be elevated . DNA sequence analysis showed that the pta and ack genes are adjacent in the clostridial chromosome, with pta upstream . The pta gene encodes a protein of 333 amino acid residues with a calculated molecular mass of 36.2 kDa, and ack encodes a polypeptide of 401 residues with a molecular mass of 44.3 kDa . Primer extension analysis identified a single transcriptional start site located 70 bp upstream of the start codon for the pta gene, suggesting an operon arrangement for these tandem genes . The results from overexpression of ack and pta in C . acetobutylicum showed that the final ratios of acetate to other major products were higher and that there was a greater proportion of two- versus four-carbon-derived products.

J Clin Invest, 1996 Aug 1, 98(3), 641 - 9
Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A; Pothoulakis C et al.; The intestinal effects of Clostridium difficile toxin A are inidated by toxin binding to luminal enterocyte receptors . We reported previously that the rabbit ileal brush border (BB) receptor is a glycoprotein with an alpha-d-galactose containing trisaccharide in the toxin-binding domain (1991 . J . Clin . Invest . 88:119-125) . In this study we characterized the rabbit ileal BB receptor for this toxin . Purified toxin receptor peptides of 19 and 24 amino acids showed 100% homology with rabbit sucrase-isomaltase (SI) . Guinea pig receptor antiserum reacted in Western blots with rabbit SI and with the purified toxin receptor . Antireceptor IgG blocked in vitro binding of toxin A to rabbit ileal villus cell BB . Furthermore, anti-SI IgG inhibited toxin A-induced secretion (by 78.1%, P < 0.01), intestinal permeability (by 80.8%, P < 0.01), and histologic injury (P < 0.01) in rabbit ileal loops in vivo . Chinese hamster ovary cells transfected with SI cDNA showed increased intracellular calcium increase in response to native toxin (holotoxin) or to a recombinant 873-amino acid peptide representing the receptor binding domain of toxin A . These data suggest that toxin A binds specifically to carbohydrate domains on rabbit ileal SI, and that such binding is relevant to signal transduction mechanisms that mediate in vitro and in vivo toxicity.

Gastroenterology, 1996 Aug, 111(2), 433 - 8
A receptor decoy inhibits the enterotoxic effects of Clostridium difficile toxin A in rat ileum; Castagliuolo I et al.; BACKGROUND & AIMS: Clostridium difficile toxin A causes secretion and intestinal inflammation in rodents by binding to a specific trisaccharide Gal alpha 1-3Gal beta 1-4 GlcNAc on enterocyte receptors . The purpose of this study was to explore the ability of Synsorb 90 (Synsorb Biotech Inc., Calgary, Alberta, Canada), and inert support carrying this trisaccharide, to bind toxin A in vitro and to inhibit its enterotoxic effects in vivo . METHODS: Binding of {3H}toxin A to Synsorb 90, Synsorb 83 (beta-mannose attached), and Chromosorb P (inert support with no sugar attached) (Synsorb Biotech Inc.) was measured . The inhibitory effects of these compounds on toxin A-mediated fluid secretion, mannitol permeability, and histological damage were measured in ileal loops in vivo . RESULTS: Toxin A showed specific binding to Synsorb 90, bearing the specific trisaccharide that binds toxin A, but not to Synsorb 83 or to Chromosorb P . Pretreatment of rats with Synsorb 90 by gavage (200 mg/kg body wt), but no Synsorb 83 or Chromosorb P at the same doses, dramatically reduced toxin A-associated fluid secretion and permeability . CONCLUSIONS: An immobilized toxin A receptor sequesters toxin A in the intestinal lumen and inhibits its effects of ileal mucosa . These results suggest a potential use for this agent in treating patients with C . difficile colitis.

Gastroenterology, 1996 Aug, 111(2), 409 - 18
Nitric oxide inhibits rat intestinal secretion by Clostridium difficile toxin A but not Vibrio cholerae enterotoxin; Qiu B et al.; BACKGROUND & AIMS: Intestinal inflammation is associated with increased synthesis of nitric oxide, whereas inhibition of NO synthase (NOS) reduces experimental chronic intestinal inflammation . The aim of this study was to test the effects of NO blockers and donors on acute intestinal inflammation induced by Clostridium difficile toxin A in rat ileum . METHODS: Rats received NOS inhibitors or NO donors before measurement of toxin-mediated ileal secretion and permeability changes . Mucosal mast cell and neutrophil activity were measured by release of rat mast cell protease II and myeloperoxidase activity, respectively . RESULTS: NOS inhibitors augmented but an NO donor inhibited toxin A-mediated ileal secretion and permeability when given before but not after toxin administration . Neither an NOS inhibitor nor an NO donor had any effect on cholera toxin-mediated secretion . Mast cell degranulation and neutrophil infiltration occurred after injection of toxin A or an NOS inhibitor, whereas the NO donor blocked both toxin A effects . CONCLUSIONS: NOS inhibitors augmented and an NO donor blocked the intestinal effects of toxin A but not of cholera toxin . NO protects against toxin A by inhibition of intestinal mast cells and neutrophils, which are activated by toxin A, but not by cholera toxin.

Mol Gen Genet, 1996 Jul 26, 251(6), 720 - 6
Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne; Katayama S et al.; The intron-encoded endonuclease I-CeuI from Chlamydomonas eugametos was shown to cleave the circular chromosomes of all Clostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE) . This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA . Using this approach, the genes for three of the four typing toxins, beta, epsilon, and tau, in addition to the enterotoxin and lambda-toxin genes, were shown to be plasmid-borne . In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins, theta and mu, were missing.

Biochem Biophys Res Commun, 1996 Jul 25, 224(3), 843 - 8
Mosaic type of the nontoxic-nonhemaggulutinin component gene in Clostridium botulinum type A strain isolated from infant botulism in Japan; Kubota T et al.; The gene encoding the nontoxic-nonhemaggulutinin (NTNH) component was amplified by the PCR technique using two primer sets and the DNA template from Clostridium botulinum type A strain 7I03-H isolated from infant botulism in Japan . The nucleotide sequence revealed that the NTNH gene was composed of 1,193 amino acids with a molecular weight of 130868.08 . Furthermore, the N-terminal half side and C-terminal half side of the NTNH component were similar to the NTNH component of type C and type A, respectively . These results indicate that the NTNH component gene codes the mosaic NTNH component composed of type A and type C . The hemaggulutinin gene, aha, and ORF-22 gene, orf-22a, were undetectable in the region upstream of the NTNH component gene, ant . Therefore, orf-22a is not thought to play a key role in the expression of botulinum type A progenitor toxin gene.

Biochim Biophys Acta, 1996 Jul 18, 1295(2), 201 - 8
The influence of conserved aromatic residues on the electron transfer reactivity of 2{4Fe-4S} ferredoxins; Quinkal I et al.; The detailed mechanism used by {4Fe-4S} ferredoxins to exchange electrons is not known . The importance of two highly conserved aromatic residues, each located close to one cluster of 2{4Fe-4S} ferredoxins has been probed by site-directed mutagenesis of Clostridium pasteurianum ferredoxin . All generated variants are less stable than the native protein and only hydrophobic residues can replace one of the two conserved aromatic residues . With leucine substituting both aromatics, Clostridium pasteurianum ferredoxin cannot even be completely purified because of its deleterious instability . The reduction potentials of Clostridium pasteurianum ferredoxin variants do not depend on the presence of aromatic residues near the clusters . However, the ferredoxin from Entamoeba histolytica which is naturally devoid of aromatic residues displays a reduction potential nearly 60 mV less negative than that of Clostridium pasteurianum ferredoxin . The rate constants for the oxidation of the reduced ferredoxins by the inorganic complexes hexaamine-cobalt(III) chloride and sodium ethylenediaminetetra-acetatecobaltate(III) are similar . This implies that electron transfer from the clusters of these molecules is not mediated by the conserved aromatic residues . These residues rather appear to be involved in maintaining the overall stability of ferredoxins.

Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 591 - 6
Effect of Clostridium difficile toxin B on IgE receptor-mediated signal transduction in rat basophilic leukemia cells: inhibition of phospholipase D activation; Ojio K et al.; Antigen (Ag)-stimulated phospholipase D (PLD) activation and secretion were almost abolished by pretreatment of rat basophilic leukemia (RBL)-2H3 cells for 4 h with 5 ng/ml Clostridium difficile Toxin B which is known to inhibit Rho family proteins (Rho, Cdc42, Rac) . The concentration-dependent inhibition of PLD activation was well correlated with the level of glucosylation of Rho family proteins . In streptolysin O-permeabilized RBL cells, Toxin B suppressed {3H} phosphatidylbutanol (PBut) formation in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) by 67 and 43%, respectively . The synergistic PLD activation by GTP gamma S and PMA was also reduced by Toxin B by 67% . These results suggest that the IgE receptor-coupled PLD activation is largely mediated by Rho proteins.

Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 508 - 15
Involvement of rho p21 in cyclic strain-induced tyrosine phosphorylation of focal adhesion kinase (pp125FAK), morphological changes and migration of endothelial cells; Yano Y et al.; The molecular mechanisms by which endothelial cells sense and respond to physical forces remain to be elucidated . Recently we reported that cyclic strain-induced morphological change and migration of EC were regulated by the tyrosine phosphorylation of focal adhesion kinase (pp125FAK) and paxillin . The aim of the present study was to clarify the role of the small GTP-binding protein rho p21 in EC exposed to cyclic strain . Bovine aortic endothelial cells (EC) were subjected to 10% average strain at 60 cycle/min . Clostridium botulinum C3 transferase (C3) was used as a specific inhibitor of rho p21 . Preincubation of EC with C3 inhibited ADP-ribosylation of rho (94%) and inhibited the morphological change, reorganization of actin filaments, and migration induced by cyclic strain . Moreover, C3 inhibited the cyclic strain-induced tyrosine phosphorylation of pp125FAK and paxillin . These results demonstrate that rho downregulates the tyrosine phosphorylation of pp125FAK and paxillin and can modulate the morphological changes and migration induced by cyclic strain.

Arch Intern Med, 1996 Jul 8, 156(13), 1449 - 54
Prevalence and pathogenicity of Clostridium difficile in hospitalized patients . A French multicenter study; Barbut F et al.; BACKGROUND . Although Clostridium difficile is the main agent responsible for nosocomial diarrhea in adults, its prevalence in stool cultures sent to hospital microbiology laboratories is not clearly established . OBJECTIVES . To determine the prevalence of C difficile in inpatient stools sent to hospital microbiology laboratories and to assess the relationship between serotypes and toxigenicity of the strains isolated and the clinical data . METHODS . From January 18, 1993, to July 31, 1993, the presence of C difficile was systematically investigated in a case-control study on 3921 stool samples sent for stool culture to 11 French hospital microbiology laboratories . The prevalence of C difficile in this population (cases) was compared with that of a group of 229 random hospital controls matched for age, department, and length of stay (controls) . Stool culture from controls was requested by the laboratory although not prescribed by the clinical staff . Serotype and toxigenesis of the strains isolated were compared . RESULTS . The overall prevalence of C difficile in the cases was twice the prevalence in the controls (9.7% vs 4.8%; P < .001) and was approximately 4 times as high in diarrheal stools (ie, soft or liquid) as in normally formed stools from controls (11.5% vs 3.3%; P < .001) . The strains isolated from diarrheal stools were more frequently toxigenic than those isolated from normally formed stools . Serogroup D was never toxigenic, and its proportion was statistically greater in the controls than in the cases (45% vs 18%; chi 2 = 5.2; P < .05) . Conversely, serogroup C was isolated only from the cases . Clostridium difficile was mainly found in older patients ( > 65 years), suffering from a severe disabling disease, who had been treated with antibiotics and hospitalized for more than 1 week in long-stay wards or in intensive care . CONCLUSIONS . This multicenter period prevalence study clearly supports the hypothesis of a common role of C difficile in infectious diarrhea in hospitalized patients . Disease associated with C difficile should therefore be systematically evaluated in diarrheal stools from inpatients.

Biochemistry, 1996 Jul 2, 35(26), 8544 - 52
Determination of the nucleotide binding site within Clostridium symbiosum pyruvate phosphate dikinase by photoaffinity labeling, site-directed mutagenesis, and structural analysis; McGuire M et al.; Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP) . The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent {32P}-8-azidoadenosine 5'-triphosphate ({32P}-8-azidoATP) . Subtilisin cleavage of the {alpha-32P}-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334 . Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling . Next, small peptide fragments of {gamma-32P}- 8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or alpha-chymotrypsin . A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in alpha-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification . Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested . K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited . Comparison of the steady-state kinetic constants measured (at pH 6.8, 25 degrees C) for wild-type PPDK (kcat = 36 s-1, AMPK(m) = 7 microM, PP(i)K(m) = 70 microM, PEPK(m) = 27 microM) to those of R337L PPDK (kcat = 2 s-1, AMPK(m) = 85 microM, PP(i)K(m) = 3700 microM, PEPK(m) = 6 microM) and G254I PPDK (kcat = 0.1 s-1, AMPK(m) = 1300 microM, PP(i)K(m) = 1200 microM, PEPK(m) = 12 microM) indicated impaired catalysis of the nucleotide partial reaction (E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i) in these mutants . The single turnover reactions of {32P}PEP to {32P}E-P.pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK . In contrast, the formation of {32P}E-PP/{32P}E-P in single turnover reactions of {beta-32P}ATP/P(i) was significantly inhibited . Finally, the location of the adenosine 5'-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain {Herzberg, O . (1996) Proc . Natl . Acad . Sci . U.S.A . 93, 2652-2657} indicates, by analogy, the location of the nucleotide binding site in PPDK . Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.

Avian Dis, 1996 Jul-Sep, 40(3), 736 - 41
Excessive mortality in market-age turkeys associated with cellulitis; Carr D et al.; Between July, 1993, and October, 1994, seven cases were examined that consisted of increased mortality in commercial turkeys due to cellulitis . The condition started at 13-16 wk of age in toms and persisted until the birds were marketed . The mortality rate was 1-2% per week . Lesions began on the ventrum of the tail and consisted of swelling and the formation of vesiclelike structures . Most of the affected birds also had an accumulation of gelatinous fluid in the subcutis of the tail and breast areas . The underlying musculature was often darkened or petechiated . Clostridium perfringens type A was isolated from two of the cases . Lesions similar to those found in the field were reproduced experimentally in turkeys injected with the subcutaneous fluid obtained from birds in field cases.

Avian Dis, 1996 Jul-Sep, 40(3), 720 - 4
Hepatic and renal ultrastructural lesions in experimental Clostridium perfringens type A enterotoxemia in chickens; Vissiennon T et al.; Hepatic and renal electron microscopic investigations were carried out in 10 chickens that had experimental intraduodenal infection with Clostridium perfringens Type A . Fourteen days postinfection, there were ultrastructural changes in hepatocytes and renal tubular epithelial cells; these included mitochondrial lesions (swelling, cristolysis, rarefication of the matrix, myelin figures), glycogen loss, and capillary endothelial cell swelling in both organs, as well as thickening of glomerular basement membrane . The findings are discussed with particular reference to the pathogenesis of Clostridium perfringens Type A enterotoxemia.

Eur J Clin Microbiol Infect Dis, 1996 Jul, 15(7), 561 - 6
Comparison of four laboratory tests for diagnosis of Clostridium difficile-associated diarrhea; Jacobs J et al.; Four different laboratory tests for diagnosis of Clostridium difficile-associated diarrhea were compared to determine the optimal one for management of patients with hospital-acquired diarrhea . Stool samples from 231 patients with diarrhea were tested by the following methods: culture for Clostridium difficile with subsequent determination of exotoxin production, with a toxigenic Clostridium difficile positive (TCP) result considered truly positive; enzyme immunoassay (EIA); latex agglutination test; and an immunobinding blot assay . The rates of positive results were as follows: EIA 5.5%, TCP 7.3%, latex agglutination 16.7%, and immunobinding blot assay 26.1% . Compared to the TCP results, the sensitivity and specificity were, respectively, 61 and 98% for EIA, 47 and 85% for latex agglutination, and 60 and 76% for the immunobinding blot assay . Samples from patients with > or = 6 stools/day were TCP and EIA positive in 27 and 17% of cases, respectively, whereas in patients with < 6 stools/day, these percentages decreased to 2 and 3%, respectively (p < 0.001) . In hospitalized patients with > or = 6 stools/day, EIA appears to be the optimal test for diagnosis of Clostridium difficile-associated diarrhea, with a 73% positive predictive value and a 97% negative predictive value . However, in patients with < 6 stools/day, the prevalence of Clostridium difficile is low, and laboratory detection of this organism remains problematic.

Naunyn Schmiedebergs Arch Pharmacol, 1996 Jul, 354(2), 87 - 94
A role for Rho in receptor- and G protein-stimulated phospholipase C . Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B; Schmidt M et al.; Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated alpha of free beta gamma subunits of the relevant G proteins . To study whether low molecular weight G proteins of the Rho family are involved in receptor signaling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype . Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation . PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, A1F-4 and guanosine 5'-{gamma-thio}triphosphate (GTP gamma S), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment . C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLC activity . Finally both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate . Accordingly, When PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca(2+)- as well as GTP gamma S- or A1F-4-stimulated PLC activities were not altered by prior toxin B treatment . In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.

FEMS Immunol Med Microbiol, 1996 Jul, 14(4), 205 - 9
Inhibition of adhesion of Clostridium difficile to Caco-2 cells; Naaber P et al.; For many microorganisms, including Clostridium difficile, mucosal association is an important factor influencing intestinal colonisation and subsequent infection . Inhibition of adhesion of C . difficile to intestinal mucosa could be a new promising strategy for prevention and treatment of antibiotic-associated diarrhoea . We investigated the possibilities of influencing the adhesion of C . difficile by xylitol and bovine colostrum whey . Caco-2 cells and C . difficile cells were incubated with 1%, 5% and 10% solutions of xylitol and colostrum . Our study revealed that both xylitol and colostrum inhibited the adhesion of C . difficile to Caco-2 cells . Inhibition by xylitol was dose-dependent . When compared to the control, the count of adherent C . difficile decreased 3.4 times when treated with 1% xylitol, 12 times when 5% xylitol was applied, and 18.7 times when treated with 10% xylitol . The inhibition of adherence by colostrum was partially dose-dependent: 3.1 times in the case of 1%, and 5.5 times in the cases of 5% and 10% colostrum . Further experimental and clinical studies are needed for the application of xylitol and colostrum in the treatment and prophylaxis of pseudomembraneous colitis.

FEMS Immunol Med Microbiol, 1996 Jul, 14(4), 187 - 93
Enzyme electrophoresis combined with serogrouping for improved differentiation of Clostridium difficile strains; Bories PN et al.; We used enzyme electrophoresis to study a set of epidemiologically related and unrelated isolates of Clostridium difficile . The 53 strains belonged to the most frequent serogroups (A1, C, G, H and K) . Nine electrophoretic profiles were defined on the basis of five enzymes, and two were characteristic of a single strain . Each serogroup was resolved into two or three different enzyme patterns . By combining the two methods we were able to resolve the strains into 12 types . There was an excellent correlation between enzyme electrophoresis and serogrouping data . This method may be of use in investigating nosocomial transmission.

Int J Food Microbiol, 1996 Jul, 30(3), 189 - 215
Antibacterial activity of Lactobacillus plantarum UG1 isolated from dry sausage: characterization, production and bactericidal action of plantaricin UG1; Enan G et al.; Lactobacillus plantarum UG1 isolated from dry sausage produced an antimicrobial substance that inhibited other strains of the genera Lactobacillus and Lactococcus, and some foodborne pathogens including Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Clostridium sporogenes . This antibacterial substance was inactivated by proteolytic enzymes and showed a bactericidal mode of action . Consequently, it was characterized as a bacteriocin, and was designated plantaricin UG1 . This bacteriocin was stable in the pH range 4.5 to 7.0, partially inactivated by amylolytic enzymes and relatively thermostable . It was not affected by organic or lipolytic enzymes . Production of plantaricin UG1 was pH- and temperature-dependant and maximum yields were obtained in MRS broth cultures maintained at initial pH 6.5, and incubated at 25 degrees C to 30 degrees C, in the exponential to the early stationary growth phase of the producer organism . Ultrafiltration studies indicated that plantaricin UG1 has a molecular weight between 3 and 10 KDa . Curing experiments with L . plantarum UG1 resulted in the appearance of variants that lost bacteriocin production ability but were still immune to the bacteriocin . Plantaricin UG1 production appeared to be chromosomal encoded . Sensitive and insensitive Gram-positive bacteria adsorbed plantaricin UG1 irrespective of their susceptibility to it . In contrast, Gram-negative bacteria did not adsorb plantaricin UG1 . The bactericidal action of plantaricin UG1 did not depend on the physiological state of the indicator culture and did not cause cell lysis . The resistance of two indicator strains to plantaricin UG1 has been studied.

Pediatr Dermatol, 1996 Jul-Aug, 13(4), 281 - 4
Aerobic and anaerobic microbiology of necrotizing fasciitis in children; Brook I; Specimens obtained from eight children with necrotizing fasciitis (NF) were cultured for aerobic and anaerobic bacteria . A total of 21 isolates were recovered, 13 anaerobic and 8 aerobic or facultatives . The facultative organism Streptococcus pyogenes was present alone in two (25%) instances, and mixed aerobic and anaerobic bacteria were isolated in six (75%) . The predominant isolates were Peptostreptococcus spp . (6 isolates, including 3 Peptostreptococcus magnus) . S . pyogenes (4), Bacteroides fragilis group (3), Clostridium perfringens (2), Escherichia coli (2), and Prevotella spp . (2) . Organisms similar to the ones isolated from the NF aspirates were recovered in the blood of all patients except one . These included S . pyogenes (3 isolates) . B . fragilis group (2), E . coli (1), and P . magnus (1) and Clostridium perfringens (1) . All patients underwent surgical fasciotomy, and four required skin grafting . Antimicrobials were administered to all children . Despite extensive resection and intense supportive therapy, three patients died from sepsis accompanied by shock acidosis and disseminated intravascular coagulation . These findings illustrate the polymicrobial aerobic-anaerobic flora of NF in children.

Toxicon, 1996 Jul, 34(7), 737 - 42
Gene probe-based detection of type E botulinum neurotoxin binding protein using polymerase chain reaction; Singh BR et al.; Using primers based on the nucleotide sequence of a neurotoxin binding protein from Type E Clostridium botulinum cultures, an amplified DNA product was obtained through polymerase chain reaction . The 400 base pair amplified DNA fragment was detectable with as low as 0.1 pg template DNA from Type E C . botulinum, and its fidelity was confirmed by Southern blotting using a DNA probe designed to detect the expected amplified DNA fragment . On the other hand, no DNA amplification was observed with as high as 10 ng template DNA from related Types A and B C . botulinum or from C . tetani, indicating the specificity of the probe.

Clin Infect Dis, 1996 Jul, 23(1), 51 - 60
Bacteriologic findings in tonsillitis and pericoronitis; Rajasuo A et al.; Bacteriologic samples from 31 young men were cultured quantitatively for aerobes and anaerobes; these samples included 31 specimens of tonsils (16 infected and 15 healthy), 16 specimens from pericoronal pockets of lower third molars (11 infected and 5 symptom-free), and 6 postoperative specimens from lower-third-molar extraction sockets . Anaerobes were isolated more often from infected third molars than from infected tonsils (14.5 isolates vs . 8.4 isolates, respectively; P < .001) . Infected tonsil samples contained significantly more anaerobic species if an adjacent partly erupted lower third molar was present rather than absent (10.3 isolates vs . 6.9 isolates, respectively; P < .05) . Eubacterium aerofaciens, Clostridium species, Peptostreptococcus micros, and Prevotella oris were frequently isolated . Streptococcus salivarius was found more frequently in tonsillar specimens, whereas Corynebacterium species, Prevotella denticola, Capnocytophaga species, Peptostreptococcus anaerobius, and Lactobacillus species were more common in pericoronal pocket samples . Thus, partial eruption of lower third molars increases the number of anaerobic bacterial species on tonsils and many species can be isolated simultaneously from both tonsils and lower third molars.

Rev Clin Esp, 1996 Jul, 196(7), 424 - 30
{Diarrhea associated with Clostridium difficile: one-year experience in a general hospital}; Bouza E et al.; Clostridium difficile is considered the most common cause of nosocomial acquired diarrhoea, with frequencies differing widely from one institution to another . So far, it is a scarcely reported condition in Spain . In the present study 129 episodes of Clostridium difficile associated diarrhoea (CDAD) occurred in 120 patients in a 2,000-bed hospital in 1994 is reported . All cases were diagnosed by demonstrating cytotoxicity on cellular lines (MRC-5) from feces or from the strain isolated from a culture medium (CCFA) . The overall incidence was 2.4 episodes every 1,000 admissions . Twenty-eight out of the 120 patients (23%) were HIV-positive patients, that is, an incidence of 30 episodes every 1,000 admissions . No significant differences were observed regarding the presentation and clinical course between HIV-positive and HIV-negative patients, with the exception of the antimicrobial agents used previously . Forty-two percent of patients had undergone surgery and 97% had received antimicrobials in the 8 weeks before the CDAD episode, with an average of 3.3 antibiotics per patient . Out of the 129 episodes, 72.8% were treated correctly . A total of 11.7% of patients responded exclusively to the discontinuation of the antimicrobials that were being administered . Eighty-three patients were treated with specific antibiotics, 59 with oral vancomycin, and 24 with metronidazole . Seventy-six patients (91.5%) responded to the initial therapy, 5 relapsed (6%), and 2 (2.5%) failed . The associated mortality rate was 0.7% . C . difficile can be a relevant cause of nosocomial diarrhoea in our setting, particularly in HIV-positive patients, but also in other patients . Its early diagnosis and appropriate therapy can contribute to decrease a relevant cause of morbidity in inpatients.

Int J Syst Bacteriol, 1996 Jul, 46(3), 753 - 8
Clostridium proteoclasticum sp . nov., a novel proteolytic bacterium from the bovine rumen; Attwood GT et al.; A novel proteolytic bacterium was isolated from rumen contents of New Zealand cattle grazing fresh forage and was designated strain B316(T) (T = type strain) . Strain B316(T) cells were straight to slightly curved rods (width, 0.4 to 0.6 microns; length, 1.3 to 3.0 microns) that were gram-positive and possessed a single subterminal flagellum . This isolate did not produce catalase, indole, ammonia, lipase, or lecithinase, or reduce nitrate, but it did produce a curd reaction with milk . Strain B316(T) was proteolytic, hydrolyzing casein and fraction I leaf protein . The crude proteinase was predominantly the serine type, but some cysteine proteinase and metallo-proteinase activities were also detected . The DNA base composition of strain B316(T) was 28 mol% G+C . A 16S ribosomal DNA sequence analysis of strain B316(T) indicated that it was most closely related to a member of clostridial cluster XIVa, viz., Clostridium aminophilum, an amino acid-fermenting organism isolated from the rumen; the similarity value was 92.2% . The results of the phenotypic characterization analysis, G+C content analysis, and phylogenetic analysis of the 16S ribosomal DNA sequence set strain B316(T) apart from all of the members of cluster XIVa . We propose that strain B316(T) should be designated a new species of the genus Clostridium, Clostridium proteoclasticum . Strain B316 is the type strain and has been deposited in the American Type Culture Collection as strain ATCC 51982.

Appl Environ Microbiol, 1996 Jul, 62(7), 2664 - 8
Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature; Graham AF et al.; Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods . Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium . The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined . No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.

Can J Microbiol, 1996 Jul, 42(7), 628 - 33
Characterization and distribution of amylases during vegetative cell growth and sporulation of Clostridium perfringens; Shih NJ et al.; Clostridium perfringens produced eight extracellular and two intracellular amylolytic activities when examined by zymograms following polyacrylamide gel electrophoresis under native conditions . The major intracellular amylase was isolated from vegetative cells of C . perfringens . It possessed an estimated molecular mass of 112 kDa . Sulfhydryl and phenol functional groups were essential to its activity . The amylase was endo-acting on starch and also hydrolyzed pullulan . Polyclonal antisera against a purified extracellular amylase did not cross-react with intracellular amylase and the two amylases were biochemically different . The distribution of extracellular amylolytic activities of sporulating cells was different from that of vegetative cells, whereas the distribution of intracellular amylolytic activities remained identical . A significant increase of a particular amylase (A8) occurred in the extracellular fluid during sporulation compared with that during vegetative growth . Regulation of the excretion of amylase(s) may be sporulation and enterotoxingenicity related.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 281 - 6
A new H2/CO2-using acetogenic bacterium from the rumen: description of Ruminococcus schinkii sp . nov; Rieu-Lesme F et al.; Two strains of H2/CO2-using acetogenic bacteria were isolated from the rumen of suckling lambs . Both strains displayed a coccobacillar morphology and possessed a Gram-positive type cell wall . Numerous organic substrates, including some O-methylated aromatic compounds, were used heterotrophically . 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa . Based on phenotypic and phylogenetic considerations a new species, Ruminococcus schinkii sp . nov., is proposed.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 185 - 91
Factors involved in the transformation of previously non-transformable Clostridium perfringens type B; Chen CK et al.; The pre-shock incubation of cells plus DNA and the methylation state of plasmid DNA were found to play a role in the electroporation-based transformation of Clostridium perfringens 3626B . Following pre-shock incubation, the highest number of C . perfringens 3626B transformants was obtained when plasmid pGK201 was both dam+ dcm+ modified, while no transformants were obtained when pGK201 was not methylated or only dcm methylated . This is consistent with the observation that plasmid pGK201 was protected against digestion by C . perfringens 3626B cell-associated nucleases for up to 3 min when methylated by both methylases . C . perfringens 3626B was successfully transformed only within a narrow cell recovery rate window . The ermAM gene associated with pGK201 and pAK102 was found to integrate into the chromosome of C . perfringens strain 13A and 3626B.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 151 - 8
Genetic characterisation of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916; Henderson I et al.; An 8 kb segment of the Clostridium botulinum NCTC 2916 genome 5' to the type A botulinum neurotoxin gene has been sequenced revealing five open reading frames . Four encode components (HA70, HA17, HA34 and NTNH/A) of the progenitor toxin complex . The product of the fifth, OrfX, possesses a putative C-terminal helix-turn-helix motif, exhibits homology with known regulatory proteins (including MsmR from Streptococcus mutans, UviA from C . perfringens and Orftxe1 located upstream of the C . difficile toxin B gene) and is also found within the vicinity of genes encoding tetanus toxin and types B, C, D and G botulinum toxins . Primer extension and Northern blotting analysis demonstrates that the genes are expressed as two divergent operons {HA34, HA17, HA70} and {NTNH/A, type A toxin gene}, with the OrfX gene expressed singly . Immediately adjacent to the transcriptional start sites of the HA34 and NTNH/A genes are two highly conserved motifs (5'-ATTTTagGTTTACAAAA-3' and 5'-ATGTTATATgTaA-3'), separated by 12 bp, that span the putative -35 and -10 promoter regions . Homologous sequences occur in the equivalent position relative to the genes at type C botulinum toxin gene and the tetanus toxin gene loci . It is likely that these sequence motifs, together with OrfX, are involved in the co-ordinate expression of the genes encoding the various components of the botulinum toxin complex in groups I, III and IV C . botulinum strains and in that of the tetanus toxin gene.

J Bacteriol, 1996 Jul, 178(14), 4166 - 75
Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements; Gutierrez JA et al.; New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM) . In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S . mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7% . Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E . coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19 . Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E . coli dfp (43% identity), and B . subtilis ylxM-ffh (58% identity), icd (citC {69% identity}), and argD (61% identity) . Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements . This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S . mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization . This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.

J AOAC Int, 1996 Jul-Aug, 79(4), 861 - 5
Molecular detection of Clostridium botulinum type E neurotoxin gene in smoked fish by polymerase chain reaction and capillary electrophoresis; Sciacchitano CJ et al.; The polymerase chain reaction (PCR), a rapid, sensitive technique for amplifying target DNA sequences of pathogenic microorganisms, was used to amplify Clostridium botulinum type E neurotoxin gene fragments in smoked fish . Other botulinal neurotoxin-producing strains, nontoxigenic strains, and food-related microorganisms did not yield nonspecific amplification products with this PCR assay . PCR products were analyzed by capillary electrophoresis (CE) using a low-viscosity entangled polymer system . Resolution, sensitivity, and DNA sizing accuracy were improved, and analytical times were markedly shortened . The PCR/CE assay detected the C . botulinum type E neurotoxin gene in as few as 10 cells . The technique to other foods may also be a valuable tool for detecting foodborne pathogens.

J Cell Biol, 1996 Jul, 134(1), 205 - 15
Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth; Igarashi M et al.; The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur . Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear . We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth . Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite . The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth . The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1 . The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1 . Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.

Infect Immun, 1996 Jul, 64(7), 2440 - 4
Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens; Guillouard I et al.; The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available . This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced . Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities . By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.

Lett Appl Microbiol, 1996 Jul, 23(1), 13 - 7
Detection by polymerase chain reaction of Clostridium perfringens producing epsilon toxin in faeces and in gastrointestinal contents of goats; Uzal FA et al.; A polymerase chain reaction (PCR) was used to identify the gene-encoding epsilon toxin production in Clostridium perfringens types B and D in faeces and in gastrointestinal contents of goats . The samples were cultured in thioglycollate broth and centrifuged . The upper layer of the pellet was used as a template for PCR, obviating the need for DNA extraction . This technique specifically differentiated Cl . perfringens types B and D from Cl . perfringens types A and C and from Escherichia coli . When used to identify Cl . perfringens type D in samples artificially spiked with the micro-organism, the PCR detected as few as 1.4 x 10(2) cfu g-1 of sample . Gastrointestinal contents and faeces were collected from 20 goats at slaughter and processed by PCR . Several positive results were obtained from the first five goats that were slaughtered and sampled a few days after their arrival at the abattoir, but only a few samples gave positive results during the following weeks, after the goats had been fed a concentrated ration containing monensin . A possible role of this drug in control of enterotoxaemia is suggested.

Am J Gastroenterol, 1996 Jul, 91(7), 1453 - 4
Albendazole-induced pseudomembranous colitis; Shah V et al.; We report a patient with AIDS and intestinal microsporidiosis . While undergoing treatment with albendazole, he developed worsening diarrhea with abdominal pain and fever . The diagnosis of pseudomembranous colitis was made by flexible sigmoidoscopy and a positive stool specimen for Clostridium difficile toxin . The patient's symptoms resolved with oral vancomycin and his stool C . difficile toxin became negative . Albendazole is an antibiotic that is chemically related to metronidazole . Although a few case reports link metronidazole with the development of pseudomembranous colitis, albendazole has not been associated with the development of this condition . The spectrum of antimicrobial activity of albendazole and its efficacy in the treatment of intestinal microsporidiosis are reviewed . Pathogenic mechanisms for the development of pseudomembranous colitis and the epidemiology of this condition in patients with AIDS are discussed.

Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 712 - 7
Is actin polymerization relevant to neurosecretion? A study on neuroblastoma cells; Viviani B et al.; We have investigated the relevance of actin rearrangement to neurotransmitter release, in neuroblastoma cell line SH-SY5Y stimulated with carbachol (1 mM) . Carbachol reversibly polymerizes actin and releases norepinephrine in undifferentiated SH-SY5Y cells as well as in tetradecanoylphorbol 13-acetate (16 nM) and retinoic acid (5 microM) differentiated SH-SY5Y cells . Microscopic analysis of F-actin distribution indicates polymerization of cortical actin . Prior treatment with iota toxin E from Clostridium perfringens inhibits both carbachol-induced actin polymerization and norepinephrine release, slightly affecting the basal actin network . These data suggest that actin polymerization is associated with norepinephrine release.

Biochem J, 1996 Jun 15, 316 ( Pt 3), 777 - 86
Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis . Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-beta-trypsin complex; Sreedharan SK et al.; 1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit beta-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin . The K1, value (3 x 10(-5) M) determined by kinetic analysis of the hydrolysis of N alpha-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 degrees C, I = 0.1, catalysed by beta-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low Mr inhibitors of this enzyme . Computer modelling of the beta-trypsin-E64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson-Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (beta-trypsin numbering from Ile-1) in the S1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group . The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and alpha-gingivain (one of the extracellular enzymes from phyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.

J Biol Chem, 1996 Jun 14, 271(24), 14548 - 53
Phosphorylation of 25-kDa synaptosome-associated protein . Possible involvement in protein kinase C-mediated regulation of neurotransmitter release; Shimazaki Y et al.; Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells . Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced {3H}-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues . The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25 . A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins . SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation . These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.

Biochim Biophys Acta, 1996 Jun 7, 1307(2), 123 - 6
Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms; Moriishi K et al.; We isolated the gene encoding a botulinum neurotoxin (BoNT) of 1285 amino acids with a molecular weight of 147,364 from the toxigenic bacteriophage d-sA of Clostridium botulinum type D strain South African (Dsa) . The BoNT of Dsa (BoNT/Dsa) is composed of three regions on the basis of the homology to BoNT types C1 (BoNT/C1) and D (BoNT/D) . The N-terminal (Met-1 to Val-522) and the C-terminal regions (Trp-945 to Glu-1285) have high identity to corresponding regions of BoNT/D (96% identity) and BoNT/C1 (74% identity), respectively . The core region (Pro-523 to Lys-944) is common to three toxins (83% to 92% identity) . These results suggest that neurotoxins produced from Clostridium botulinum types C and D are composed in a mosaic-like fashion.

Biochemistry, 1996 Jun 4, 35(22), 7188 - 96
Evidence for electron transfer from the nitrogenase iron protein to the molybdenum-iron protein without MgATP hydrolysis: characterization of a tight protein-protein complex; Lanzilotta WN et al.; MgA TP hydrolysis has been proposed to be absolutely required for electron transfer from the nitrogenase iron (Fe) protein to the molybdenum-iron (MoFe) protein . This work presents evidence for primary electron transfer from the Azotobacter vinelandii nitrogenase Fe protein to the MoFe protein in the absence of MgATP hydrolysis . Deletion of an amino acid (Leu 127) in a signal transduction pathway in the Fe protein resulted in an Fe protein conformation resembling the MgATP-bound state . This altered Fe protein (L127delta) was found to bind to the MoFe protein in the absence of MgATP, forming a tight protein complex . Both steady state and stopped-flow transient kinetic measurements suggest that two L127delta Fe proteins bind to one MoFe protein with an extremely high affinity . From pre-steady state kinetic determinations of the rate of complex dissociation, the affinity was found to be at least 350 times tighter than that of the wild-type A . vinelandii nitrogenase complex and at least 20 times tighter than that of the heterologous Clostridium pasteurianum Fe protein-A . vinelandii MoFe protein complex . The L127delta Fe protein-MoFe protein complex was isolated by gel filtration liquid chromatography . Scanning densitometry of an SDS gel of the complex isolated from the gel filtration column revealed a stoichiometry of 1.7 L 127 delta Fe proteins bound per MoFe protein . The L 127 delta Fe protein was found to transfer a single electron from its {4Fe-4S} cluster to the MoFe protein at a rate of 0.2s-1 . This compares with the MgATP dependent electron transfer rate of 140 s-1 observed for transfer of an electron from the wild-type Fe protein to the MoFe protein . No substrate reduction (H+ or C2H2) was detected when wild-type MoFe protein was complemented with L 127 delta Fe protein . The MgATP-independent electron transfer from the L 127 delta Fe protein to the MoFe protein required active MoFe protein and was not inhibited by MgADP . EPR spectroscopy of the complex was employed to confirm the electron transfer reaction . These results show that Fe protein in a conformation resembling the MgATP-bound state can transfer at least one electron to the MoFe protein without the need for MgATP hydrolysis.

J Ind Microbiol, 1996 Jun, 16(6), 354 - 9
The effect of novobiocin on solvent production by Clostridium acetobutylicum; Wong J et al.; Cells of Clostridium acetobutylicum treated with novoblocin, a DNA gyrase inhibitor, produced higher butyrate levels and lower solvent levels with acetone being the most affected . Seven enzyme activities involved in acid and solvent production were analyzed . Among them, only CoA transferase, required for acetone formation and acid uptake, experienced a significant decrease in activity . As in Escherichia coli and Bacillus subtilis, DNA from C . acetobutylicum became less negatively supercoiled in the early stationary phase (solventogenic stage), as shown by analysis of linking number of a reporter plasmid by agarose gel electrophoresis in the presence of chloroquine.

Gastroenterology, 1996 Jun, 110(6), 1704 - 12
A human antibody binds to alpha-galactose receptors and mimics the effects of Clostridium difficile toxin A in rat colon; Pothoulakis C et al.; BACKGROUND & AIMS: Nearly all human sera contain an immunoglobulin G antibody (antigalactose) that binds the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc expressed on cells from most mammals but not humans . Because the Clostridium difficile toxin A receptor in rodents contains this trisaccharide, the aim of this study was to examine whether antigalactose could mimic the enterotoxic effects of toxin A and bind to receptors containing this trisaccharide . METHODS: Fluid secretion, {3H}-mannitol permeability, and release of rat mast cell protease II and prostaglandin E2 were measured after luminal exposure of rat colon to either purified human anti-galactose, control immunoglobulin G, toxin A, or buffer . RESULTS: Toxin A (5 micrograms) and antigalactose (250 micrograms) but not control immunoglobulin (250 micrograms) stimulated colonic fluid secretion and caused increased mannitol permeability and rat mast cell protease II release . Antigalactose and toxin A and, to a lesser degree, control immunoglobulin G also stimulated release of prostaglandin E2, but only toxin A produced acute inflammation of rat colonic mucosa . Antigalactose and toxin A bound specifically to a single class of colonic brush border receptors with dissociation constants of 10(-6) mol/L and 5.4 x 10(-8) mol/L, respectively . CONCLUSIONS: Fluid secretion, increased permeability, and mast cell activation occur in rat colon when toxin A or human antigalactose immunoglobulin G bind to receptors bearing the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc.

Crit Rev Biochem Mol Biol, 1996 Jun, 31(3), 201 - 36
The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation; Beguin P et al.; Clostridium thermocellum produces a highly active cellulase system that consists of a high-M(r) multienzyme complex termed cellulosome . Hydrolytic components of the cellulosome are organized around a large, noncatalytic glycoprotein termed CipA that acts both as a scaffolding component and a cellulose-binding factor . Catalytic subunits of the cellulosome bear conserved, noncatalytic subdomains, termed dockerin domains, which bind to receptor domains of CipA, termed cohesin domains . CipA includes nine cohesin domains, a cellulose-binding domain, and a specialized dockerin domain . Proteins of the cell envelope carrying cohesin domains that specifically bind the dockerin domain of CipA have been identified . These proteins may mediate anchoring of the cellulosomes to the cell surface . Cellulase complexes similar to the cellulosome of C . thermocellum are produced by several cellulolytic clostridia . High-M(r) multienzyme complexes have also been identified in anaerobic rumen fungi . The architecture of the fungal complexes also seems to rely on the interaction of conserved, noncatalytic docking domains with a scaffolding component . However, the sequence of the fungal docking domains bears no resemblance to the clostridial dockerin domains, suggesting that the fungal and clostridial complexes arose independently.

Nutr Clin Pract, 1996 Jun, 11(3), 117 - 20
Diarrhea associated with lorazepam solution in a tube-fed patient; Shepherd MF et al.; A 43-year-old patient with adult respiratory distress syndrome, alcoholic hallucinosis, and delirium required significant amounts of lorazepam, morphine, and midazolam for management of agitation and increased peak airway pressures . Broad-spectrum antibiotics and intermittent pancuronium therapy were instituted . A nasoenteral feeding tube was placed for nutrition and medication administration during mechanical ventilation . Tube feedings were well tolerated except for intermittent bouts of large amounts of diarrhea . Clostridium difficile culture and toxin results were negative . Lorazepam and morphine administration were converted from the IV to enteral route to decrease the amount of fluid administered . The tube feeding was changed to an electrolyte rehydration solution and eventually discontinued . A search for drug-related contributing factors to the diarrhea revealed polyethylene glycol present in the lorazepam solution . It was postulated that this could be a contributing cause to the diarrhea . The lorazepam solution was changed to enterally administered crushed tablets with subsequent resolution of diarrhea.

Appl Environ Microbiol, 1996 Jun, 62(6), 2174 - 6
New gene from nine Bacillus sphaericus strains encoding highly conserved 35.8-kilodalton mosquitocidal toxins; Liu JW et al.; A new gene encoding a 35.8-kDa mosquitocidal toxin (Mtx3; 326 amino acids) was isolated from Bacillus sphaericus SSII-1 DNA . Mtx3 is a new type of mosquitocidal toxin with homology to the Mtx2 mosquitocidal toxin of B . sphaericus SSII-1, the epsilon-toxin of Clostridium perfringens, and the cytotoxin of Pseudomonas aeruginosa . The mtx3 gene is highly conserved and widely distributed in both high- and low-toxicity mosquito larvicidal strains of B . sphaericus.

Appl Environ Microbiol, 1996 Jun, 62(6), 2059 - 65
Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA; Rolleke S et al.; Medieval wall paintings are often affected by biodecay . An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process . This stems from the lack of effective means for such a stocktaking . Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism . Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings . We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions . To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers . The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE) . By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria . Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia . To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings.

Pediatr Infect Dis J, 1996 Jun, 15(6), 514 - 9
Prospective study of toxigenic Clostridium difficile in children given amoxicillin/clavulanate for otitis media; Mitchell DK et al.; OBJECTIVE: Evaluate antibiotic-associated diarrhea and toxigenic Clostridium difficile in stool specimens obtained from children before and after 10 days of amoxicillin/clavulanate for otitis media . DESIGN: Children, 12 to 47 months of age, treated with amoxicillin/clavulanate for otitis media in an outpatient setting were enrolled . Stool specimens were obtained at enrollment, when diarrhea occurred and at the end of therapy . All stool specimens were tested for C . difficile toxins A and B by enzyme immunoassay . RESULTS: Seventy-six children who had stool specimens collected at enrollment and after therapy were included in the analysis . None had C . difficile toxin in stool specimens at enrollment . Six (27%) of 22 children with diarrhea, and 4 (7%) of 54 children without diarrhea had C . difficile toxin present at completion of therapy (P = 0.03) . CONCLUSION: Toxigenic C . difficile was identified in 13% of children at the conclusion of amoxicillin/clavulanate therapy with a significantly higher frequency in children with diarrhea.

Protein Expr Purif, 1996 Jun, 7(4), 415 - 22
Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99; Kruse S et al.; A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101 . Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed . The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein . In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity . The sialidase production was increased about 2.5-fold when compared with that of the original clone . The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE . The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1 . A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture . For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K . The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions . The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.

J Clin Pathol, 1996 Jun, 49(6), 500 - 3
The potential role of Clostridium perfringens alpha toxin in the pathogenesis of acute pancreatitis; Holdsworth RJ et al.; BACKGROUND: Clostridium perfringens is a bowel commensal that can colonise the biliary tract . It produces the alpha toxin (phospholipase C), which can induce spontaneous tissue necrosis . AIMS: To investigate whether there is any evidence that Clostridium perfringens alpha toxin can be detected in acute pancreatitis . METHODS: Serum samples from 21 patients with acute pancreatitis and 22 controls were assayed for C perfringens phospholipase C as well as anti-phospholipase C IgG and IgM; IgG and IgM anti-toxins were measured by enzyme linked immunosorbent assay . RESULTS: In normal healthy controls there is a very high level of natural anti-toxin of both the IgG and IgM class . Of the 21 patients with acute pancreatitis alpha toxin was detected in five (23.8%) . Levels of both IgG and IgM anti-toxin were significantly reduced in acute pancreatitis . CONCLUSIONS: The results suggest that there is an abnormality of the immune status to C perfringens alpha toxin in patients with acute pancreatitis . This may be the result of a release of alpha toxin, although it is difficult to state whether this is a primary or secondary phenomenon in these patients . These preliminary results merit further investigation.

Protein Sci, 1996 Jun, 5(6), 1192 - 4
Subcloning of a DNA fragment encoding a single cohesin domain of the Clostridium thermocellum cellulosome-integrating protein CipA: purification, crystallization, and preliminary diffraction analysis of the encoded polypeptide; Beguin P et al.; An Escherichia coli clone encoding a single cohesin domain of the cellulosome-integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution . Crystals exhibit orthorhombic symmetry, space group P2(1)2(1)2(1), with cell dimensions a = 37.7 A, b = 80.7 A, c = 93.3 A, and four or eight molecules in the unit cell . The crystals diffract X-rays to beyond 2 A resolution and are suitable for further crystallographic studies.

J Ethnopharmacol, 1996 Jun, 52(2), 61 - 70
The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine; Cichewicz RH et al.; A survey of the Mayan pharmacopoeia revealed that tissues of Capsicum species (Solanaceae) are included in a number of herbal remedies for a variety of ailments of probable microbial origin . Using a filter disk assay, plain and heated aqueous extracts from fresh Capsicum annuum, Capsicum baccatum, Capsicum chinese, Capsicum frutescens, and Capsicum pubescens varieties were tested for their antimicrobial effects with fifteen bacterial species and one yeast species . Two pungent compounds found in Capsicum species (capsaicin and dihydrocapsaicin) were also tested for their anti-microbial effects . The plain and heated extracts were found to exhibit varying degrees of inhibition against Bacillus cereus, Bacillus subtilis, Clostridium sporogenes, Clostridium tetani, and Streptococcus pyogenes.

Appl Biochem Biotechnol, 1996 Jun, 59(3), 259 - 82
Antigen-antibody diffusion-limited binding kinetics for biosensors . A fractal analysis; Sadana A et al.; A fractal analysis is made for antigen-antibody binding kinetics for different biosensor applications available in the literature . Both types of examples are considered wherein: (1) the antigen is in solution and the antibody is immobilized on the fiberoptic surface, and (2) the antibody is in solution and the antigen is immobilized on the fiberoptic surface . For example, when the antibody is immobilized on the surface, an increase in the antigen Clostridium botulinum toxin A concentration in solution leads to (1) a decrease in the fractal dimension value or state of disorder, and (2) a higher rate constant for binding on the fiberoptic surface . An analysis of the effect of the influence of different parameters on the fractal dimension values for a particular example, such as varying treatments or incubation procedures, helps provide insights into the conformational states and reactions occurring on the fiberoptic surface . The analysis of the different example taken together provides novel physical insights into the state of "disorder" and reactions occurring on the surface . Such types of analysis should help contribute toward manipulating the reactions occurring on the fiberoptic surfaces in desired directions.

Eur J Biochem, 1996 Jun 1, 238(2), 346 - 9
Nuclear-magnetic-resonance determination of the electron self-exchange rate constant of Clostridium pasteurianum rubredoxin; Gaillard J et al.; The iron ion of rubredoxins efficiently exchanges one electron between the Fe(II) and Fe(III) oxidation states in mixtures of oxidized and reduced protein . The conditions under which the relaxation properties of the NMR signals can provide information about this exchange process have been worked out . The rate constant for the rubredoxin electron self-exchange ranges between 1.5 x 10(5) M-1 s-1 at 12 degrees C and 3 x 10(5) M-1 s-1 at 30 degrees C with an activation energy of the order of 24-30 kJ mol-1 in 50 mM potassium phosphate, pH 7 . The increase of the electron self-exchange rate constant with ionic strength suggests that neutralizing electrostatic repulsion between the active sites of two molecules further accelerates the already fast electron exchange.

J Immunol, 1996 Jun 1, 156(11), 4146 - 53
Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4; Moorman JP et al.; The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton . Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho . Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells . We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells . This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho . dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis . Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed . In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells . To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest . As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3 . dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

J Clin Oncol, 1996 Jun, 14(6), 1922 - 7
Response of recurrent medulloblastoma to low-dose oral etoposide; Ashley DM et al.; PURPOSE: The outcome for patients with recurrent medulloblastoma has historically been poor, with most patients dying of disseminated disease . Here, we report on seven patients with recurrent medulloblastoma, most heavily pretreated with a variety of chemotherapeutic agents, including parenteral etoposide (VP-16), who showed responses to the administration of repeated courses of low-dose oral VP-16 . PATIENTS AND METHODS: Seven patients age 4 to 16 years were treated with VP-16 after neuroradiographic and clinical evidence of tumor progression . Six had received prior irradiation . All seven had been pretreated with a variety of chemotherapeutic agents and schedules, including parenteral VP-16 . VP-16 was administered orally as repeated 21-day courses at 50 mg/m2/d with a 7-day interval between courses . Evaluation consisted of neuroradiographic and clinical examination after completion of every two courses of therapy . Complete blood cell counts were performed weekly . RESULTS: The major toxicity of oral VP-16 was hematologic, with two patients requiring platelet transfusions due to thrombocytopenia and two requiring RBC transfusions . All seven patients developed treatment-related neutropenia . Two patients were supported with granulocyte colony-stimulating factor (G-CSF) between courses . One patient developed infectious epididymitis after course 2 and required intravenous antibiotics; this illness was complicated by Clostridium difficile colitis . There was one episode of fever associated with neutropenia . There were no treatment-related deaths . Of seven patients assessed, six have demonstrated partial responses (PRs) and the remaining patient had stable disease (SD) . CONCLUSION: This report demonstrates the activity of oral VP-16 in the treatment of a small cohort of pretreated patients with recurrent medulloblastoma . This form of administration of oral VP-16 was well tolerated and produced modest toxicity.

J Bacteriol, 1996 Jun, 178(12), 3539 - 43
Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum; He Z et al.; A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized . The estimated molecular mass of the enzyme was 270 kDa . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits . The temperature and pH optima were 50 degrees C and pH 7.0, respectively . The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C . The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C . The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0 . The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate . The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate . Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.

J Bacteriol, 1996 Jun, 178(11), 3099 - 105
Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp . malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins; Barloy F et al.; A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp . malaysia CH18 with two gene-specific oligonucleotide probes . The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C . bifermentans subsp . malaysia . Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks . However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein . The cbm71 gene was expressed in a nontoxic strain of B . thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture . Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.

J Bacteriol, 1996 Jun, 178(11), 3077 - 84
A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA; Leibovitz E et al.; The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain . This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA . The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa . SAA comprises an NH2-terminal leader peptide followed by three distinct regions . The NH2-terminal region is similar to the NH2-terminal repeats of C . thermocellum OlpB and ORF2p . The central region is rich in lysine and harbors a motif present in Streptococcus M proteins . The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins . The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA . Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined . Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.

J Bacteriol, 1996 Jun, 178(11), 3015 - 24
Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824; Boynton ZL et al.; The enzymes beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase (BHBD), crotonase, and butyryl-CoA dehydrogenase (BCD) from Clostridium acetobutylicum are responsible for the formation of butyryl-CoA from acetoacetyl-CoA . These enzymes are essential to both acid formation and solvent formation by clostridia . Clustered genes encoding BHBD, crotonase, BCD, and putative electron transfer flavoprotein alpha and beta subunits have been cloned and sequenced . The nucleotide sequence of the crt gene indicates that it encodes crotonase, a protein with 261 amino acid residues and a calculated molecular mass of 28.2 kDa; the hbd gene encodes BHBD, with 282 residues and a molecular mass of 30.5 kDa . Three open reading frames (bcd, etfB, and etfA) are located between crt and hbd . The nucleotide sequence of bcd indicates that it encodes BCD, which consists of 379 amino acid residues and has high levels of homology with various acyl-CoA dehydrogenases . Open reading frames etfB and etfA, located downstream of bcd, encode 27.2- and 36.1-kDa proteins, respectively, and show homology with the fixAB genes and the alpha and beta subunits of the electron transfer flavoprotein . These findings suggest that BCD in clostridia might interact with the electron transfer flavoprotein in its redox function . Primer extension analysis identified a promoter consensus sequence upstream of the crt gene, suggesting that the clustered genes are transcribed as a transcriptional unit and form a BCS (butyryl-CoA synthesis) operon . A DNA fragment containing the entire BCS operon was subcloned into an Escherichia coli-C . acetobutylicum shuttle vector . Enzyme activity assays showed that crotonase and BHBD were highly overproduced in cell extracts from E . coli harboring the subclone . In C . acetobutylicum harboring the subclone, the activities of the enzymes crotonase, BHBD, and BCD were elevated.

Gynecol Oncol, 1996 Jun, 61(3), 369 - 72
Clostridium difficile colitis associated with cisplatin-based chemotherapy in ovarian cancer patients; Emoto M et al.; The purpose of this study was to examine the incidence and cause of Clostridium difficile colitis occurring after cisplatin-based combination chemotherapy in ovarian cancer patients . Thirty-three patients with primary ovarian malignancy were treated with cisplatin-based combination chemotherapy ranging from 1 to 12 (mean 4.6) cycles . All patients who developed diarrhea after undergoing the cancer chemotherapy were examined to determine whether or not they were complicated by C difficile colitis . The diagnosis of C . difficile was confirmed by a stool-cytotoxin test and endoscopic examination . Severe C . difficile colitis occurred in 2 patients (6.1%) after receiving cisplatin-based combination chemotherapy for ovarian malignancies . Although both patients recovered from the colitis after the administration of vancomycin, the first case demonstrated a relapse of the colitis after receiving a subsequent course of the same chemotherapy with cisplatin . Both patients were then treated with a carboplatin alternative to cisplatin in the following courses, which resulted in neither a relapse of the colitis nor a recurrence of the malignancies up to this time . This report suggests the importance of searching for the presence of C . difficile and its toxin in patients with diarrhea after undergoing cancer chemotherapy since C . difficile may cause severe colitis . Based on our findings, it is thus concluded that cisplatin may cause C . difficile colitis.

Curr Microbiol, 1996 Jun, 32(6), 349 - 56
Recombination-induced variants of Clostridium acetobutylicum ATCC 824 with increased solvent production; Wong J et al.; Three sporulation-specific genes (orfA, sigE, sigG) from clostridium acetobutylicum ATCC 824 are arranged in a cluster, encoding the putative sigma E-processing enzyme, sigma E, and sigma sigma G respectively . When they were transformed into Clostridium acetobutylicum while on a plasmid functional in this organism, transformants did not survive . Three kinds of recombinations were then attempted with nonreplicative plasmids: duplication of orfA and sigE, replacement of all of the three genes, and inactivation of orfA . While the wild-type strain ceased to grow and produce solvents in batch cultures after approximately 24 h, mutant strains were isolated that showed sustained growth for a much longer time and produced a threefold increase in acetone and butanol in test tube cultures . In addition, one of the derived strains showed a significantly higher growth rate . Features of the restriction maps of the recombinants did not correlate with expected maps, indicating possible complications occurring during the recombination events.

J Biol Chem, 1996 May 31, 271(22), 13135 - 9
Evidence for Rho-mediated agonist stimulation of phospholipase D in rat1 fibroblasts . Effects of Clostridium botulinum C3 exoenzyme; Malcolm KC et al.; Small GTP-binding proteins of the Rho family are implicated in the in vitro regulation of phosphatidylcholine hydrolysis by phospholipase D (PLD) . However, their role in agonist-stimulated PLD activity in whole cells is not clear . The ribosyltransferase C3 from Clostridium botulinum modifies Rho proteins and inhibits their function . When introduced into rat1 fibroblasts by scrape-loading, C3 inhibited PLD activity stimulated by lysophosphatidic acid (LPA), endothelin-1, or phorbol ester . Neither the time course nor agonist dose response for LPA-stimulated PLD activity was altered in C3-treated cells . In contrast to the effects of C3 on PLD activity, agonist-stimulated phosphatidylinositol-phospholipase C activity was not altered in C3-treated cells . Surprisingly, C3 treatment led to a decrease in the amount of RhoA protein, indicating that the loss of PLD activity in response to agonist was partly due to the loss of Rho proteins . As described previously, C3 treatment led to the inhibition of LPA-stimulated actin filament formation . However, disruption of actin filaments with cytochalasin D caused only a minor inhibition of LPA-stimulated PLD activity . Interestingly, stimulation of cells with LPA caused a rapid enrichment of RhoA in the particulate fraction of cell lysates . These data support an in vivo role for RhoA in agonist-stimulated PLD activity that is separate from its role in actin fiber formation.

J Biol Chem, 1996 May 31, 271(22), 12951 - 5
Inhibition of NAD+ glycohydrolase and ADP-ribosyl cyclase activities of leukocyte cell surface antigen CD38 by gangliosides; Hara-Yokoyama M et al.; We have recently reported that gangliosides act as inhibitors of ADP-ribosyltransferases and NAD+ glycohydrolases (NADase) of pertussis toxin and the C3 exoenzyme from Clostridium botulinum (Hara-Yokoyama, M., Hirabayashi, Y., Irie, F., Syuto, B., Moriishi, K., Sugiya, H., and Furuyama, S . (1995) J . Biol . Chem . 270, 8115-8121) . Here, we investigated the effect of gangliosides on the enzymatic activity of leukocyte cell surface antigen CD38, which is identified as an ecto-NADase (Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T . (1993) J . Biol . Chem . 268, 16895-16898) . Gangliosides GM1a and GQ1balpha inhibited the NADase activity in the immunoprecipitate of anti-CD38 antibody from the membrane extract of retinoic acid-treated human leukemic HL-60 cells . Gangliosides also inhibited the NADase activity of the extracellular domain of CD38 antigen that was deprived of the transmembrane domain and was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP-CD38) . The order of the inhibitory effect of purified ganglioside species on the NADase activity on MBP-CD38 was as follows: GQ1balpha > GT1b, GQ1b > GD1a, GD1b, GM1a, GM1b, GD3, GM3 . GQ1balpha inhibited the NADase of MBP-CD38 in a noncompetitive manner versus NAD+ with a Ki value of about 0.3 microM . Neither ceramide nor the oligosaccharide moiety of GQ1balpha had an effect on the NADase activity . GQ1balpha, GT1b, and GQ1b also efficiently inhibited the ADP-ribosyl cyclase activity of MBP-CD38 . At present, gangliosides are the only endogenous species that can block the enzymatic activity of CD38 antigen . The present results suggest a potential role of gangliosides as inhibitors of the ecto-NADases.

J Biol Chem, 1996 May 24, 271(21), 12542 - 8
ADP-ribosylation of the G protein Rho inhibits integrin regulation of tumor cell growth; Udagawa T et al.; Using a gastric derived tumor line, we investigated the involvement of beta 1 integrin and Rho in cell growth regulation in response to collagen . The addition of C3 exoenzyme from clostridium botulinum to specifically ribosylate and inhibit the function of the rho gene products inhibited cellular proliferation in a dose-dependent fashion . C3 exoenzyme exhibited broad cytostatic activity toward a number of tumor lines and induced G0/G1 accumulation, cyclin A inhibition, and pronounced alterations in cell morphology . Integrin-mediated adhesion to collagen led to the expression of the cyclin A gene whose expression could be blocked using anti-beta 1 integrin monoclonal antibodies . Phospholipid levels were induced upon beta 1 integrin-mediated adhesion to collagen, and the phospholipid induction was inhibited by either antibodies to beta 1 integrin or pretreatment of cells with C3 exoenzyme . Significant reduction in phospholipid levels correlated with proliferation for a panel of tumor lines deprived of adhesion to substrate . These results implicate a novel role for integrins and Rho in the regulation of tumour growth in response to matrix.

FEBS Lett, 1996 May 20, 386(2-3), 133 - 6
Substrate residues N-terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity; Wictome M et al.; Clostridium botulinum type B neurotoxin is a highly specific zinc-endopeptidase which cleaves vesicle-associated membrane protein (VAMP/synaptobrevin), a critical component of the vesicle docking/fusion mechanism . In this study, substrate residues flanking the N-terminal side of the cleavage site are shown to play a key role in enzyme substrate recognition . Two aspartate residues in this region are identified as critical determinants of the neurotoxin's specificity . These findings are discussed in relation to the mechanism by which botulinum type B neurotoxin cleaves its substrate.

Ann N Y Acad Sci, 1996 May 15, 782, 241 - 51
Identification and characterization of cellulose-binding domains in xylanase A of Clostridium stercorarium; Sakka K et al.; The xynA gene encoding a major xylanase of Clostridium stercorarium F-9 was sequenced . The structural gene consists of an open reading frame of 1533 bp encoding a protein of 511 amino acids with an M(r) of 56,519 . XynA consists of a catalytic domain belonging to family G at the NH2-terminus and two direct repeats of about 90 amino acids with a short spacing at the COOH-terminus . The repeated sequences, CBDI and CBDII, were not homologous with amino acid sequences of the CBDs classified into families I to V . Nevertheless, XynA showed an affinity for insoluble cellulose such as Avicel . Binding of XynA to Avicel was strongly dependent on the concentration of the incubation buffer and was inhibited by Triton X-100 . XynA bound to Avicel (2.4 nmol/g-cellulose) and acid-swollen cellulose (180 nmol/g-cellulose), suggesting that this enzyme has higher affinity for amorphous cellulose than for crystalline cellulose . Functions of CBDI and CBDII were investigated by constructing the mutant enzymes and evaluating the cellulose-binding ability of each of them . XynA4 lacking CBDI and XynA5 lacking CBDII bound to Avicel to a lesser extent than the parental enzyme XynA; but XynA6, devoid of both CBDs, did not bind at all, indicating that CBDI and CBDII each functioned independently as CBD in XynA and their binding capacity was additive . Although the Ruminococcus albus endoglucanase EgIV that was joined to CBDs of XynA acquired cellulose-binding ability, the substrate specificity of EgIV was not altered in the presence or absence of CBDs.

FEMS Microbiol Lett, 1996 May 15, 139(1), 43 - 50
Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein 1 (CBP1) of Fibrobacter succinogenes S85; Mitsumori M et al.; The nucleotide sequence of the gene encoding the Fibrobacter succinogenes S85 cellulose-binding protein 1 (CBP1) has been determined . The gene encodes a protein of 1054 amino acids with a molecular mass of 118614 . The deduced amino acid sequence of CBP1 showed an extensive similarity to the cellulose-binding domain of an endoglucanase (EGCCD) from Clostridium cellulolyticum and contained the reiterated regions . The cloned gene was inserted into an expression vector, pRSETA, and was expressed in E . coli as a fused protein with the peptide consisting of six consecutive histidine residues . The fused protein was detected by immunoblotting using antiserum against CBP1, and exhibited the cellulose-binding activities.

Biochem J, 1996 May 15, 316 ( Pt 1), 239 - 45
Leukotriene D4-induced mobilization of intracellular Ca2+ in epithelial cells is critically dependent on activation of the small GTP-binding protein Rho; Gronroos E et al.; We have previously shown that the leukotriene D4 (LTD4)-induced mobilization of intracellular Ca2+ in epithelial cells is mediated by a G-protein that is distinctly different from the pertussis toxin-sensitive G-protein that regulates the subsequent influx of Ca2+ . In the present study, we attempted to gain further knowledge about the mechanisms involved in the LTD4-induced mobilization of intracellular Ca2+ in epithelial cells by investigating the effects of compactin, an inhibitor of the isoprenylation pathway, on this signalling event . In cells preincubated with 10 microM compactin for 48 h, the LTD4-induced mobilization of intracellular Ca2+ was reduced by 75% in comparison with control cells . This reduction was reversed by co-administration of mevalonate (1 mM) . The effect of compactin occurred regardless of whether or not Ca2+ was present in the extracellular medium, suggesting that isoprenylation must occur before Ca2+ is released from intracellular stores . In accordance with this, we also found that both the LTD4-induced formation of inositol 1,4,5-trisphosphate and the LTD4-induced phosphorylation of phospholipase C gamma 1 (PLC gamma 1) on tyrosine residues were significantly reduced in compactin-pretreated cells . These results open up the possibility that the activation of PLC gamma 1 is related to a molecule that is sensitive to impaired activity of the isoprenylation pathway, such as a small monomeric G-protein . This idea was supported by the observation that Clostridium botulinum C3 exoenzyme-induced inhibition of Rho proteins abolished the LTD4-induced intracellular mobilization of Ca2+ . A regulatory role of Rho proteins in the LTD4-induced activation of PLC gamma 1 is unlikely to be indirectly mediated via an effect on the cytoskeleton, since cytochalasin D had no major effect on the LTD4-induced mobilization of Ca2+ . Although the mechanism of interaction remains to be elucidated, the present findings indicate an important role of an isoprenylated protein such as Rho in the LTD4-induced Ca2+ signal.

J Biol Chem, 1996 May 3, 271(18), 10786 - 92
Genetic characterization of Clostridium botulinum type A containing silent type B neurotoxin gene sequences; Hutson RA et al.; A recent study detected genes encoding type B botulinum neurotoxin in some type A strains of Clostridium botulinum that exhibit no type B toxin activity . In this study, we investigated the presence, structure, linkage, and organization of genes encoding botulinum neurotoxin (BoNT) and other components of the progenitor complex . Sequence analysis showed that the silent BoNT/B gene is highly related to that from authentic proteolytic type B C . botulinum . However, a stop signal and deletions were found within the sequence . A non-toxin nonhemagglutinin gene (NTNH) was mapped immediately upstream of both the BoNT/A and silent BoNT/B genes . Significantly the NTNH gene adjacent to the defective BoNT/B gene was "chimeric, " the 5'- and 3'-regions of the gene had high homology with corresponding regions of the type B NTNH gene, while the 471-amino acid sequence in the central region was identical to NTNH of type A . Hemagglutinin genes HA-33 and HA-II were not found adjacent to the NTNH/A gene, but instead there was an unidentified open reading frame previously reported in strains of C . botulinum types E and F . By contrast HA-II, HA-33, and NTNH genes were located immediately upstream of the silent BoNT/B gene . Pulsed-field gel electrophoretic analysis of chromosomal DNA digests indicated the distance between type A and B gene clusters to be less than 40 kilobases.

Vet Surg, 1996 May-Jun, 25(3), 195 - 8
Detection of bacteria in equine synovial fluid by use of the polymerase chain reaction; Crabill MR et al.; Equine synovial fluid aliquots were inoculated with Salmonella enteritidis, Escherichia coli, Actinobacillus equuli, Staphylococcus aureus, and Streptococcus zooepidemicus to obtain approximate concentrations of 1000, 100, 10, and 1 colony forming U/mL . Synovial fluid aliquots were also inoculated with an unquantitated inoculum of Bacteroides fragilis and Clostridium perfringens . Inoculated synovial fluid was incubated in trypticase-soy broth or Columbia broth for approximately 12 hours . Then aliquots were removed for DNA extraction and polymerase chain reaction (PCR) analysis for detection of a 531 base-pair segment of bacterial DNA corresponding to a region of the 16S ribosomal gene . Duplicate samples of inoculated synovial fluid were prepared for microbial culture . Bacteria were detected in all samples inoculated with bacteria but not in control synovial fluid samples . Under experimental conditions there was no difference between microbial culture and PCR analyses for detection of bacteria . Experimentally, PCR was able to detect bacteria in synovial fluid within 24 hours of inoculation.

Zentralbl Veterinarmed B, 1996 May, 43(3), 137 - 46
Plasmid profiling for strain differentiation and characterization of Clostridium perfringens isolates; Eisgruber H et al.; Plasmid profiling was used for the characterization of 13 Clostridium perfringens collection strains as well as 85 clinical and food isolates . Methodological details and limitations of the plasmid isolation procedure are outlined and discussed . For 19 clinical isolates obtained during seven group outbreaks, a close connection between at least some strains from each individual outbreak was established for six of them; only results from one outbreak were completely inconclusive, due to missing plasmids in one of the two isolates . The presence of multiple plasmid types within seven out of 12 given food samples, from which at least two C . perfringens isolates were obtained, indicates the importance of multiple isolates for meaningful typing results in epidemiological investigations . By including results from a previous report from this laboratory, baseline data on plasmid profiles for a total of 133 isolates are provided . The results of this study revealed that 36% of the food isolates unrelated to disease outbreaks carried no plasmids, as compared with 19-25% among disease-related isolates . A high prevalence (24.8%) of a 8.9 +/- 0.5 MDa plasmid was found among the 133 isolates, which contributed to one of four occurrences of identical plasmid profiles among strains that were initially considered unrelated . Two of these identical plasmid profiles were found among strains from the same culture collection, indicating the possibility of a common ancestor strain or cross-contamination.

Biol Chem Hoppe Seyler, 1996 May, 377(5), 293 - 9
Isolation and characterization of sialate lyase from pig kidney; Schauer R et al.; Sialate lyase (sialate aldolase; systematic name N-acetylneuraminate pyruvate-lyase, EC 4.1.3.3) was isolated as soluble enzyme from pig kidney and purified 630-fold using a heating step, gel filtration, and chromatography on immobilized neuraminic acid beta-methyl glycoside in 14% yield to apparent homogeneity as tested by SDS-gel electrophoresis . The molecular mass is 58 kDa and the pH-optimum is at pH 7.2 . Kinetic parameters were determined with N-acetyl-neuraminic acid as substrate: Km 3.7 mM and Vmax 37.1 mU . The lyase cleaves only free sialic acids with relative rates of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for N-acetyl-9-O-acetylneuraminic acid, whereas N-acetyl-4-O-acetylneuraminic acid or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid are not substrates . Enzyme activity was inhibited with p-chloromercuribenzoate, o-phenanthroline, cyanide, 5-diazonium-1-H-tetrazole, 5,5'-dithiobis(2-nitrobenzoic acid), diethylpyro-carbonate, and Rose Bengal in the presence of light and O2 . Reduction with sodium borohydride in the presence of N-acetylneuraminic acid or pyruvate resulted in irreversible inhibition of enzyme activity . The inhibition experiments suggest the involvement of histidine, lysine and SH-residues in enzyme catalysis . Thus, this mammalian lyase most probably belongs to the Class I aldolases, and has properties similar to the same enzyme from Clostridium perfringens and is active with the alpha-form of N-acetylneuraminic acid.

Mol Biochem Parasitol, 1996 May, 77(2), 217 - 23
Sequence of a Giardia lamblia gene coding for the glycolytic enzyme, pyruvate,phosphate dikinase; Nevalainen L et al.; The sequence of the gene coding for pyruvate,phosphate dikinase (EC 2.7.9.1) in Giardia lamblia (syn . G . duodenalis) has been established . The deduced amino acid sequence is very similar to all of its known homologs from the protist, Entamoeba histolytica, the eubacterium, Clostridium symbiosum and plant chloroplasts . Phylogenetic reconstruction with neighbor-joining and maximum parsimony methods reveals that the sequences form two clades, one comprising the anaerobic microorganisms, and the other the chloroplast enzymes.

Neuromuscul Disord, 1996 May, 6(3), 177 - 85
Nerve terminal sprouting in botulinum type-A treated mouse levator auris longus muscle; Juzans P et al.; The marked outgrowth of the motor nerve terminal arborization triggered by an in vivo local injection of Clostridium botulinum type-A toxin in the mouse levator auris longus muscle was studied with morphological and immunochemical approaches . The increase in total nerve terminal length depended on the time elapsed after toxin administration and was due to both increased number of terminal branches and branch length as revealed by a quantitative morphological analysis of whole mounts using the combined cholinesterase-silver stain . Nerve terminal sprouts increased in number, length and complexity even after the functional recovery of neuromuscular transmission had occurred as revealed by electrophysiological examination . Although we cannot exclude that transmitter release sites from the original nerve terminal arborization may still be functional after botulinum type-A toxin (BoTx-A) treatment, it is likely that newly formed functional release sites on the sprouts play a major role in the functional recovery of neuromuscular transmission . The presence of an immunoreactivity to synaptophysin and synaptotagmin-II, integral proteins of synaptic vesicles, gives support to our previous findings suggesting that nerve terminal sprouts have the molecular machinery for acetylcholine release.

Pathol Biol (Paris), 1996 May, 44(5), 358 - 62
Use of the E-test for determining antimicrobial susceptibility of anaerobic bacteria; Pierard D et al.; Routine determination of antimicrobial susceptibility of anaerobic clinical isolates is difficult . The E-test is a practical alternative technique that we evaluated while testing clinical isolates in a multicenter study . The susceptibility to 9 antibiotics (penicillin, amoxycillin/clavulanate, ticarcillin/clavulanate, piperacillin/tazobactam, imipenem, cefoxitin, metronidazole, clindamycin, chloramphenicol) of 351 strains belonging to 63 different species was determined by the NCCLS reference agar dilution procedure using Wilkins-Chalgren agar medium with 5% sheep blood and was compared to the E-test performed on the same medium and using manufacturer's recommendations . The MIC values obtained with the E-test were generally one dilution lower than those obtained with the reference technique, 87.1% of the results being within two dilutions . In terms of susceptibility categories, 95.1% agreement was observed with 3.8% minor errors and only 0.5% major and 0.6% very major errors . With some Fusobacterium spp . and Clostridium spp . strains, the E-test was difficult to read or not interpretable because of the presence of growth within the inhibition zone of all beta-lactam antibiotics, representing a trailing phenomenon . We conclude that, if some interpretation difficulties are taken into account, the E-test is a convenient and reliable technique that can be applied in all clinical laboratories . It could be used for the individual testing of important anaerobes in certain clinical situations but cannot yet be considered as a reference technique . Its utility is emphasised by the increased resistance rate against clindamycin and the appearance of a few strains in the B . fragilis group with a reduced susceptibility against metronidazole.

Pathology, 1996 May, 28(2), 178 - 81
Comparison of two commercial enzyme immunoassays with cytotoxicity assay and culture for the diagnosis of Clostridium difficile related diarrhea; Siarakas S et al.; 184 stool samples were analysed for the presence of Clostridium difficile and toxins using the Meridian Premier Toxin A and TechLab Tox-A EIA kits, selective culture and cytotoxin assay . Of the 184 samples 36 stools tested positive for cytotoxin . In comparison the sensitivity and specificity of the EIAs and culture were as follows: Meridian, 72 and 87, TechLab, 64 and 95, and selective culture, 83 and 96%, respectively . The positive predictive values and negative predictive values for the various methods were: Meridian, 58 and 93, TechLab, 77 and 92, and selective culture, 83 and 96%, respectively . Discrepant results to those obtained by cytotoxicity assay were encountered with both EIA kits evaluated and less so by culture . In this study direct isolation of Clostridium difficile from stool samples most closely paralleled the findings of the "gold standard" cell line cytotoxicity assay . It appears that a single test for the determination of Clostridium difficile disease is adequate, although a second method improves the predictability of the diagnosis . Direct culture of feces provided a reliable secondary procedure to cytotoxicity assay . The EIAs were simple to use, labour efficient and provided a rapid result . However the lack of sensitivity and relative expense did not justify their routine use in our laboratory.

Pharmacol Toxicol, 1996 May, 78(5), 283 - 8
Refinement and validation of an alternative bioassay for potency testing of therapeutic botulinum type A toxin; Sesardic D et al.; The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurones . This property has led to the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms . At present, the only recognised assay with the specificity and sensitivity to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the end point . Refinement of this assay, with respect to the end point, was explored on the basis of the development of flaccid paralysis of muscles following subcutaneous injection of the toxin at the inguinocrural region . Potency estimates, relative to in house reference preparations, for different therapeutic preparations obtained using flaccid paralysis as a scored response gave excellent agreement with estimates obtained in independent assay using the currently required control method . This study demonstrates that an alternative, more humane bioassay for potency testing of clostridia neurotoxins gives valid estimates equivalent to those currently in use.

Blutalkohol, 1996 May, 33(3), 113 - 41
{Formation of so-called byproducts (propanols, butanols et al.) from ethanol by microorganisms}; Stohlmacher P; Microbiological literature implies and furnishes evidence that aliphatic alcohols and the corresponding carboxylates as well as acetone can be produced from ethanol during microbial metabolic processes . Propionate/propanol-1 followed by butyrate can be obtained by means of step-by-step reductive carboxylation of acetyl-CoA . Both butyrate/butanol-1 and caproate/hexanol-1 are typical fermentation products of Clostridium kluyvery . In cases where butyrate decomposition is disrupted up to 50% of butyrate is isomerised to isobutyrate . In addition to ethanol, butyrate and butanol-1, isopropanol and acetone are characteristic products of commercially used Clostridia . One would expect that saccharolytic organisms producing ethanol in addition to other "solvents" (butanol-1, acetone, isopropanol) can also synthesise the solvents if the substrate is changed (ethanol instead of carbohydrate).Under carbon monoxide, formiate and hydrogen, some CODH-active Clostridia can, very efficiently, convert various carboxylates into the corresponding alcohols . There are several groups of organisms present in human intestinal tract that can utilise ethanol and other alcohols.

Protein Sci, 1996 May, 5(5), 883 - 94
2D 1H and 3D 1H-15N NMR of zinc-rubredoxins: contributions of the beta-sheet to thermostability; Richie KA et al.; Based on 2D 1H-1H and 2D and 3D 1H-15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc-containing Clostridium pasteurianum rubredoxin (Cp ZnRd) . Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N-terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (-), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcus furiosus (Pf Rd) . The secondary structure of both wild-type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same . However, the NMR data indicate an extension of the three-stranded beta-sheet in the mutated Cp ZnRd to include the N-terminal Ala residue and Glu 15, as occurs in Pf Rd . The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the beta-sheet and, in fact, throughout the mutated Rd . However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild-type Cp Rds are much less thermostable than Pf Rd . These correlations strongly suggest that, contrary to a previous proposal {Blake PR et al., 1992, Protein Sci 1:1508-1521}, the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N-terminal strand of the beta-sheet . The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability.

J Clin Microbiol, 1996 May, 34(5), 1153 - 7
Comparison of PCR-based approaches to molecular epidemiologic analysis of Clostridium difficile; Collier MC et al.; Representative isolates of the 10 serogroups of Clostridium difficile and 39 clinical isolates (30 toxigenic and 9 nontoxigenic), including 5 isolates from a confirmed nosocomial outbreak, were analyzed by using two previously described arbitrary-primer PCR (AP-PCR) molecular typing methodologies (AP-PG05 and AP-ARB11) and PCR ribotyping . The two AP-PCR methods investigated gave comparable results; AP-PG05 and AP-ARB11 identified 8 and 7 groups among the serogroup isolates and classified the clinical isolates into 21 and 20 distinct groups, respectively . PCR ribotyping also identified 8 unique groups among the serogroup isolates but classified the clinical isolates into 23 groups . In addition, when results obtained by the PCR methods were compared with typing data generated by pulsed-field gel electrophoresis (PFGE), PCR ribotyping and PFGE were found to be in agreement for 83% (29 of 35) of isolates typeable by both techniques while AP-PG05 was in agreement with PFGE for 60% (20 of 33) and AP-ARB11 was in agreement with PFGE for only 44% (17 of 36) . These results indicate that PCR ribotyping is a more discriminatory approach than AP-PCR for typing C . difficile and, furthermore, that this technique generates results that are in higher concordance with those obtained by using an established method for differentiating isolates of this organism on a molecular level than are results generated by using AP-PCR.

Proteins, 1996 May, 25(1), 134 - 6
Crystallization of a family 8 cellulase from Clostridium thermocellum; Souchon H et al.; The catalytic domain of cellulase CelA, a family 8 glycohydrolase from C . thermocellum, has been crystallized in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 50.12 A, b = 63.52 A, c = 104.97 A . The diffraction pattern extends beyond 1.5 A resolution.

J Neurosci Res, 1996 May 1, 44(3), 263 - 71
Productive and non-productive binding of botulinum neurotoxin A to motor nerve endings are distinguished by its heavy chain; Daniels-Holgate PU et al.; Botulinum neurotoxin type A, a di-chain protein produced by Clostridium botulinum and responsible for botulism, blocks acetylcholine release from peripheral nerves by binding to the terminals, undergoing internalization and proteolyzing a protein essential for exocytosis . As butolinum neurotoxin is being used clinically for the treatment of dystonias and certain spasticities, deciphering the details of its specific targeting to cholinergic nerve endings has assumed great importance . Thus, interaction of butolinum neurotoxin type A with murine motor nerve terminals-a prime target in vivo-was investigated . Autoradiographic analysis revealed saturable, high-affinity interaction of radioiodinated toxin (0.4 nM) with two ecto-acceptor types, distinguished by an excess of the toxin's heavy chain which prevented only a fraction of this binding . Botulinum neurotoxin was also biotinylated through its free sulfhydryl groups, known not to be essential for neurotoxicity . Similar binding of this active derivative was, likewise, partially blocked by heavy chain, confirming the above results . This binding that is resistant to heavy chain equates to botulinum neurotoxin interacting with productive ecto-acceptors, leading to delivery to its cytosolic site of action, because heavy chain proved unable to antagonize toxin-induced neuromuscular paralysis . In contrast, it is deduced that botulinum neurotoxin bound to heavy chain-susceptible sites has a different fate, presumably due to trafficking via another route, and thus would be inefficient in causing neuroparalysis.

Clin Infect Dis, 1996 May, 22(5), 813 - 8
Comparison of vancomycin, teicoplanin, metronidazole, and fusidic acid for the treatment of Clostridium difficile-associated diarrhea; Wenisch C et al.; We conducted a prospective, randomized study to compare the efficacy of oral fusidic acid, oral metronidazole, oral vancomycin, and oral teicoplanin for the treatment of Clostridium difficile-associated diarrhea . Treatment resulted in clinical cure for 94% of the patients who were treated with vancomycin, 96% of those treated with teicoplanin, 93% of those treated with fusidic acid, and 94% of those treated with metronidazole . Clinical symptoms recurred in 16% of patients treated with vancomycin, 7% of those treated with teicoplanin, 28% of those treated with fusidic acid, and 16% of those treated with metronidazole . There was asymptomatic carriage of C . difficile toxin in 13% of patients treated with vancomycin, 4% of those treated with teicoplanin, 24% of those treated with fusidic acid, and 16% of those treated with metronidazole . No adverse effects related to therapy with vancomycin or teicoplanin were observed . Considering the costs of treatment, our findings suggest that metronidazole is the drug of choice for C . difficile-associated diarrhea and that glycopeptides should be reserved for patients who cannot tolerate metronidazole or who do not respond to treatment with this drug.

Clin Diagn Lab Immunol, 1996 May, 3(3), 270 - 4
Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination; Kimura A et al.; To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E . histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis . A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined . The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively . In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223 . The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls . The recombinant protein had potent and specific antigenicity . In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42 . In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value . These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E . histolytica.

Am J Med, 1996 May, 100(5), 487 - 95
Clinical prediction rules to optimize cytotoxin testing for Clostridium difficile in hospitalized patients with diarrhea; Katz DA et al.; BACKGROUND: Although routine testing of hospitalized patients with diarrhea for Clostridium difficile cytotoxin has been advocated as a high-yield procedure, the rationale for this practice has been questioned . To target a low-yield subgroup for whom routine testing could be deferred, we derived a clinical decision rule for predicting results of the C difficile cytotoxin assay in hospitalized adults with diarrhea . METHODS: We hypothesized a priori that two variables, antibiotic use (within 30 days prior to testing) and history of significant diarrhea (new onset of > 3 partially formed or watery stools per 24 hour period), would be highly predictive of cytotoxin results, and obtained these data on 480 consecutive patients who underwent diagnostic testing for C difficile at a university hospital and affiliated Veterans Affairs medical center . For more detailed modelling, we recorded symptoms, signs, comorbidity, and other potential causes of diarrhea for 68 test positive patients (cases) and 265 randomly selected test negative patients (controls) within the study cohort . RESULTS: The overall prevalence of positive cytotoxin assays was 14% . Prior antibiotic therapy (OR = 9.0, 95% CI 2.1-38.4), significant diarrhea (OR = 2.2, 95% CI 1.1-4.7), and abdominal pain (OR = 1.9, 95% CI 0.96-3.7) were independent predictors of cytotoxin assay results . The model discriminated patients with positive and negative assays with a receiver operating characteristic (ROC) area of 0.68; observed and predicted probabilities of a positive cytotoxin assay were well correlated over the entire range of observed probabilities (r2 = 0.86) . A decision rule (defined as positive if prior antibiotic use and either significant diarrhea or abdominal pain are present) demonstrated sensitivity and specificity of 86 and 45% . When applied to the entire dataset (N = 480), a simplified a priori rule, defined as positive if both prior antibiotic use and history of significant diarrhea are present, demonstrated sensitivity, specificity, positive and negative predictive value of 80, 45, 18 and 94%, respectively (6% of those predicted to be cytotoxin-negative actually tested positive) . Use of this rule would have averted 39% of cytotoxin assays in our study population . CONCLUSIONS: Patients without prior antibiotic use and either significant diarrhea or abdominal pain are unlikely to have positive C difficile cytotoxin assay results, and may not routinely require cytotoxin testing.

FEMS Microbiol Rev, 1996 May, 18(2-3), 105 - 17
Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus glutamate dehydrogenase; Rice DW et al.; The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 A resolution . The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa . This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues . Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited . The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures.

Appl Environ Microbiol, 1996 May, 62(5), 1842 - 6
Degradation of 2-sec-butyl-4,6-dinitrophenol (dinoseb) by Clostridium bifermentans KMR-1; Hammill TB et al.; A strain of Clostridium bifermentans, KMR-1, degraded 2-sec-butyl-4,6-dinitrophenol (dinoseb) to a level below the limit of detection by high-performance liquid chromatography (0.5 mg/liter) within 96 h, with no accumulation of aromatic intermediates . KMR-1 could not utilize dinoseb as a sole carbon or energy source, and degradation occurred via cometabolism in the presence of a fermentable carbon source . KMR-1 mineralized some dinoseb in anaerobic cultures, evolving 7.2% of the radioactive label in U-ring 14C-labeled dinoseb as 14CO2 . The remaining anaerobic degradation products were incubated with aerobic soil bacteria, and 35.4% of this residual radioactive label was evolved as 14CO2 . During this mineralization experiment, 38.9% of the initial label was evolved as 14CO2 after both anaerobic and aerobic phases . This is the first demonstration of dinoseb degradation by a pure microbial culture.

Appl Environ Microbiol, 1996 May, 62(5), 1741 - 6
Distribution of sewage indicated by Clostridium perfringens at a deep-water disposal site after cessation of sewage disposal; Hill RT et al.; Clostridium perfringens, a marker of domestic sewage contamination, was enumerated in sediment samples obtained from the vicinity of the 106-Mile Site 1 month and 1 year after cessation of sewage disposal at this site . C . perfringens counts in sediments collected at the disposal site and from stations 26 nautical miles (ca . 48 km) and 50 nautical miles (ca . 92 km) to the southwest of the site were, in general, more than 10-fold higher than counts from an uncontaminated reference site . C . perfringens counts at the disposal site were not significantly different between 1992 and 1993, suggesting that sewage sludge had remained in the benthic environment at this site . At stations where C . perfringens counts were elevated (i.e., stations other than the reference station), counts were generally higher in the top 1 cm and decreased down to 5 cm . In some cases, C . perfringens counts in the bottom 4 or 5 cm showed a trend of higher counts in 1993 than in 1992, suggesting bioturbation . We conclude that widespread sludge contamination of the benthic environment has persisted for at least 1 year after cessation of ocean sewage disposal at the 106-Mile Site.

J Bacteriol, 1996 May, 178(9), 2668 - 75
Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824; Gorwa MF et al.; A 2.8-kbp DNA region of Clostridium acetobutylicum ATCC 824 containing the putative hydrogenase gene (hydA) was cloned and sequenced . The 1,745-bp hydA encodes a 64,415-Da protein and presents strong identity with the {Fe} hydrogenase genes of Desulfovibrio and Clostridium species . The level of the putative hydA mRNA was high in cells from an acidogenic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate-limited continuous culture . These results were in agreement with the hydrogenase protein level, indicating that expression of hydA is regulated at the transcriptional level . Primer extension analysis identified a major transcriptional start site 90 bp upstream of the hydA start codon . The position of a putative rho-independent transcription terminator immediately downstream of the termination codon is in agreement with the size of the hydA transcript (1.9 kb) determined by Northern (RNA) blot experiments and confirms that the gene is transcribed as a monocistronic operon . Two truncated open reading frames (ORFs) were identified downstream and upstream of hydA and in opposite directions . The amino acid sequence deduced from ORF2 presents strong identity with ortho phosphoribosyl transferases involved in pyrimidine synthesis . The amino acid sequence deduced from ORF3 presents no significant similarity to any sequence in various available databases.

J Bacteriol, 1996 May, 178(9), 2551 - 8
Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions; Li T et al.; A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture . Clostridium ghonii was inhibited by chloramphenicol (10 micrograms/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 micrograms/ml), without the transformation of phenol being affected . Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C hastiforme strain (strain 2) (width, 1 micron) and an unidentified strain 6 (width, 0.6 micron) which was not detected on solid medium . Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6 . C hastiforme 2 was eliminated from this culture by dilution . The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone . Both of these activities are inducible . This strain is a gram- variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol . It has a cellular fatty acid composition like that of C . hastiforme . However, strain 6 does not hydrolyze gelatin or produce indole . The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86% . Tbe evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium-related species revealed that it does not emerge from any of these groups . Strain 6 most likely belongs to a new species closely related to Clostridium species.

J Bacteriol, 1996 May, 178(9), 2514 - 20
The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens; Ba-Thein W et al.; Extracellular toxin production in Clostridium perfringens is positively regulated by the two-component regulatory genes virR and virS . Northern (RNA) blots carried out with RNA preparations from the wild-type strain 13 and the isogenic virR and virS mutants TS133 and JIR4000 showed that the virR and virS genes composed an operon and were transcribed as a single 2.1-kb mRNA molecule . Primer extension analysis led to the identification of two promoters upstream of virR . Hybridization analysis of the mutants and their complemented derivatives showed that the virR/virS system positively regulated the production of alpha-toxin (or phospholipase C, theta-toxin (perfringolysin O), and kappa-toxin (collagenase) at the transcriptional level . However, the modes of regulation of these genes were shown to differ . The theta-toxin structural gene, pfoA, had both a major and a very minor promoter, with the major promoter being virR/virS dependent . The colA gene, which encodes the kappa-toxin, had two major promoters, only one of which was virR/virS-dependent . In contrast, the alpha-toxin structural gene, p1c, had only one promoter, which was shown to be partially regulated by the virR and virS genes . Comparative analysis of the virR/virS-dependent promoters did not reveal any common sequence motifs that could represent VirR-binding sites . It was concluded that either the virR/virS system modulates its effects via secondary regulatory genes that are specific for each toxin structural gene or the VirR protein does not have a single consensus binding sequence.

Infect Immun, 1996 May, 64(5), 1589 - 94
Molecular composition of Clostridium botulinum type A progenitor toxins; Inoue K et al.; The molecular composition of progenitor toxins produced by a Clostridium botulinum type A strain (A-NIH) was analyzed . The strain produced three types of progenitor toxins (19 S, 16 S, and 12 S) as reported previously . Purified 19 S and 16 S toxins demonstrated the same banding profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that they consist of the same protein components . The nontoxic components of the 19 S and 16 S toxins are a nontoxic non-hemagglutinin (HA) (molecular mass, 120 kDa) and HA . HA could be fractionated into five subcomponents with molecular masses of 52, 35, 20, 19, and 15 kDa in the presence of 2-mercaptoethanol . The molar ratios of neurotoxins, nontoxic non-HAs, and each HA subcomponent of the 19 S and 16 S toxins showed that only HA-35 of the 19 S toxin was approximately twice the size of that of the 16 S toxin, suggesting that the 19 S toxin is a dimer of the 16 S toxin cross-linked by the 35-kDa subcomponent . The nontoxic non-HA of the 12 S toxin, but not those of the 19 S and 16 S toxins, demonstrated two bands with molecular masses of 106 and 13 kDa on SDS-PAGE with or without 2-mercaptoethanol . It was concluded from the N-terminal amino acid sequences that 106- and 13-kDa proteins were generated by a cleavage of whole nontoxic non-HA . This may explain why the 12 S and 16 S (and 19 S) toxins exist in the same culture . We also found that the HA and its 35-kDa subcomponent exist in a free state in the culture fluid along with three types of progenitor toxins.

J Biol Chem, 1996 Apr 26, 271(17), 10217 - 24
Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation; Popoff MR et al.; Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins . On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers . We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo . LT glucosylates Ras, Rap, and Rac . In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT . Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT . Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo . We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology . LT is thus a powerful tool to inhibit Ras function in vivo.

J Biol Chem, 1996 Apr 26, 271(17), 10149 - 53
Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation; Just I et al.; The lethal toxin (LT) from Clostridium sordellii belongs to the family of large clostridial cytotoxins causing morphological alterations in cultured cell lines accompanied by destruction of the actin cytoskeleton . C . sordellii LT exhibits 90% homology to Clostridium difficile toxin B, which has been recently identified as a monoglucosyltransferase (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K . (1995) Nature 375, 500-503) . We report here that LT too is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases . LT selectively modifies Rac and Ras, whereas the substrate specificity of toxin B is confined to the Rho subfamily proteins Rho, Rac, and Cdc42, which participate in the regulation of the actin cytoskeleton . In Rac, both toxin B and LT share the same acceptor amino acid, threonine 35 . Glucosylation of Ras by LT results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway . LT is the first bacterial toxin to inactivate Ras in intact cells.

Neurosci Lett, 1996 Apr 19, 208(2), 105 - 8
Binding of botulinum type B neurotoxin to Chinese hamster ovary cells transfected with rat synaptotagmin II cDNA; Nishiki T et al.; We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin . In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied . In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium . Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin . However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a . In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis . These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.

J Mol Biol, 1996 Apr 19, 257(5), 1042 - 51
The crystal structure of a family 5 endoglucanase mutant in complexed and uncomplexed forms reveals an induced fit activation mechanism; Dominguez R et al.; The structures of the Glu140-->Gln mutant of the Clostridium thermocellum endoglucanase CelC in unliganded form (CelC(E140Q)) and in complex with cellohexaose (CelC(E140Q)-Gl(C6)) have been refined to crystallographic R-factors of 19.4% at 1.9 A and 17.8% at 2.3 A resolution, respectively . The structure of CelC(E140Q)-Gl(C6) complex shows two D-glucosyl residues bound to the non-reducing end of the substrate-binding cleft . Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138-141 which carries the general acid/base catalyst Glu140 in wild-type CelC . Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (beta/alpha)8-fold with an additional subdomain of 54 amino acids inserted between beta-strand 6 and alpha-helix 6 . Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases . Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues . Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond . An aromatic residue occurs at an equivalent position in many other (beta/alpha)8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 beta-glucosidases, family 2 beta-galactosidases, family 5 cellulases, family 17 beta-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (beta/alpha)8- architecture.

Gene, 1996 Apr 17, 170(1), 85 - 9
A Bacillus sphaericus gene encoding a novel type of mosquitocidal toxin of 31.8 kDa; Thanabalu T et al.; A size-fractionated genomic library of Bacillus sphaericus strain SSII-1 was constructed and screened for toxicity against larvae of the mosquito Culex quinquefasciatus (Cq) . One toxin-producing clone, pS35, was identified and a 2.7-kb subclone was completely sequenced . An open reading frame of 879 bp encoding a 31.8-kDa protein (designated Mtx2) was identified . Purified, recombinant Mtx2 was toxic to Cq larvae . Mtx2 shows no significant homology to known insecticidal toxins, but has homology to two toxins active against mammalian cells, namely the epsilon-toxin of Clostridium perfringens and the cytotoxin of Pseudomonas aeruginosa . Thus, Mtx2 represents a new type of mosquitocidal toxin.

Biochemistry, 1996 Apr 16, 35(15), 4906 - 10
Structural role of calcium for the organization of the cellulosome of Clostridium thermocellum; Choi SK et al.; The cellulosome of Clostridium thermocellum is a multipolypeptide complex of structural and catalytic subunits . Several of the catalytic subunits have at the carboxyl end a conserved duplicated region (CDR) which interacts with internally repeated elements (IREs) of scaffolding subunits such as CipA . This interaction requires calcium . The two parts of the CDR region here designated CDR1 and CDR2 (closest to the carboxyl end) each consist of about 20 amino acids residues . As shown in our previous paper {Choi, S.K., & Ljungdahl, L.G . (1996) Biochemistry 35, 4897-4905}, treatment of the cellulosome with ethylenediaminetetraacetic acid (EDTA) under aerobic conditions disintegrates the cellulosome with formation of truncated catalytic subunits . The cleavage is at a specific asparagine residue located within CDR1 and occurs with complete loss of CDR2 . Two branched peptides containing the amino acid sequences of CDR1 and CDR2 (designated bCDR1 and bCDR2) were synthesized, and specific antibodies were raised against them . These antibodies did not cross react with bCDR1 or bCDR2, respectively . After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, it was observed that about 15 subunits of the cellulosome reacted with anti-bCDR1 and anti-bCDR2 . In a similar experiment with EDTA-treated cellulosomes, these subunits reacted with anti-bCDR1 but not with anti-bCDR2, showing that they lost the bCDR2 epitope and were truncated . The peptide bCDR1 binds calcium, whereas bCDR2 does not . Furthermore, bCDR1 but not bCDR2 binds to CipA, presumably at IRE regions . This binding requires calcium . A model is proposed for the binding of the catalytic subunits to CipA which involves CDR1, an IRE, and calcium.

Biochemistry, 1996 Apr 16, 35(15), 4897 - 905
Dissociation of the cellulosome of Clostridium thermocellum in the presence of ethylenediaminetetraacetic acid occurs with the formation of trucated polypeptides; Choi SK et al.; The cellulosome of Clostridium thermocellum JW20 consists of 14-26 different polypeptides as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The intact cellulosomes hydrolyzes crystalline cellulose in the presence of Ca2+ and thiols . This activity is inhibited by ethylenediaminetetraacetic acid (EDTA) . Ca is incorporated into the cellulosome and tightly bound as demonstrated using 45Ca added to the growth medium . Upon incubation in 50 mM Tris (pH 7.5), 0.1 M NaCl, and 5 mM EDTA at 37 degrees C, Ca is released from the cellulosome, which disintegrates into polypeptides . The SDS-PAGE pattern of cellulosomal polypeptides is remarkably different after the EDTA treatment when compared to this pattern of untreated cellulosomes . Polypeptide bands corresponding to molecular masses of 160, 98, 76, 91, and 54 kDa disappear, and new bands of masses 150, 132, 91, 71, 57, and 46 kDa appear . N-terminal analyses of the 98, 76, 91, and 71 kDa polypeptides show that the 91 and 71 kDa polypeptides are truncated products of the 98 and 76 kDa polypeptides, respectively . The 76 and 71 kDa polypeptides correspond to CelS {Wang, W . K., Kruus K., & Wu, J . H . D . (1993) J . Bacteriol . 175, 1293-1302} . The 71 kDa polypeptide is formed from the 76 kDa polypeptide during the EDTA treatment, by a cleavage that occurs at asparagine residue 681 . It involves the removal of 60 amino acid residues from the C-terminal end . All catalytic subunits so far characterized contain an asparagine residue corresponding to residue 681 of CelS . This residue is part of the conserved duplicated region found in catalytically active subunits, and it is postulated that several of these subunits also are truncated by the EDTA treatment . The polypeptides truncated by the EDTA treatment reduced Ca binding capacities compared to their native subunits, indicating a Ca-binding site within the conserved duplicated region . This region has been implicated in the binding of the catalytic peptides to the scaffolding polypeptide CipA, which is a structural protein of the cellulosome and has a strong affinity for calcium.

Biochim Biophys Acta, 1996 Apr 3, 1280(1), 120 - 6
Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes; Nagahama M et al.; The effect of Clostridium perfringens alpha-toxin on multilamellar liposomes prepared from various phospholipids and cholesterol was investigated . The toxin induced carboxyfluorescein leakage from liposomes composed of the choline-containing phospholipids such as egg-yolk phosphatidylcholine and bovine brain sphingomyelin in dose-dependent manner, but did not induce leakage from those liposomes composed of bovine brain phosphatidylethanolamine, egg-yolk phosphatidylserine or phosphatidylglycerol . The toxin-induced carboxyfluorescein leakage from egg-yolk phosphatidylcholine liposomes was increased by addition of divalent cations . The toxin induced carboxyfluorescein release from liposomes composed of phosphatidylcholine containing unsaturated fatty acyl residues or shorter chain length saturated fatty acyl residues (12 or 14 carbon atoms), but did not induce such release from liposomes composed of phosphatidylcholine containing saturated fatty acyl residues of between 16 and 20 carbon atoms . Furthermore, the toxin-induced carboxyfluorescein release decreased with increasing chain length of acyl residues of phosphatidylcholine used . The toxin bound to liposomes composed of phospholipids which are hydrolyzed by the toxin, but did not bind to those composed of phospholipids which are not attacked by the toxin . The toxin-induced carboxyfluorescein release from liposomes composed of dipalmitoleoyl-L-alpha-phosphatidylcholine and cholesterol and the toxin binding to the liposomes decreased with decreasing cholesterol contents . These observations suggest that the specific binding site formed by the choline-containing phospholipids and cholesterol, and membrane fluidity in liposomes are essential for the membrane-damaging activity of alpha-toxin.

Pediatr Pol, 1996 Apr, 71(4), 373 - 6
{Acute clostridium difficile gastroenteritis infection in children: report of three cases}; Prokopowicz D et al.; The causes and clinical manifestations of Clostridium difficile infection in children are described in this report . The studies were performed on three children aged up to 3 years . Risk factors as well as possible diagnostic and therapeutic procedures are discussed.

Pol Tyg Lek, 1996 Apr, 51(14-18), 212 - 4
{Current diagnosis, clinical course and treatment of acute colitis infection with Clostridium difficile}; Prokopowicz D et al.; Detection rate of G . intestionalis in feces with direct microscopy has been compared with the immuno-enzymatic technique detecting protein GSA 65 with Alexon Inc., ProSpec T/Giardia reagents kit . The results obtained with both methods have further been compared with those obtained by microscopic examination of the duodenal content . Detectability of Giardia intestinalis with EIA technique with the use of ready-made kit has been assessed . Feces have been collected from 371 patients . Protein GSA 65 has been present in 170 samples, 45.8%, examined with the use of ProSpec T/Gardia kit . Giardia intestinalis cysts have been detected microscopically in the feces of 37 patients, i.e . in 22.3% . Microscopic examinations carried out by three independent examiners have shown marked diversity in the rate of positive results, being 0.1% (examiner A), 28.6% (B), and 45.2% (C) . Comparison of G . intestinale detectability of all 3 techniques used have shown absence of protein GSA 65 in 2 out of 9 examined patients . However, trophozoits have been present in the duodenal content . Test performed with kits made by Alexon Inc . and DIALAB have given 45.8% and 60.7% of the positive results, respectively.

Nippon Ika Daigaku Zasshi, 1996 Apr, 63(2), 106 - 16
{Effect of green tea polyphenol fraction on 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis in the rat}; Kan H et al.; We studied the anti-tumor effect of green tea polyphenol fraction (Sunphenon, SF: provided by Taiyo Kagaku Inc., Mie, Japan) on DMH-induced colorectal carcinogenesis in male Wistar rats . DMH was subcutaneously administered weekly at 20 mg/kg for 14 weeks . The rats in group I (20 rats) were given tap water for the whole of the study period . The rats in group II (15 rats) were given tap water from weeks 0-14, and 0.1% SF from weeks 15-35 . The rats in group III (21 rats) were given 0.1% SF during the whole period . The rats were sacrificed at week 35 . The cecal contents were aseptically removed and examined microbiologically to obtain the counts of four bacteria species (including Clostridium perfringens) per 1 g of cecal contents . The incidence of tumors production was significantly decreased (Group I: 100% vs Group II: 57.1%, Group III: 62.5%, p < 0.05), and the frequency of occurrence of C . perfringens (which is thought to yield harmful products which may be carcinogenic) was decreased in the SF-treated groups . These results suggest that SF prevents DMH-induced carcinogenesis in rats, and that its effect may be somehow related to its ability to preserve the composition of the colonic microflora.

Microbiology, 1996 Apr, 142 ( Pt 4), 1013 - 23
Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli; Reverbel-Leroy C et al.; The CelF-encoding sequence was isolated from Clostridium cellulolyticum genomic DNA using the inverse PCR technique . The gene lies between cipC (the gene encoding the cellulosome scaffolding protein) and celC (coding for the endoglucanase C) in the large cel cluster of this mesophilic cellulolytic Clostridium species . Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other beta-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases) . The protein was overproduced in Escherichia coli using the T7 expression system . It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 degrees C . Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the Ion protease-deficient strain BL21(DE3) . The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable . It was collected using the osmotic shock method . The enzymic degradation of various cellulosic substrates by CelFp was studied . CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan . Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.

Infect Control Hosp Epidemiol, 1996 Apr, 17(4), 232 - 5
Risk factors associated with Clostridium difficile diarrhea in hospitalized adult patients: a case-control study--sucralfate ingestion is not a negative risk factor; Watanakunakorn PW et al.; OBJECTIVES: To assess risk factors associated with Clostridium difficile diarrhea in hospitalized adult patients, and to test the hypothesis that sucralfate ingestion is associated with nondetection of C difficile cytotoxin in stool specimens . DESIGN: A retrospective case-control study of hospitalized adult patients who had stool specimens assayed for C difficile cytotoxin . For each patient who had positive C difficile cytotoxin, a patient who had negative C difficile cytotoxin was used as a control . The study period was January to December 1993 . SETTING: A community teaching hospital affiliated with a medical school in northeastern Ohio . RESULTS: There were 91 case patients and 91 control patients . Cephalosporin exposure was identified as a risk factor in patients with C difficile diarrhea . The number of patients who had sucralfate ingestion was comparable in both groups of patients . CONCLUSIONS: Administration of cephalosporins was identified as a risk factor in patients with C difficile diarrhea . We were not able to confirm a recent report of the association between ingestion of sucralfate and nondetection of C difficile cytotoxin in stool specimens . Our findings suggest that sucralfate ingestion is not associated with nondetection of C difficile cytotoxin in stool specimens . Assay of C difficile cytotoxin in stool specimens remains a valid method of diagnosing C difficile diarrhea, even in patients who ingest sucralfate.

Lett Appl Microbiol, 1996 Apr, 22(4), 271 - 4
Influence of bile salts on beta-glucuronidase activity of intestinal bacteria; Fujisawa T et al.; The beta-glucuronidase activity of intact cells of Escherichia coli and Clostridium perfringens was increased in the presence of bile salts . In contrast, bile salts had inhibitory effects on the activity of beta-glucuronidase extracted from the lysed cells . These results suggest that the permeability of the bacterial cells is increased by the presence of bile salts, and that bile salts may significantly enhance bacterial beta-glucuronidase activity in the intestinal tract.

Appl Environ Microbiol, 1996 Apr, 62(4), 1441 - 3
Sporulation-promoting ability of Clostridium perfringens culture fluids; Shih NJ et al.; The culture supernatant fluids (CSFs) of 12 strains of Clostridium perfringens types A, B, C, and D stimulated sporulation of test strains NCTC 8238 and NCTC 8449 of this organism . The sporulation-promoting ability was present in vegetative and sporulating CSFs of both enterotoxin-positive (Ent+) and Ent- strains . The sporulation factor possessed a molecular weight between 1,000 and 5,000 and was heat and acid stable . This study suggests a potential role for Ent- strains in food-borne disease outbreaks caused by Ent+ strains of C . perfringens type A.

Biosci Biotechnol Biochem, 1996 Apr, 60(4), 726 - 8
An inhibitor of S-hemolysin, SHI, produced by streptomyces sp . strain No . 6288; Suzuki K et al.; Streptomyces sp . strain No . 6288 produces S-Hemolysin, which is a unique phospholipase C with a high substrate specificity for sphingomyelin . Moreover, the strain also produced two kinds of phospholipase inhibitors, designated as SHI and S-PLI, in different phases of cultivation . The purified SHI was shown to be a basic substance containing an amino group and glycoside moiety, and it was a more effective inhibitor of S-Hemolysin and sphingomyelinase from Streptomyces sp . with a higher substrate specificity for sphingomyelin than alpha-toxin from Clostridium perfringens.

J Clin Microbiol, 1996 Apr, 34(4), 882 - 5
PCR for detection of Clostridium botulinum type C in avian and environmental samples; Franciosa G et al.; A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay . PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment . The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification . Eleven isolates of C . botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak.

Aust N Z J Public Health, 1996 Apr, 20(2), 119 - 22
Clostridium perfringens food-borne outbreak: an epidemiological investigation; Hook D et al.; On 3 April 1994, the Western Sector Public Health Unit was notified of an outbreak of gastroenteritis at a Christian youth camp . Attending the camp were 820 people; 241 (42 per cent) of 574 camp participants who completed a questionnaire reported illness . Of these, 230 met the case definition . Main symptoms reported were stomach cramps (78 per cent), diarrhoea (67 per cent) and nausea (46 per cent) . Bacterial analysis of leftover chicken grew 2.3 x 10(7) and 3.3 x 10(7) colonies/g of Clostridium perfringens, and we identified Type A enterotoxin of C . perfringens in four of seven stool samples collected from ill people . Camp participants who consumed chicken at lunch on the second day of the camp were nearly four times as likely to be ill than those who did not (relative risk 3.81, 95 per cent confidence interval 3.07 to 4.72) . There were deficiencies in hygiene and food preparation . We highlight the importance of time and temperature controls in food preparation and storage to prevent contamination and subsequent poisoning by C . perfringens or other food pathogens . This investigation demonstrates the importance of a multidisciplinary team when investigating disease outbreaks.

Int J Food Microbiol, 1996 Apr, 29(2-3), 371 - 8
Association of psychrotrophic Clostridium spp . with deep tissue spoilage of chilled vacuum-packed lamb; Broda DM et al.; Early spoilage of commercial vacuum-packed chilled lamb legs was manifested as an objectionable 'cheesy', deep tissue odour that became evident when a cut was made into the stifle joint . Investigation of the probable causative agents led to the isolation of two psychrotrophic strains of clostridia . The isolates could not be identified using traditional identification schemes . One isolate was able to produce strong, objectionable 'cheesy' odours in deep tissues of artificially inoculated beef.

Int J Food Microbiol, 1996 Apr, 29(2-3), 335 - 52
Psychrotrophic Clostridium spp . associated with 'blown pack' spoilage of chilled vacuum-packed red meats and dog rolls in gas-impermeable plastic casings; Broda DM et al.; 'Blown pack' spoilage of vacuum-packed chilled beef, lamb and venison, and of a cooked meat product, chilled dog rolls packed in an oxygen-impermeable plastic casing, was characterised by sensory, chemical and microbiological analysis . Investigation of the probable causative agents led to the isolation of eight strains of psychrotrophic clostridia . Three strains have been provisionally identified as C . difficile, C . beijerinckii and C . lituseburense; the other five remain unidentified . In inoculation studies only one isolate produced significant amount of gas on meat, causing pack 'blowing' . It is, therefore, possible that 'blown pack' spoilage involves a synergism with one or more other organisms.

Int J Food Microbiol, 1996 Apr, 29(2-3), 255 - 70
Production and characterization of enterocin 900, a bacteriocin produced by Enterococcus faecium BFE 900 from black olives; Franz CM et al.; Enterococcus faecium BFE 900 isolated from black olives produced a bacteriocin termed enterocin 900, which was antagonistic towards Lactobacillus sake, Clostridium butyricum, enterococci as well as Listeria spp . including Listeria monocytogenes . Enterocin 900 was inactivated by pepsin, alpha-chymotrypsin, proteinase K and trypsin but not by catalase, alpha-amylase, or other non-proteolytic enzymes tested . The bacteriocin was heat stable, retaining activity after heating at 121 degrees C for 15 min . Enterocin 900 was active at pH values ranging from 2.0-10.0, with highest activity at pH 6.0 . Bacteriocin production occurred in the late logarithmic growth phase when culture density was ca . log 8.0 CFU ml-1 . Enterocin 900 was produced in media with initial pH ranging from 6.0-10.0, but not in media with a pH lower than 6.0 . Medium composition, especially the concentrations of peptone and yeast extract influenced bacteriocin production, with no bacteriocin being produced in the absence of either of these compounds . No plasmids could be isolated from Enterococcus faecium BFE 900, indicating that the gene for bacteriocin activity is located on the chromosome.

Int J Food Microbiol, 1996 Apr, 29(2-3), 241 - 54
Evaluation of citric acid and GDL in the recovery at different pH levels of Clostridium sporogenes PA 3679 spores subjected to HTST treatment conditions; Silla Santos MH et al.; Spores of Clostridium sporogenes PA 3679 were treated at different temperatures (121, 126, 130 and 135 degrees C) in white asparagus puree (pH 5.8) and acidified with glucono-delta-lactone (GDL) and citric acid to pH levels of 5.5, 5.0 and 4.5 . Afterwards, the spores were recovered in MPA3679 medium in various conditions: unacidified (pH 7.5), acidified with GDL (500 ppm) and acidified with citric acid (500 and 250 ppm) to pH levels of 6.5, 6.0 and 5.0 . The results indicated that the pH levels, concentration and type of acid used act synergistically rather than independently . Citric acid has a stronger inhibiting effect than GDL on the recovery of C . sporogenes PA 3679 spores . At the higher heat treatments (130 and 135 degrees C) the major injury on the spores sensitize more than against the acids and low pH values.

Int J Food Microbiol, 1996 Apr, 29(2-3), 141 - 8
An epidemiological study of food poisoning in Korea and Japan; Lee WC et al.; The purpose of this study was to make a comparative epidemiological observation on outbreaks of food poisoning between Korea and Japan, during the period from 1971 to 1990 . The mean morbidity rate by fiscal year from 1971 to 1990 was 3.0 per 100,000 population in Korea, and 29.2 in Japan . The mean mortality rate, in Korea was 2.48%, and in Japan, 0.07% . When both morbidity and mortality rates in Korea and Japan were compared during same period, the morbidity rate in Japan was much higher than that in Korea . However, the mortality rate of patients in Korea was much higher than that in Japan . Comparison of food poisoning rates according to food preparation facilities in Korea and Japan was also performed; outbreaks in Korea most frequently involved home-made foods (48% of the total cases), while in Japan, restaurants accorded for 32.7% . Foods most commonly incriminated in Korea were seafood, meat and animal products, grains and vegetables, including mushrooms . In Japan, however, unknown causes, followed by seafood, vegetables, and meat and animal products were the most common . Food poisoning of bacterial origin in Korea was 58.6% of the total case, including Vibrio spp . (37.6%), Salmonella spp . (23.1%), Staphylococcus spp . (14.9%), pathogenic E . coli (6.8%) Clostridium spp . (0.5%) and other species (17.1%) . In Japan, the majority of bacterial causes were Vibrio spp . (47.3%), Staphylococcus spp . (24.6%), Salmonella spp . (14.8%), E . coli (3.5%), Clostridium spp . (0.2%) and other species (9.6%) . In conclusion, the outbreaks of food poisoning in Korea and Japan may be mainly caused by food handling, and their occurrence may be different according to food sources.

Eur J Clin Microbiol Infect Dis, 1996 Apr, 15(4), 330 - 6
Role of culture and toxin detection in laboratory testing for diagnosis of Clostridium difficile-associated diarrhea; Peterson LR et al.; Two variations of an egg yolk agar base medium containing cycloserine, cefoxitin, and fructose (CCFA), one with 250 micrograms and other with 500 micrograms of cycloserine/ml of agar medium were compared to study the effect of the cycloserine concentration on recovery of Clostridium difficile from stool samples . In addition, the role of prior anaerobic reduction of these media in the detection of Clostridium difficile-associated diarrhea (CDAD) was tested . Each medium was studied over a two-month period, with outcome compared between the testing periods and to historical data from our institution . Clinical correlation of test results was performed . The use of the originally described formulation of CCFA with 500 microgram of cycloserine/ml of agar combined with 4 h of anaerobic reduction prior to specimen inoculation increased the rate of isolation of toxigenic Clostridium difficile from clinical specimens from 6 to 17% (p < 0.001) . Combining direct detection of stool toxin and properly performed culture for toxigenic Clostridium difficile enhances the potential for diagnosis of CDAD . For optimal performance the culture medium should contain the originally proposed cycloserine concentration of 500 microgram/ml of agar and should be anaerobically reduced at least 4 h prior to specimen inoculation.

FEMS Immunol Med Microbiol, 1996 Apr, 13(4), 279 - 85
Investigation of animal botulism outbreaks by PCR and standard methods; Fach P et al.; A double PCR procedure is proposed for identification of Clostridium botulinum C and D . This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment) . This method was found to be specific for C . botulinum C and D, amongst 81 strains of C . botulinum and 21 different species of other Clostridium and bacteria tested . The detection limit ranged from 10 to 10(3) bacteria in the reaction volume according to the C . botulinum C and D strains . In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay . A clear distinction between botulism type C and D was obtained . The double PCR provides a reliable alternative for detection and identification of C . botulinum C and D in clinical and food samples.

Bioorg Med Chem, 1996 Apr, 4(4), 521 - 2
New drugs--reports of new drugs recently approved by the FDA . Dirithromycin; Shinkai I et al.; Dirithromycin is a semisynthetic derivative of erythromycin, a 14-membered ring macrolide antibiotic . The drug is converted during absorption and distribution, to an active metabolite 9-(S)-erythromycylamine, which is the predominant compound found in plasma and extravascular tissues . High tissue concentration of erythromycylamine is achieved after oral doses of dirithromycin, with slow release back into the circulation . The mechanism of action of dirithromycin is like that of erythromycin and other macrolides . These compounds inhibit RNA-dependent protein synthesis . It has recently been suggested that all macrolides stimulate dissociation of peptidyl-tRNA from ribosomes during the elongation phase, leading to inhibited protein synthesis . The antimicrobial spectrum of dirithromycin is similar to that of erythromycin, although the drug offers no significant advantage with regard to MIC values . In vitro against Gram-positive isolates, dirithromycin exhibits similar potency to that of clarithromycin, erythromycin, roxithromycin, and clindamycin . In vivo, dirithromycin is active against penicillin-susceptible Staphylococcus aureus, beta-hemolytic streptococci, and Streptococcus pneumoniae . Dirithromycin is as effective as penicillin VK against streptococcal pharyngitis and tonsilitis, and as effective as erythromycin against acute superimposed chronic bronchitis and skin and soft-tissue infections . In comparison with other newer macrolides, dirithromycin has shown similar or lesser in vitro activity . In particular, Haemophilus influenzae, Bacteroides spp., Peptococcus-Peptostreprococcus spp., Clostridium perfringens, Legionella spp., Neisseria gonorrhoeae, and Chlamydia trachomatis were all less sensitive to dirithromycin than azithromycin or clarithromycin . Once-daily oral administration of dirithromycin (500 mg) has been demonstrated to be similar in efficacy to erythromycin (250 mg, 4 times daily), each for approximately 7 days, in the treatment of acute bronchitis or acute-exacerbations of chronic bronchitis in controlled studies . Proven or presumed pathogen eradication rates were 83 and 86% for acute bronchitis patients treated with dirithromycin and erythromycin, respectively . Corresponding bacteriological response rates in acute exacerbations of chronic bronchitis were 75 to 84% with dirithromycin and 75 to 82% with erythromycin . Both agents achieved clinical cure or improvement in over 85% of the patients with either condition . The main advantage of dirithromycin over erythromycin appears to be once-daily administration . Lilly launched dirithromycin in September 1993, in Spain, received approval from FDA in August 1995, and launched it during October 1995.

Aliment Pharmacol Ther, 1996 Apr, 10(2), 157 - 63
Sulphasalazine treatment and the colorectal mucosa-associated flora in ulcerative colitis; Hartley MG et al.; AIM: To study the influence of sulphasalazine treatment on the mucosa-associated bacterial flora of rectal biopsy tissue specimens in patients with ulcerative colitis . PATIENTS: Twenty-four patients had newly diagnosed active ulcerative colitis; 20 patients had acute relapse of ulcerative colitis (10 not taking maintenance sulphasalazine); (40 patients had quiescent ulcerative colitis; 21 not taking maintenance sulphasalazine) . The influence of 3 weeks of sulphasalazine treatment on the mucosa-associated flora was studied in the patients presenting with active disease . RESULTS: Comparison of patients according to sulphasalazine usage revealed few differences in the mucosal flora . In patients with quiescent ulcerative colitis, Escherichia coli was found at lower counts in patients taking maintenance sulphasalazine; however, this effect was not evident in patients with active disease . Inconsistent changes in other facultatives were seen between the two active disease groups, particularly for a miscellaneous group of unidentified Gram-positive rods . Three patients, all receiving sulphasalazine, were colonized with Clostridium difficile, but this did not appear to influence their disease . CONCLUSION: Sulphasalazine treatment in ulcerative colitis causes only minor disturbance to the populations of bacteria colonizing the colorectal mucosa.

J Clin Gastroenterol, 1996 Apr, 22(3), 186 - 9
Whole-bowel irrigation as an adjunct to the treatment of chronic, relapsing Clostridium difficile colitis; Liacouras CA et al.; We report the successful treatment of two patients with chronic, intractable Clostridium difficile infection using whole-bowel irrigation with a polyethylene glycol solution (Golytely) as adjunctive therapy . Before this treatment, both patients had recurrent symptoms of diarrhea, weight loss, abdominal pain, and documented C . difficile toxin-positive stools despite multiple pharmacologic treatments . Each child was prescribed myriad drug therapies, including vancomycin, metronidazole, bacitracin, and rifampin . Cholestyramine and lactobacillus were also tried alone and in combination with antibiotics . In each case, symptoms recurred shortly after cessation of therapy . Whole-bowel irrigation was subsequently administered until profuse, clear liquid stools were produced . This treatment was followed by a 3-week course of oral vancomycin and lactobacillus . In both cases, the patient became asymptomatic within 3 days of therapy; they have remained symptom-free for 36 and 48 months, respectively . We suggest that whole-bowel irrigation clears active C . difficile organisms, toxins, and spores from the intestine and is effective as an adjunct to routine therapy for chronic, relapsing C . difficile infections.

Am J Vet Res, 1996 Apr, 57(4), 496 - 501
Hybridization of 2,659 Clostridium perfringens isolates with gene probes for seven toxins (alpha, beta, epsilon, iota, theta, mu, and enterotoxin) and for sialidase; Daube G et al.; OBJECTIVE--To genetically characterize Clostridium perfringens isolates for association of pathologic type with various diseases . DESIGN--Prospective study . SAMPLE POPULATION--2,659 C perfringens isolates from various nonhuman animals species, human beings, and foods . PROCEDURE--Colony hybridization with DNA probes for 7 toxin (alpha, beta, epsilon, iota (subunits a and b), theta, mu, and enterotoxin) genes and 1 sialidase gene were performed to group the isolates by pathologic type . RESULTS--Enterotoxin-negative type-A isolates were the most common (2,575/2,659), were isolated from all sources, and were separated into 5 pathologic types . In cattle and horses with enterotoxemia, essentially only these pathologic types were identified . The enterotoxin-negative isolates of types C or D each had a single pathologic type . Type-C isolates were isolated only from swine with necrotic enteritis and type-D isolates from small ruminants with enterotoxemia, except that 1 type-D isolate was also found from a healthy fish . Type-B or -E isolates were not found . Among the 47 enterotoxin-positive isolates, 5 isolates from sheep or deer were type D and the other 42 were type A . These 42 isolates were grouped into 3 pathologic types: 1 type was isolated from samples of almost all origins, but the other 2 types were found in only 5 fish, 4 human beings, and 1 dog . CONCLUSIONS AND CLINICAL RELEVANCE--Genetic characterization of these isolates allowed identification of 11 different pathologic types . This approach may be useful in molecular diagnosis and prophylaxis of clostridial disease.

Zentralbl Veterinarmed B, 1996 Apr, 43(2), 119 - 27
Seasonal variations in the isolation of Salmonella typhimurium, Salmonella enteritidis, Bacillus cereus and Clostridium perfringens from environmental samples; Davies RH et al.; Calf carcasses contaminated with S . typhimurium, B . cereus and Cl . Perfringens were placed in either a decomposition pit or in a deep burial pit . Salmonella was isolated from the soil around the decomposition pit for 27 weeks and for 15 weeks around the burial site . Salmonella re-appeared in soil samples during cold winter weather after an apparent 9-week absence from the decomposition pit and after 68 weeks in the burial site (a total of 88 weeks after the start of the experiment) . There was also an annual rise in the isolation rate of B . cereus from the soil during the colder winter months, but Cl . perfringens appeared to be more prevalent in samples taken during spring of the second year of the study . A similar apparent rise in the prevalence of S . enteritidis during a cold winter period occurred in an empty poultry house that had previously held a naturally infected broiler-breeder flock.

J Bacteriol, 1996 Apr, 178(8), 2440 - 4
Molecular characterization of the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum; Weidner G et al.; Formate is the major source of C1 units in many species of the genus Clostridium . In this study we have cloned and characterized the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum . The genetic and transcriptional organizations of the genes and the high level of homology exhibited by the respective gene products to their Escherichia coli counterparts indicate strong evolutionary conservation of these enzymes.

J Bacteriol, 1996 Apr, 178(8), 2279 - 86
Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome; Pages S et al.; The 5' end of the cipC gene, coding for the N-terminal part of CipC, the scaffolding protein of Clostridium cellulolyticum ATCC 35319, was cloned and sequenced . It encodes a 586-amino-acid peptide, including several domains: a cellulose-binding domain, a hydrophilic domain, and two hydrophobic domains (cohesin domains) . Sequence alignments showed that the N terminus of CipC and CbpA of C . cellulovorans ATCC 35296 have the same organization . The mini-CipC polypeptide, containing a cellulose-binding domain, hydrophilic domain 1, and cohesin domain 1, was overexpressed in Escherichia coli and purified . The interaction between endoglucanase CelA, with (CelA2) and without (CelA3) the characteristic clostridial C-terminal domain called the duplicated-segment or dockerin domain, and the mini-CipC polypeptide was monitored by two different methods: the interaction Western blotting (immunoblotting) method and binding assays with biotin-labeled protein . Among the various forms of CelA (CelA2, CelA3, and an intermediary form containing only part of the duplicated segment), only CelA2 was found to interact with cohesin domain 1 of CipC . The apparent equilibrium dissociation constant of the CelA2-mini-CipC complex was 7 x 10(-9)M, which indicates that there exists a high affinity between these two proteins.

J Vasc Surg, 1996 Apr, 23(4), 714 - 8
Primary Clostridium septicum aortitis: a rare cause of necrotizing suprarenal aortic infection . A case report and review of the literature; Sailors DM et al.; A 74-year old woman sought medical attention for general symptoms of nausea, vomiting, and back pain . A computed tomographic scan showed gas in the wall of the descending thoracic and suprarenal aortas . Emergency thoracoabdominal exploration revealed a necrotizing infection of the thoracic aorta extending to the origin of the celiac axis . After surgery Clostridium septicum was identified in tissue culture . Surgical management consisted of in-situ graft replacement of the thoracoabdominal aorta . Three months later, a pseudoaneurysm developed at the distal anastomosis . The patient refused further surgery and died 3 days later . The cause of death was presumed to be a ruptured mycotic aneurysm as a result of recurrent C . septicum infection . The relationship of C . septicum with occult gastrointestinal and hematologic malignancy has been documented . This patient represents the 10th reported case of C . septicum arteritis . Including the nine previous case reports of C . septicum arteritis, the mortality rate is 70% . When evaluating a patient with a mycotic aneurysm or aortitis, C . septicum should be considered . If it is found, a search should be carried out for an associated gastrointestinal or hematologic malignancy . Surgical repair should include extraanatomic revascularization and wide debridement of the infected field . Consideration should be given to lifelong antimicrobial therapy for this potentially fatal infection.

J Bacteriol, 1996 Apr, 178(7), 1990 - 5
Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes; Newton GL et al.; Mycothiol {2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl- (1-->1)-myo-inositol} (MSH) has recently been identified as a major thiol in a number of actinomycetes (S . Sakuda, Z.-Y . Zhou, and Y . Yamada, Biosci . Biotech . Biochem . 58:1347-1348, 1994; H . S . C . Spies and D . J . Steenkamp, Eur . J . Biochem . 224:203-213, 1994; and G . L . Newton, C . A . Bewley, T . J . Dwyer, R . Horn, Y . Aharonowitz, G . Cohen, J . Davies, D . J . Faulkner, and R . C . Fahey, Eur . J . Biochem . 230:821-825, 1995) . Since this novel thiol is more resistant than glutathione to heavy-metal ion-catalyzed oxidation, it seems likely to be the antioxidant thiol used by aerobic gram-positive bacteria that do not produce glutathione (GSH) . In the present study we sought to define the spectrum of organisms that produce MSH . GSH was absent in all actinomycetes and some of the other gram-positive bacteria studied . Surprisingly, the streptococci and enterococci contained GSH, and some strains appeared to synthesize it rather than import it from the growth medium . MSH was found at significant levels in most actinomycetes examined . Among the actinobacteria four Micrococcus species produced MSH, but MSH was not found in representatives of the Arthrobacter, Agromyces, or Actinomyces genera . Of the nocardioforms examined, Nocardia, Rhodococcus, and Mycobacteria spp . all produced MSH . In addition to the established production of MSH by streptomycetes, we found that Micromonospora, Actinomadura, and Nocardiopsis spp . also synthesized MSH . Mycothiol production was not detected in Propionibacterium acnes or in representative species of the Listeria, Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Clostridium genera . Examination of representatives of the cyanobacteria, purple bacteria, and spirochetes also gave negative results, as did tests of rat liver, bonito, Candida albicans, Neurospora crassa, and spinach leaves . The results, which indicate that MSH production is restricted to the actinomycetes, could have significant implications for the detection and treatment of infections with actinomycetes, especially those caused by mycobacteria.

Postgrad Med, 1996 Apr, 99(4), 217 - 20, 224
Gas gangrene: potential for hyperbaric oxygen therapy; Stephens MB; Clostridial myonecrosis (gas gangrene) is an uncommon sequela of traumatic injury . Infection with Clostridium perfringens in devitalized tissue is the most common cause . Wide surgical debridement and appropriate antibiotic therapy remain the standard of care . However, the addition of hyperbaric oxygen therapy to standard management has been shown to have a synergistic effect in reducing morbidity and mortality in both canine and murine models . Although no prospective human data are available, retrospective data indicate that concomitant hyperbaric oxygen therapy has resulted in a twofold reduction in mortality . Where feasible, hyperbaric oxygen therapy should routinely be incorporated into the treatment plan for gas gangrene . Primary care physicians are in a unique position not only to make an early diagnosis but also to have a central role in coordinating multidisciplinary care often needed for this potentially fatal infection.

Am Surg, 1996 Apr, 62(4), 326 - 7
Clostridium perfringens sepsis with intravascular hemolysis following laparoscopic cholecystectomy: a newly reported complication; Bush GW et al.; Clostridium perfringens sepsis with hemolysis following cholecystectomy is a rare complication that has a very high mortality . The best chance for survival is ensured by early diagnosis, prompt initiation of antibiotics, and hyperbaric oxygen therapy if readily available . To our knowledge, this is the first reported case following laparoscopic cholecystectomy.

Schweiz Med Wochenschr, 1996 Mar 30, 126(13), 528 - 34
{Side effects and consequences of frequently used antibiotics in clinical practice}; Cerny A; Oral antimicrobial substances belonging to the beta-lactams, quinolones, macrolides, tetracyclines and the trimethoprim-sulfamethoxazole combination are among the most prescribed classes of drugs in private practice . Knowledge of the potential side effects considered in the light of various patient-associated factors such as genetic makeup, renal and liver function, underlying diseases, drug allergies and coadministered drugs, is important in order to minimize the risk of adverse reactions . This article reviews important side effect patterns and focuses on more recent aspects of antibiotic-associated diarrhea and beta-lactam allergy relevant to the practicing physician . Diarrhea occurring during antibiotic treatment raises the possibility of Clostridium-difficile-associated disease, which may evolve into life-threatening toxic megacolon . Mild cases with resolving symptoms after discontinuation of the antibiotic usually do not require further workup . More severe cases with watery diarrhea, abdominal pain, dehydration and electrolyte abnormalities warrant rapid diagnosis, cessation of antibiotic treatment and specific treatment including oral metronidazole . The use of oral vancomycin as a first line drug is discouraged because of the possibility of selecting vancomycin-resistant enterococci . Hypersensitivity reactions to beta-lactams are the most important type of side effects which can often be prevented . Patients with a history of beta-lactam associated IgE-mediated hypersensitivity (hives, wheezing or hypotension) should undergo penicillin skin testing . The frequently observed maculopapular rash associated with aminopenicillins without hives is in most cases not caused by an IgE-mediated mechanism . Patients with previous life-threatening penicillin allergy such as anaphylaxis or Lyell's syndrome should not undergo skin testing . Currently available tests do not reliably predict cephalosporin hypersensitivity . More recent data suggest that crossreactivity between penicillins and cephalosporins is infrequent . It thus seems safe to administer a cephalosporin to a penicillin-allergic patient, though excluding patients with previous life-threatening penicillin reactions.

Commun Dis Rep CDR Rev, 1996 Mar 29, 6(4), R57 - 63
General outbreaks of infectious intestinal disease in England and Wales 1992 to 1994; Djuretic T et al.; Data from the surveillance scheme of general outbreaks of infectious intestinal disease in England and Wales, reported to the PHLS Communicable Disease Surveillance Centre (CDSC), were used to review 1280 of the 1594 outbreaks identified between 1 January 1992 and 31 December 1994 for which a minimum data set was captured . The number of outbreaks reported in each regional health authority ranged from 31 in Mersey to 221 in Yorkshire . The commonest pathogens reported were salmonellas in 32% (412) of outbreaks, small round structured virus (SRSV) in 27% (342), Clostridium perfringens in 7% (90), and Shigella sonnei in 4% (46) . The main mode of transmission was described as foodborne in 50% (642), over half of which were caused by salmonellas, and person to person in 39% (496), over half of which were caused by SRSV . Most outbreaks transmitted from person to person occurred in hospitals and in residential institutions for elderly people . Outbreaks lasted from one to 217 days (median five days) and their duration varied with the pathogen . The median attack rate was 37% . Illness was reported in 34,158 people, 751 of whom (2%) were admitted to hospital . There were 55 deaths, 28 of which were associated with salmonella and 12 with SRSV . Most of the outbreaks reported and the associated morbidity and mortality could have been prevented by following standard food hygiene practices, implementing infection control policies, and ensuring that food entering kitchens was of the highest microbiological quality possible.

J Biol Chem, 1996 Mar 29, 271(13), 7324 - 9
Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase)
Prepens U, Just I, von Eichel-Streiber C, Aktories K.
Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked {3H}serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60% . In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein . By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B . In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml . Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin . Depolymerization of the microfilament cytoskeleton of RBL cells by C . botulinum C2 toxin or cytochalasin D resulted in an increased {3H}serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation . The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

Vet Rec, 1996 Mar 23, 138(12), 281 - 3
Effect of salinomycin in the control of Clostridium perfringens type C infections in sucklings pigs; Kyriakis SC et al.; The ability of salinomycin to control Clostridium perfringens type C infection in sows and their offspring was examined under field conditions . Two groups of sows and their offspring were offered feed either medicated with 60 ppm salinomycin or free of antibiotics, and their performance was compared . The number of piglets with diarrhoea, the duration of the diarrhoea, and the mortality of the piglets during the lactation period were markedly lower in the group given salinomycin . In addition, laboratory examinations showed that the numbers of carrier piglets and sows were reduced after treatment with Salinomycin . Finally, the sows treated with salinomycin lost less weight during the lactation period and weaned more and heavier piglets than the untreated sows . It was concluded that salinomycin incorporated in the diet can be used for controlling C perfringens type C infection in sows and their offspring.

J Biol Chem, 1996 Mar 22, 271(12), 6925 - 32
UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii; Chaves-Olarte E et al.; We have previously isolated a fibroblast mutant cell with high resistance to the two Rho-modifying glucosyltransferase toxins A and B of Clostridium difficile . We demonstrate here a low level of UDP-glucose in the mutant, which explains its toxin resistance since: (i) to obtain a detectable toxin B-mediated Rho modification in lysates of mutant cells, addition of UDP-glucose was required, and it promoted the Rho modification dose-dependently; (ii) high pressure liquid chromatography analysis of nucleotide extracts of cells indicated that the level of UDP-glucose in the mutant (0.8 nmol/10(6) cells) was lower than in the wild type (3.7 nmol/10(6) cells); and (iii) sensitivity to toxin B was restored upon microinjection of UDP-glucose . Using the mutant as indicator cell we also found that the related Clostridium sordellii lethal toxin is a glucosyltransferase which requires UDP-glucose as a cofactor . Like toxin B it glucosylated 21-23-kDa proteins in cell lysates, but Rho was not a substrate for lethal toxin.

Oncogene, 1996 Mar 21, 12(6), 1357 - 60
Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers; Taniguchi T et al.; Specific receptors for brain-gut peptide hormones, cholecystokinin (CCK) and gastrin, are expressed in a variety of human tumor cells . CCK and gastrin promote the growth of NIH3T3 cells into which the CCK-B/gastrin receptor had been introduced via a eukaryotic expression vector . In this study, we have examined the effect of CCK-8 on the actin cytoskeleton by using two mouse fibroblast cell lines expressing human CCK-B/gastrin receptors . Treatment with very low concentration of CCK-8 (10(-10) M) induced the formation of actin stress fibers within one minute . Stress fiber formation increased for 30 min . In contrast, a potent mitogen for fibroblasts, platelet-derived growth factor (PDGF), initially induced membrane ruffling and, later, a weak formation of stress fibers . Microinjection of rho GDP dissociation inhibitor or Clostridium botulinum ADP-ribosyltransferase C3 which is known to impair the function of a small GTP-binding protein, rho p21, inhibited the stress fiber formation by CCK-8 as well as by PDGF . These results indicate that CCK-B/gastrin receptor could regulate stress fiber formation in a rho p21-dependent manner . The signals from CCK-B/gastrin receptor might affect cell growth as well as cell motility or adhesion by regulating the actin cytoskeleton.

Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 353 - 9
Characterization of component-I gene of botulinum C2 toxin and PCR detection of its gene in clostridial species; Fujii N et al.; Botulinum C2 toxin is composed of two nonlinked protein components, component-I (light chain) and component-II (heavy chain) . It is produced by Clostridium botulinum types C and D, and is thought to play a lethal pathogenic role . These biological activities of C2 toxin may be due to the ADP-ribosylation of non-muscle actin by component-I of the toxin . We were able to isolate two overlapping gene fragments encoding component-I from the chromosomal DNA of Clostridium botulinum type C strain (C)-203U28, and determine the complete nucleotide sequence of component-I gene . The gene for component-I, bc21, consists of one open reading frame (ORF) encoding 431 amino acid residues (1293 nucleotides) without signaling peptide sequence . The molecular mass calculated from the deduced amino acid sequence was 49400.37 Da . Mono-ADP-ribosyltransferase activity was demonstrated in the lysate from E . coli transformed by the recombinant plasmid, pGEM-C2 encompassing whole component-I gene with its own promoter.

Structure, 1996 Mar 15, 4(3), 265 - 75
The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum; Alzari PM et al.; BACKGROUND . Cellulases, which catalyze the hydrolysis of glycosidic bonds in cellulose, can be classified into several different protein families . Endoglucanase CelA is a member of glycosyl hydrolase family 8, a family for which no structural information was previously available . RESULTS . The crystal structure of CelA was determined by multiple isomorphous replacement and refined to 1.65 A resolution . The protein folds into a regular (alpha/alpha)6 barrel formed by six inner and six outer alpha helices . Cello-oligosaccharides bind to an acidic cleft containing at least five D-glucosyl-binding subsites (A-E) such that the scissile glycosidic linkage lies between subsites C and D . The strictly conserved residue Glu95, which occupies the center of the substrate-binding cleft and is hydrogen bonded to the glycosidic oxygen, has been assigned the catalytic role of proton donor . CONCLUSIONS . The present analysis provides a basis for modeling homologous family 8 cellulases . The architecture of the active-site cleft, presenting at least five glucosyl-binding subsites, explains why family 8 cellulases cleave cello-oligosaccharide polymers that are at least five D-glycosyl subunits long . Furthermore, the structure of CelA allows comparison with (alpha/alpha)6 barrel glycosidases that are not related in sequence, suggesting a possible, albeit distant, evolutionary relationship between different families of glycosyl hydrolases.

Presse Med, 1996 Mar 2-9, 25(8), 385 - 92
{Epidemiology of Clostridium difficile nosocomial infections}; Barbut F et al.; Clostridium difficile accounts for 15-25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudo-membranous colitis (PMC) . This anaerobic bacterium is also carried in the gastro-intestinal tract of less than 3% of the normal adult population and can be isolated from the feces of 50-70% asymptomatic neonates . Since recent years, C . difficile has been identified as the leading cause of nosocomial diarrhea in adults . Pathogenesis relies on a disruption of the normal bacteria flora of the colon, a colonization with C . difficile and the release of toxins that cause mucosal damage and inflammation . Incidence of C . difficile intestinal disorders varies between 1 to 30 per thousand patient admissions . Risk factors for C . difficile-associated diarrhea include antimicrobial therapy, older age (> 65 years), intensive care, nasogastric tube, anti-acid use, and length of hospital stay . Nosocomial transmission of C . difficile via orofecal route occurs in 3-30% of total patient admissions but it often remains asymptomatic . Environmental contamination and carriage of the organism on the hands of hospital staff are common . Measures that are recommended to reduce cross-infection rely on an accurate and rapid diagnosis, implementation of enteric isolation, use of disposable gloves, hand washing with a suitable disinfectant (e.g . chlorhexidine) and daily environmental disinfection . C . difficile is a common cause of infectious diarrhea and should be therefore systematically investigated in patients with nosocomial diarrhea.

Appl Environ Microbiol, 1996 Mar, 62(3), 815 - 21
An rRNA approach for assessing the role of obligate amino acid-fermenting bacteria in ruminal amino acid deamination; Krause DO et al.; Ruminal amino acid degradation is a nutritionally wasteful process that produces excess ruminal ammonia . Monensin inhibited the growth of monensin-sensitive, obligate amino acid-fermenting bacteria and decreased the ruminal ammonia concentrations of cattle . 16S rRNA probes indicated that monensin inhibited the growth of Peptostreptococcus anaerobius and Clostridium sticklandii in the rumen . Clostridium aminophilum was monensin sensitive in vitro, but C . aminophilum persisted in the rumen after monensin was added to the diet . An in vitro culture system was developed to assess the competition of C . aminophilum, P . anaerobius, and C . sticklandii with predominant ruminal bacteria (PRB) . PRB were isolated from a 10(8) dilution of ruminal fluid and maintained as a mixed population with a mixture of carbohydrates . PRB did not hybridize with the probes to C . aminophilum, P . anaerobius, or C . sticklandii . PRB deaminated Trypticase in continuous culture, but the addition of C . aminophilum, P . anaerobius, and C . sticklandii caused a more-than-twofold increase in the steady-state concentration of ammonia . C . aminophilum, P . anaerobius, and C . sticklandii accounted for less than 5% of the total 16S rRNA and microbial protein . Monensin eliminated P . anaerobius and C . sticklandii from continuous cultures, but it could not inhibit C . aminophilum . The monensin resistance of C . aminophilum was a growth rate-dependent, inoculum size-independent phenomenon that could not be maintained in batch culture . On the basis of these results, we concluded that the feed additive monensin cannot entirely counteract the wasteful amino acid deamination of obligate amino acid-fermenting ruminal bacteria.

Can J Microbiol, 1996 Mar, 42(3), 298 - 304
A comparative study on the conditions of growth and sporulation of three strains of Clostridium perfringens type A; Decaudin M et al.; Different conditions of growth and sporulation of a strain of Clostridium perfringens type A (NCTC 8798) and two derived mutant strains, the lysozyme-germination dependent strain 8-6 and the revertant strain R3, have been determined . No sporulation was detected for the three strains in the Duncan and Strong (DS) medium; 100% sporulation was routinely obtained for the two mutant strains in the defined (D) medium . Factors promoting in vitro sporulation of C . perfringens type A were assayed: the volume of the culture, the type of preculture, and the addition of lysozyme in precultures . The paper also provided additional information on growth and sporulation of the mutant strains 8-6 and R3 . Glucose concentrations up to 11 mM produced high percentages of sporulation . However, strain R3 still sporulated at 20% with 56 mM of glucose . A high volume of D medium led to slow growth kinetics and favoured sporulation . Faster kinetics of growth and the best percentage of sporulation were obtained with a young inoculum of the two mutant strains . On the other hand, the type of medium in the precculture (fluid thioglycollate (FTG) or basal carbonate yeast trypticase (BCYT)) did not influence the percentage of sporulation . However, while strain R3 was not affected by the addition of lysozyme in D medium, kinetics of growth were strongly influenced by this addition in strain 8-6, and the percentage of sporulation increased with a preculture in FTG medium and decreased when BCYT medium was used.

J Appl Bacteriol, 1996 Mar, 80(3), 283 - 90
The application of a log-logistic model to describe the thermal inactivation of Clostridium botulinum 213B at temperatures below 121.1 degrees C; Anderson WA et al.; In this work, the death of Clostridium botulinum 213B was measured at temperatures between 101 degrees C and 121 degrees C . It was found that at all temperatures tested, survivor curves deviated from log-linearity which prevented their description using traditional first order kinetics . The survivor curves were better described using a vitalistic approach and the log-logistic transformation proposed by Cole et al . (1993) . A single equation was derived to describe all survivor curves over the temperature range tested and a comparison of predicted and measured data showed good correlation . The implications of the use of the vitalistic approach to the validity of the 'minimum botulinum cook' is discussed.

Poult Sci, 1996 Mar, 75(3), 335 - 8
The effects of nonautoclaved and autoclaved water-soluble wheat extracts on the growth of Clostridium perfringens; Branton SL et al.; Clostridium perfringens is the causative agent of necrotic enteritis, a commonly diagnosed disease in chickens that is also observed in turkeys and geese . Two trials were conducted to determine the in vitro effect of filter-sterilized, water-soluble wheat extracts on the growth of C . perfringens . The extracts were either nonautoclaved or autoclaved at 121 C for 40 min and were used to reconstitute thioglycolate broth media . Results of this study suggest that growth of C . perfringens is suppressed in vitro by inclusion of either extract . Glycosyl composition analysis revealed no significant differences in arabinose, xylose, or mannose content between the nonautoclaved and autoclaved extracts . Galactose, glucose, and total glycosyl content were significantly higher in the nonautoclaved extract.

Res Microbiol, 1996 Mar-Apr, 147(3), 193 - 9
Identification of spore-forming strains involved in biodegradation of acifluorfen; Fortina MG et al.; We isolated and identified four spore-forming bacteria from activated sludges and soil, three of which were able to degrade acifluorfen . Biochemical characteristics, DNA base composition and DNA-DNA homology indicated that the degrading strains belonged to the species Bacillus thuringiensis, Clostridium perfringens and Clostridium sphenoides . The fourth strain, identified as C . sphenoides and showing the same characteristics of the corresponding degrading strain, was unable to metabolize acifluorfen . Thus, the plasmid content of these strains was analysed to study the possible correlation between the presence of extrachromosomal elements and the ability to degrade this herbicide.

Clin Exp Rheumatol, 1996 Mar-Apr, 14(2), 137 - 44
The reactivity of sera from patients with systemic lupus erythematosus to seven different species of single and double stranded deoxyribonucleic acids; Yu CL et al.; OBJECTIVE: Anti-DNA antibodies are frequently found in the serum of patients with systemic lupus erythematosus (SLE) . To understand whether the avidity of SLE sera to different species of single-stranded (ss) and double-stranded (ds) DNA is different or not, the reactivity of active SLE sera to seven species of DNA from viral, bacterial, piscine, and mammalian sources was compared . METHODS: Nineteen sera from patients with active SLE were studied for their reactivity to different ssDNA and dsDNA from Escherichia coli (EC), Micrococcus lysodeikticus (ML), Clostridium perfringens (CP), calf thymus (CT), salmon testis (ST), human placenta (HP) and lambda phage by ELISA . The dsDNA was purified by treating it with S1 nuclease and proteinase K, followed by Sephacryl S-300 gel filtration . The ssDNA was purified by absorption on a hydroxyapatite column after heat-cleavage of the dsDNA . RESULTS: The reactivity of SLE sera to 7 species of dsDNA was not significantly different and they recognized a more widely shared epitope . In contrast, the reactivity of these sera to 7 species of ssDNA was erratic and the antigens could be grouped into high (CP and HP), medium (EC, ML, CT, and ST) and low (lambda-phage) antigenicities . CONCLUSION: The anti-ssDNA and anti-dsDNA antibodies of SLE patients recognize more widely shared determinants on the DNA of seven different species . Lambda-phage DNA shows the poorest immunogenicity among them.

J Pediatr Surg, 1996 Mar, 31(3), 394 - 6
Pyomyositis in children, caused by anaerobic bacteria; Brook I; The author describes the microbiology and clinical features of six pyomyositis infections in children, which yielded anaerobic bacteria . Anaerobic bacteria alone were recovered in four instances, and they are mixed with facultative bacteria in two . There were 15 bacterial isolates (13 anaerobic, 2 facultative) . The bacteria were Peptostreptococcus sp (5 isolates), Bacteroides fragilis (3), Clostridium sp (2), Fuso-bacterium nucleatum (1), Prevotella sp (1), Bateroides sp (1), Streptococcus pyogenes (1), and Escherichia coli (1) . Recent trauma or injury had occurred in five cases; three such injuries were from penetrating objects . This study highlights the potential importance of anaerobic bacteria in children with pyomyositis.

Infect Control Hosp Epidemiol, 1996 Mar, 17(3), 180 - 2
Clostridium difficile contamination of blood pressure cuffs: a call for a closer look at gloving practices in the era of universal precautions; Manian FA et al.; We report an outbreak of Clostridium difficile-associated diarrhea at our medical center following adoption of Universal Precautions . Environmental cultures revealed unexpected contamination of blood pressure cuffs at a rate similar to that for bedside commodes (10% and 11.5%, respectively) . An observational survey revealed that healthcare workers in the patient care areas not infrequently failed to remove their potentially stool-contaminated gloves prior to touching clean surfaces, which might have contributed to contamination of blood pressure cuffs.

Plasmid, 1996 Mar, 35(2), 91 - 7
Structural organization of pRAM4, a cryptic plasmid from Prevotella ruminicola; Ogata K et al.; A total of 530 strains of rumen bacteria were screened for the presence of plasmid DNA . The percentage of plasmid-bearing strains was found to be the highest among the Bacteroides/ Prevotella group (9.9%), while it was less than 1% in the Butyrivibrio (0.2%) and Clostridium (0.6%) genera . A small cryptic plasmid pRAM4 from Prevotella ruminicola T31 was subcloned in Escherichia coli and completely sequenced . Two open reading frames, encoding potential polypeptides of M(r) 32,322 (ORF1) and 32,122 (ORF2) with limited sequence similarity to replication initiation and mobilization proteins, respectively, could be identified within the sequence . The region upstream from ORF1 had an AT-rich (75%) region followed by four 22-bp direct repeats, a structure characteristic of replication origins . The plasmid hybridized at high stringency with plasmids from Bacteroides/Prevotella and Butyrivibrio, and with pBR322, suggesting that at least regions of the plasmid are widespread.

Gut, 1996 Mar, 38(3), 337 - 47
Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment; Mahida YR et al.; Clostridium difficile is the aetiological agent of pseudomembranous colitis, and animal studies suggest the essential role of secreted toxin A in inducing disease . This study examined the biological responses to toxin A by human intestinal epithelial cells . Confluent monolayers of Caco2, HT29, and T84 cells and primary epithelial cells in organ cultures of human colonic biopsy specimens and after detachment with EDTA were studied . Interleukin 8 was assayed using enzyme linked immunosorbent assay (ELISA) . Purified C difficile toxin A induced cell rounding and detachment of monolayers of the epithelial cell lines . Cells in detached monolayers initially remained viable while adherent to each other . Subsequently, an increasing number of apoptotic cells appeared in suspension . Exposure to toxin A for 24 hours induced interleukin 8 production in T84 and HT29 cells . Toxin A also induced epithelial cell rounding, detachment, and apoptosis in organ cultures of human colonic biopsy specimens . During culture (in medium only), EDTA detached colonic epithelial cells produced interleukin 8 and cell death occurred by apoptosis . Colonic disease by C difficile may be initiated by toxin A mediated induction of epithelial cell interleukin 8 production and apoptosis after cell detachment from the basement membrane . Studies on isolated (toxin untreated) colonic epithelial cells suggest that interleukin 8 production and apoptosis occur as a consequence of cell injury and detachment.

FEMS Microbiol Rev, 1996 Mar, 18(1), 5 - 63
Tungsten in biological systems; Kletzin A et al.; Tungsten (atomic number 74) and the chemically analogous and very similar metal molybdenum (atomic number 42) are minor yet equally abundant elements on this planet . The essential role of molybdenum in biology has been known for decades and molybdoenzymes are ubiquitous . Yet, it is only recently that a biological role for tungsten has been established in prokaryotes, although not as yet in eukaryotes . The best characterized organisms with regard to their metabolism of tungsten are certain species of hyperthermophilic archaea (Pyrococcus furiosus and Thermococcus litoralis), methanogens (Methanobacterium thermoautotrophicum and Mb . wolfei), Gram-positive bacteria (Clostridium thermoaceticum, C . formicoaceticum and Eubacterium acidaminophilum), Gram-negative anaerobes (Desulfovibrio gigas and Pelobacter acetylenicus) and Gram-negative aerobes (Methylobacterium sp . RXM) . Of these, only the hyperthermophilic archaea appear to be obligately tungsten-dependent . Four different types of tungstoenzyme have been purified: formate dehydrogenase, formyl methanufuran dehydrogenase, acetylene hydratase, and a class of phylogenetically related oxidoreductases that catalyze the reversible oxidation of aldehydes . These are carboxylic reductase, and three ferredoxin-dependent oxidoreductases which oxidize various aldehydes, formaldehyde and glyceraldehyde 3-phosphate . All tungstoenzymes catalyze redox tungsten in these enzymes is bound by a pterin moiety similar to that found in molybdoenzymes . The first crystal structure of a tungsten- or pterin-containing enzyme, that of aldehyde ferredoxin oxidoreductase from P . furiosus, has revealed a catalytic site with one W atom coordinated to two pterin molecules which are themselves bridged by a magnesium ion . The geochemical, ecological, biochemical and phylogenetic basis for W- vs . Mo-dependent organisms is discussed.

Ann Plast Surg, 1996 Mar, 36(3), 309 - 12
Late Clostridium perfringens breast implant infection after dental treatment; Hunter JG et al.; Late infection is rare after breast augmentation . Pathogenesis is usually implant seeding caused by bacteremia as a consequence of antecedent distant infections or medical/dental procedures . Reported is the first case of late implant infection, after extensive dental treatment, caused by Clostridium perfringens, an anaerobic pathogen commonly present in the human gastrointestinal tract . Prompt diagnosis and early antibiotic treatment of all bacterial infections, and serious consideration of antibiotic prophylaxis for all bacteremia-producing procedures, is essential for breast implant patients.

Infect Immun, 1996 Mar, 64(3), 933 - 7
Analysis of the specificity of bacterial immunoglobulin A (IgA) proteases by a comparative study of ape serum IgAs as substrates; Qiu J et al.; Immunoglobulin A (IgA) proteases are bacterial enzymes with substrate specificity for human serum and secretory IgAs . To further define the basis of this specificity, we examined the ability of IgA proteases of Clostridium ramosum, Streptococcus pneumoniae (EC 3.4.24.13), Neisseria meningitidis (EC 3.4.21.72), and Haemophilus influenzae (EC 3.4.21.72) to cleave serum IgAs of gorillas, chimpanzees, and orangutans . All enzymes cleaved the IgAs of the three apes despite differences in ape IgA1 hinge sequence relative to the human prototype . To directly compare the ape and human hinge cleavage sites, the sites were identified in eight ape IgA digests . This analysis confirmed that ape proteins were all cleaved in the IgA hinge region, in all but one case after proline residues . The exception, C . ramosum protease, cleaved gorilla and chimpanzee IgAs at peptide bonds having no proline, but the scissile bonds were in the same hinge location as the Pro-221-Val-222 cleaved in human IgA1 . These data indicate that proline is not an invariant substrate requirement for all IgA proteases and that the location of the scissile bond, in addition to its composition, is a critical determinant of cleavage specificity.

Infect Immun, 1996 Mar, 64(3), 1020 - 5
Evidence that a region(s) of the Clostridium perfringens enterotoxin molecule remains exposed on the external surface of the mammalian plasma membrane when the toxin is sequestered in small or large complexes; Kokai-Kun JF et al.; In studies performed to investigate the topology of Clostridium perfringens enterotoxin (CPE) when this toxin is associated with intestinal brush border membrane (BBMs), it was shown that radiolabeled CPE antibodies react more strongly against intact CPE-treated BBMs than against control BBMs . Immunoprecipitation studies then demonstrated that CPE antibodies are able to react with both small and large CPE-containing complexes while these complexes are still present in intact BBMs . Therefore, at least a portion of the CPE molecule appears to remain surface exposed in BBMs throughout the action of this toxin.

Am J Gastroenterol, 1996 Mar, 91(3), 460 - 4
Prognostic criteria in Clostridium difficile colitis; Ramaswamy R et al.; OBJECTIVE: To determine the prognostic factors in Clostridium difficile (CD) colitis . METHODS: We conducted a retrospective study of proven cases of CD colitis in l8 months . Seventy six patients (from a 605-bed community hospital in the Bronx, NY) with proven CD colitis were studied . Mortality in patients with CD colitis was also examined . RESULTS: Seventy six patients with proven CD colitis were admitted between January 1993 and June 1994 . Eighteen patients died during the same admission . Upon admission, serum albumin was less than 25 g/L in 12 (20.6%) of the survivors and in eight (44%) of the deceased patients (p <0.05) . A fall in serum albumin levels was noted with the onset of symptoms of CD colitis in those who survived as well as in those who died, with a greater fall of 11.2 g/L (range 10-20 g/L) in patients who died compared with a fall of 6 g/L (range 5-10 g/L)in those who survived (p <0.05) . Use of more than three antibiotics was noted in 13 (72%) of those who died and in 18 (31%) of those who survived (p <0.05) . Persistence of CD cytotoxin 7 or more days after initiation of treatment was present in 14 (77%) of those who died and in eight (13%) of the survivors (p <0.01) . Duration of hospitalization correlated with the development of CD colitis (35.89 vs 11.7 days) with no significant difference between survivors and deceased patients with CD colitis . Factors such as age, sex, residence, past medical history score, mean score of presenting complaints of CD colitis, history of prior episodes CD colitis, and mean number of recurrent episodes showed no difference in mortality . CONCLUSION: Factors predictive of an increased mortality in patients with CD colitis include a serum albumin of less than 25 g/L on admission, a fall in serum albumin level of greater than 11 g/L at the onset of symptoms of CD colitis, use of three or more antibiotics, and persistence of positive CD cytotoxin in the stool after completion of 7 or more days of treatment.

Dig Dis Sci, 1996 Mar, 41(3), 614 - 20
Increased substance P receptor expression by blood vessels and lymphoid aggregates in Clostridium difficile-induced pseudomembranous colitis; Mantyh CR et al.; Pseudomembranous colitis is most often caused by toxins secreted by Clostridium difficile following bowel flora overgrowth after antibiotic use . The secretory and inflammatory effects observed in C . difficile toxin A-induced enterocolitis in the rat ileum are inhibited by CP-96,345, a substance P (SP) receptor antagonist . To determine if SP plays a role in the pathogenesis of human pseudomembranous colitis, SP receptor distribution was examined in a toxin A-positive specimen of bowel . Quantitative receptor autoradiography was used to examine SP receptors in tissue from a patient who tested positive for C . Difficile toxin . SP receptors were massively increased in small blood vessels and lymphoid aggregates in the pseudomembranous colitis bowel in comparison to control specimens . The SP binding was saturable and exhibited similar affinities for SP and CP-96,345 . SP may contribute to the inflammatory response in pseudomembranous colitis via a massive increase in SP receptor antagonists may offer a novel therapeutic intervention for pseudomembranous colitis.

Virology, 1996 Mar 1, 217(1), 323 - 31
A new form of particulate single and multiple immunogen delivery system based on recombinant bluetongue virus-derived tubules; Mikhailov M et al.; The development of particular vector systems for the presentation of immunogenic epitopes provides a powerful approach for the delivery of antigens . These include the core-like particles formed by recombinant bluetongue virus (BTV) capsid proteins VP3 and VP7 synthesized in insect cells by recombinant baculoviruses . Previously we have reported localization of an antigenic site on the surface of tubular structures formed by the nonstructural protein NS1 of BTV, and their potential use for epitope presentation . In this study foreign sequences ranging form 44 to 116 aa in length and representing 44 aa sequence from Clostridium difficile toxin A, 48 aa of the hepatitis B virus preS2 region, and the whole of bovine leukemia virus p15 protein were inserted at the C-terminus of BTV-10 NS1 . The chimeric NS1 genes were expressed using recombinant baculoviruses and the ability of the mutated NS1 proteins to form tubules was investigated . All chimeric constructs formed tubular structures which carried the foreign antigenic sequences exposed on the surface of the tubules and were highly immunogenic . When Sf cells were coinfected with three recombinant baculoviruses expressing chimeric NS1 proteins with different epitopes, their simultaneous assembly into the same tubule was demonstrated . This observation opens up the possibility of using recombinant NS1 tubules as carriers for the delivery of multiple epitopes.

Int J Cancer, 1996 Mar 1, 65(5), 627 - 32
rho-Mediated protein tyrosine phosphorylation in lysophosphatidic-acid-induced tumor-cell invasion; Imamura F et al.; Rat ascites hepatoma cell (MM1) invade a mesothelial cell monolayer in vitro in assay medium containing serum, but not in serum-free medium . Serum could be completely replaced by 1-oleoyl lysophosphatidic acid (LPA) in inducing invasion . LPA-induced invasion was inhibited by genistein, a tyrosine-kinase inhibitor . Protein tyrosine phosphorylation in response to LPA was thus analyzed in order to determine the molecular mechanism of invasion . LPA of invasion-inducible concentrations evoked a transient increase in tyrosine phosphorylation, mainly of 110- to 130-kDa proteins in MM1 cells but not in mesothelial cells . These concentrations of LPA were over 10 times higher (10 to 25 micron) than those necessary to produce a variety of biological actions, such as tyrosine phosphorylation in fibroblasts, neurite retraction and platelet aggregation . Protein tyrosine phosphorylation and invasion by MM1 cells induced by LPA are largely regulated by rho p21, because both were inhibited by Clostridium botulinum C3 exo-enzyme, which is known to specifically inactivate rho p21 . Invasion of MCL by MM1 cells induced by serum and that by B16FE7 cells induced by LPA were inhibited by genistein or C3 as well . By immunoprecipitation, we detected p 125 focal adhesion kinase (FAK) as a major protein of 110- to 130-kDa tyrosine phosphorylated in response to LPA . Tyrosine phosphorylation of paxillin by LPA was also detected.

Biochemistry, 1996 Feb 20, 35(7), 2476 - 81
A conformational change in the methyltransferase from Clostridium thermoaceticum facilitates the methyl transfer from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein in the Acetyl-CoA pathway; Zhao S et al.; The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the methylation of a corrinoid/iron-sulfur protein (C/Fe-SP) by the N5 methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) . This is an important reaction in the reductive acetyl-CoA pathway . The forward and reverse reactions of MeTr have a pH dependence that appears to reflect protonation of a group on the protein {Zhao, S., Roberts, D . L., & Ragsdale, S . W . (1995) Biochemistry 34, 15075-15083} . In the work reported here, fluorescence and rapid reaction kinetics were used to demonstrate that this protonation elicits a rate-limiting conformational change . As the pH was lowered, the emission maximum for intrinsic tryptophan fluorescence underwent a red shift (pK(a) = 5.4) and the emission intensity increased (pK(a) = 5.1) . The extrinsic fluorescence probe, 4,4'-bis-1-phenylamino-8-napthalenesulfonate (bis-ANS) was used to report on the conformational change . The bis-ANS fluorescence was strongly enhanced upon binding MeTr . As the pH was decreased, the fluorescence was further enhanced and the emission maximum underwent a 14 nm blue shift (pK(a) = 5.0) . By stopped-flow fluorescence studies, it was shown that these fluorescence changes occur at rates similar to the k(cat) for the MeTr reaction and thus reflect catalytically competent events . The combined results indicate that CH3-H4folate binds to a hydrophobic region in MeTr that includes a tryptophan residue(s) . MeTr undergoes a pH-dependent conformational change that exposes this region to solvent and facilitates substrate binding.

FEBS Lett, 1996 Feb 19, 380(3), 291 - 5
Analysis of the catalytic site of the actin ADP-ribosylating Clostridium perfringens iota toxin; van Damme J et al.; The enzyme component of actin ADP-ribosylating Clostridium perfringens iota toxin was affinity labelled by UV irradiation in the presence of {carbonyl-14C}NAD . A peptide containing the radiolabel was generated by CNBr cleavage and subsequent proteolysis with trypsin . Its amino acid sequence is Gly-Ser-Pro-Gly-Ala-Tyr-Leu-Ser-Ala-Ile-Pro-Gly-Tyr-Ala-Gly-X-Tyr-Glu-Va l-Leu-Leu-Asn-His-Gly-Ser-Lys corresponding with the region Gly-363 through Lys-388 in the C . perfringens iota toxin . Mass spectrometric data as well as results of the PTH-amino acid analysis are in line with a modification of a glutamic acid side chain located at position 378 . Therefore, in addition to Glu-380, as could be concluded by analogy with other ADP-ribosyltransferases, Glu-378 may play a pivotal role in the active site of C . perfringens iota toxin.

FEBS Lett, 1996 Feb 19, 380(3), 287 - 90
Molecular mechanism of pyruvate-ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme; Moulis JM et al.; Pyruvate-ferredoxin oxidoreductase oxidises pyruvate in many fermentative microorganisms . The enzyme from Clostridium pasteurianum is an air-sensitive homodimer of 2x120000 daltons, for which pyruvate is the best substrate found among several alpha-ketoacids . Each subunit contains eight iron atoms in two {4Fe-4S} clusters . Two distinct EPR signals, possibly associated with two ligand environments, arise from one of these clusters . Binding of pyruvate does not generate a radical . The results reported suggest a scheme for the electron flow in pyruvate ferredoxin oxidoreductases according to which the detailed reaction mechanism depends on the number (even or odd) of {4Fe-4S} clusters present in a given enzyme.

FEMS Microbiol Lett, 1996 Feb 15, 136(2), 163 - 8
Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750; Holck A et al.; Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus . Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited . The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography . The complete amino acid sequence was determined by Edman degradation . The peptide consisted of 34 amino acid residues . The calculated M(r) from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry . Divergicin 750 did not show any sequence similarities to other known bacteriocins . The plasmid-located structural gene encoding divergicin 750 (dvn750) was cloned and sequenced . The gene encoded a primary translation product of 63 amino acids with a deduced M(r) = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.

Biochemistry, 1996 Feb 13, 35(6), 1965 - 71
Carbon monoxide dehydrogenase from Clostridium thermoaceticum: quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form; Xia J et al.; The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300,000 +/- 30,000 Da . Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82,000 Da for alpha and 73,000 Da for beta), this indicates an alpha 2 beta 2 quaternary structure . The enzyme was previously thought to have an alpha 3 beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420,000 Da . The disproportionately fast migration rate suggests that the enzyme is nonspherical . SDS induces the dissociation of an alpha subunit, yielding a stable species called FM-CODH . FM-CODH had a molecular mass of 210,000 +/- 30,000 Da, indicating an alpha 1 beta 2 structure . It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha 1 beta 2, exhibited S-->Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals . Quantitation of the 1.82 and (1.94 +/- 1.86) signals afforded 0.35 and 1.9 spin/alpha 1 beta 2, respectively . FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity . Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme . These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the beta subunit . The alpha subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity . FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity . Incubating FM-CODH and alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha 2 beta 2 structure . Thus, the SDS-induced dissociation of the enzyme appears to be reversible.

J Biol Chem, 1996 Feb 2, 271(5), 2422 - 6
Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B . Role of Rho proteins; Schmidt M et al.; Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations . To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) . Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD . In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced . Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD . Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins . In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively . Finally, C . botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity . In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.

Tokai J Exp Clin Med, 1996 Feb, 21(1), 33 - 6
Acute emphysematous cholecystitis associated with pneumobilia: a case report; Ohtani Y et al.; This report describes a rare case of acute emphysematous cholecystitis with pneumobilia in the common bile duct . The patient was a 66-year-old woman with a part history of diabetes mellitus, and operations for gastric and breast carcinoma . The chief complaint was pain in the right hypochondrium with severe right hypochondrial tenderness and distention of the gallbladder detected on examination . Laboratory tests showed leukocytosis, marked elevation of CRP, jaundice, liver dysfunction, and hyperglycemia . Gas was detected in the gallbladder on plain abdominal X-rays and CT scans of the abdomen, and a small amount of gas was also observed in the common bile duct . On the day of admission, percutaneous transhepatic gallbladder drainage (PTGBD) was carried out under ultrasound guidance, and Clostridium perfingens and E . coli were detected in the bile . Imaging after PTGBD showed no cystic duct obstruction . On the 12th day after PTGBD, cholecystectomy and choledochotomy with primary closure were performed . The postoperative course was good and the patient was discharged on the 15th day after surgery.

Gene Ther, 1996 Feb, 3(2), 173 - 8
Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia; Fox ME et al.; Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis . We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery . Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil . The E . coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium . When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold . This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.

Antimicrob Agents Chemother, 1996 Feb, 40(2), 373 - 9
Anti-Clostridium difficile bovine immunoglobulin concentrate inhibits cytotoxicity and enterotoxicity of C . difficile toxins; Kelly CP et al.; Clostridium difficile diarrhea and colitis result from the actions of bacterial exotoxins on the colonic mucosa . This study examined the ability of hyperimmune bovine colostral antibodies to neutralize the biological effects of these toxins . Anti-C . difficile bovine immunoglobulin concentrate was prepared from the colostral milk of Holstein cows previously immunized with C . difficile toxoids . The anti-C . difficile bovine immunoglobulin concentrate contained high levels of bovine immunoglobulin G specific for C . difficile toxins A and B, as evaluated by enzyme-linked immunosorbent assay . Anti-C . difficile bovine immunoglobulin concentrate neutralized the cytotoxic effects of purified toxin A and toxin B on cultured human fibroblasts, whereas control bovine immunoglobulin concentrate had little toxin-neutralizing activity . Anti-C . difficile bovine immunoglobulin concentrate also blocked the binding of toxin A to its enterocyte receptor and inhibited the enterotoxic effects of C . difficile toxins on the rat ileum, as measured by an increased rat ileal loop weight/length ratio (63% inhibition; P < 0.01), increased mannitol permeability (92% inhibition; P < 0.01), and histologic grading of enteritis (P < 0.01 versus nonimmune bovine immunoglobulin concentrate) . Thus, anti-C . difficile bovine immunoglobulin concentrate neutralizes the cytotoxic effects of C . difficile toxins in vitro and inhibits their enterotoxic effects in vivo . This agent may be clinically useful in the prevention and treatment of C . difficile diarrhea and colitis.

Med Microbiol Immunol (Berl), 1996 Feb, 184(4), 175 - 80
Cytotoxic effects by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence of Clostridium perfringens iota toxin; Kiefer G et al.; The ADP-ribosylating toxins Clostridium botulinum C2 toxin and C . perfringens iota toxin, which ADP-ribosylate monomeric G-actin at Arg-177 but not the polymeric F-actin, induce depolymerization of the actin cytoskeleton in cultured cells . Since ADP-ribosylated G-actin has properties of a barbed-end-capping protein, we studied whether the ADP-ribosylated actin affects the actin cytoskeleton of PtK2 cells even in the absence of ADP-ribosylating toxin . Skeletal muscle actin was ADP-ribosylated by C . perfringens iota toxin and the toxin was removed using an anti-iota toxin antibody . Microinjection of ADP-ribosylated actin caused retraction of the cell body, redistribution and depolymerization of the actin cytoskeleton in a concentration- and time-dependent manner . The finding that ADP-ribosylated actin affects per se the actin cytoskeleton explains the cytopathic effects of ADP-ribosylating toxins on microfilaments, although F-actin is not directly modified by the toxins.

Int J Food Microbiol, 1996 Feb, 29(1), 59 - 73
Thermotolerance of meat spoilage lactic acid bacteria and their inactivation in vacuum-packaged vienna sausages; Franz CM et al.; Heat resistance of three meat spoilage lactic acid bacteria was determined in vitro . D-values at 57, 60 and 63 degrees C were 52.9, 39.3 and 32.5 s for Lactobacillus sake, 34.9, 31.3 and 20.2 s for Leuconostoc mesenteroides and 22.5, 15.6 and 14.4 s for Lactobacillus curvatus, respectively . The three lactic acid bacteria were heat sensitive, as one log reductions in numbers were achieved at 57 degrees C in less than 60 s . Z-values could not be accurately determined as D-values did not change by a factor of 10 over the temperature range studied . In-package pasteurization processes were calculated using the highest in vitro D-value and applied to vacuum-packaged vienna sausages . Microbiological shelf life (time for lactic acid bacteria count to reach 5 x 10(6) CFU/g) increased from 7 days for non-pasteurized samples to 67, 99 and 119 days for samples of the three pasteurization treatments at 8 degrees C storage . Enterobacteriaceae were detected at levels of log 4.0 CFU/g in non-pasteurized samples, but were reduced to < log 1.0 CFU/g in pasteurized samples . The incidence of listeriae in non-pasteurized samples was low as only one Listeria innocua strain was isolated . No Listeria spp . were isolated from pasteurized samples . Numbers of Clostridium isolates increased from one in non-pasteurized samples to 25 in pasteurized samples . Increasing incidences of clostridia, and the presence of C . perfringens in pasteurized samples indicated that in-package pasteurization could compromise product safety.

Enferm Infecc Microbiol Clin, 1996 Feb, 14(2), 96 - 100
{Clostridium difficile and diarrhea associated with the use of antibiotics in the origin of nosocomial and community-acquired diarrhea}; Knobel H et al.; BACKGROUND: Clostridium difficile is currently recognized as an important nosocomial enteric pathogen . The significance as etiologic agent of community and nosocomial diarrhea is not well known in Spain . METHODS: Retrospective study of all cases of community diarrhea that required admission in the hospital and all nosocomial diarrhea observed in a period of three months in a 450-bed university hospital . We performed conventional coprocultives and detection of toxin-A of C . difficile with the method ELISA Premier . RESULTS: During the period of study were included 66 patients, 19 pediatrics and 47 adults patients (23 males, 24 females, age: 54.5 +/- 21.8 years) . Three cases (15.8%) of pediatrics patients were diagnosticated of antibiotic-associated diarrhea, only one case were of nosocomial origin . Toxin A of C . difficile were detected in 6 cases, all were patients under two years old, represented 60% of these patients . The origin of diarrhea were: community in 32 cases and nosocomial in 15 of adults patients, in 18 cases (38.3%) were diagnosticated antibiotic-associated diarrhea, 11 were nosocomial . Toxin A of C . difficile were detected in 12 patients, 25.5% of adults, and 4 cases had criteria of C . difficile associated diarrhea, representing 8.5% of the diarrhea . None of this cases were suspected during admission . CONCLUSIONS: Antibiotic-associated diarrhea and C . difficile associated diarrhea were not infrequent cause of diarrhea of nosocomial and of community origin in our environment . We recommended culture and/or detection of toxins of C . difficile in patients who were treated with antibiotics and diarrhea of more than 72 hours of evolution.

Arch Pathol Lab Med, 1996 Feb, 120(2), 206 - 11
The use and abuse of routine stool microbiology: a College of American Pathologists Q-probes study of 601 institutions; Valenstein P et al.; OBJECTIVE: To examine the efficiency with which physicians use routine stool microbiology tests . DESIGN: Questionnaire and structured review of 100 consecutive stool bacteriology and parasitology examinations at each participating institution . SETTING: Six hundred one institutions enrolled in the College of American Pathologists Q-probes Program . RESULTS: Of 59500 bacteriology specimens, 3808 (6.4%) contained a pathogen . The vast majority (99%) of bacterial pathogens were detected in either the first or second specimen submitted . Almost 40% of inpatient specimens were collected after the third day of hospitalization, but only 0.6% of these specimens were positive for enteric pathogens that had not been previously recovered . More than half of the laboratories reported having no limits on the number of bacteriology specimens per patient that could be submitted for testing, and fewer than 8% of laboratories rejected specimens from inpatients after a certain number of days in the hospital . The frequency with which laboratories performed tests for Clostridium difficile varied widely . Of 58500 parasitology specimens, 1463 (2.5%) contained a pathogen; 97.6% of pathogens were detected by the second stool specimen, and 99.8% were detected by the third specimen . Only 0.7% of specimens from inpatients hospitalized more than 4 days contained a new pathogen . CONCLUSIONS: We recommend that no more than two bacteriology specimens and no more that two or three parasitology specimens be processed per patient without consultation . Standard stool examination for a bacterial pathogens has a low yield and should not be performed after 3 days of hospitalization . Likewise, parasitology examinations should not be performed after 4 days of hospitalization.

J Antimicrob Chemother, 1996 Feb, 37(2), 209 - 22
The comparative efficacy and safety of teicoplanin and vancomycin; Wood MJ; Glycopeptide antibiotics, such as teicoplanin and vancomycin, are active against staphylococci (including methicillin resistant strains), streptococci, enterococci and Clostridium spp . Vancomycin and teicoplanin are both widely used in the treatment of infections caused by Gram-positive organisms . Vancomycin can, however, provoke a number of side-effects, and serum concentrations should be monitored during treatment . Teicoplanin has a longer half-life than vancomycin, it can be given as an intravenous bolus or by intramuscular injection, and nephrotoxicity and ototoxicity are relatively uncommon . Treatment with teicoplanin might, therefore, offer advantages over treatment with vancomycin-provided that similar clinical efficacy can be shown . At least 11 clinical trials comparing the efficacy and safety of teicoplanin and vancomycin have been carried out worldwide . Meta-analysis of the combined results from these studies indicates that more than three-quarters of the patients in each of the treatment groups had a clinical response to therapy . Meta-analysis of the numbers of adverse events occurring in each treatment group shows significantly fewer reports of adverse events in patients receiving teicoplanin (13.9%) than in those receiving vancomycin (21.9%) . Direct comparisons are difficult because of inherent differences between studies, but available data suggest that teicoplanin is as effective as vancomycin and that its superior tolerability together with advantages such as once-daily bolus administration, intramuscular use and lack of requirement for routine serum monitoring, give it considerable potential for use in clinical practice.

Lab Anim Sci, 1996 Feb, 46(1), 21 - 5
A novel presentation of Clostridium piliforme infection (Tyzzer's disease) in nude mice; Livingston RS et al.; Clostridium piliforme infection (Tyzzer's disease) was diagnosed in a colony of nude mice . Because spontaneous Tyzzer's disease had not been reported in nude mice, a study was undertaken to better define the clinicopathologic features of this disease outbreak . Sixty homozygous nude (nu/nu) females, 10 nu/nu males, and 10 heterozygous nude (nu/+) females were observed for signs of disease . Over a 3-month period, 43% of the nu/nu mice died or manifested clinical signs of disease and were euthanized, but nu/+ mice remained healthy . Clinical signs of disease were infrequently observed in nu/nu mice and, when evident, were followed by rapid deterioration and death . Gross and histologic lesions, including severe hepatic and intestinal necrosis associated with C . piliforme, were observed only in clinically affected animals . Clostridium piliforme isolated from diseased livers had marked cytotoxicity in in vitro assays . This outbreak is unique in that, contrary to a previous experimental report, nu/nu mice had increased susceptibility to Tyzzer's disease, suggesting that T cells may play an important role in host defenses against C . piliforme infection . In addition, this is the first report of a toxigenic isolate of C . piliforme recovered from mice . The cytotoxin produced by the isolate may have contributed to the severity of clinical disease and lesions.

Aust Vet J, 1996 Feb, 73(2), 55 - 61
Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle; Gregory AR et al.; The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described . Partially purified type C and D toxins were used as antigens to develop these ELISAs . Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds . The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic . The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism . Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic . Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests . The use of these tests in investigations of botulism in cattle is discussed.

Scott Med J, 1996 Feb, 41(1), 15 - 6
Community-acquired toxigenic Clostridium difficile diarrhoea in the normoxaemic elderly who have received no antimicrobials: soft evidence for ischaemic colitis?
Laing RB, Dykhuizen RS, Smith CC, Gould IW, Reid TM.
We report three examples of community-acquired toxigenic Clostridium difficile diarrhoea in elderly patients who had neither received antimicrobial therapy nor been institutionalised . These cases stimulated interest in the non-antimicrobial changes which might predispose the host to C . difficile-related disease and raised the spectre of bowel ischaemia as a possible aetiological factor.

J Clin Pathol, 1996 Feb, 49(2), 172 - 3
Nosocomial empyema caused by Clostridium difficile; Simpson AJ et al.; Pleural infection with Clostridium difficile is extremely rare . A case of nosocomial empyema following chest drain insertion in a 46 year old man is described . The potential of C difficile to cause extra-intestinal infections should be recognised and its isolation from other sites should not be ignored.

J Med Microbiol, 1996 Feb, 44(2), 115 - 23
Cell surface properties of Clostridium difficile: haemagglutination, relative hydrophobicity and charge; Krishna MM et al.; Five well characterised strains of Clostridium difficile of differing virulence and two Escherichia coli strains, a verotoxigenic O157:H7 isolate and a urinary isolate, were examined for cell surface hydrophobicity and charge, and haemagglutinating ability . Phase partition in hexadecane or octan-1-ol was similar for C . difficile and E . coli, as was retention by hydrophobic interaction chromatography (HIC), indicating moderate hydrophobicity . The salt agglutination test showed E . coli to be hydrophobic and C . difficile to be hydrophilic . Relative hydrophobicity determined by HIC when charge effects were not nullified, i.e., to reflect more closely conditions in vivo, showed C . difficile to bind less well . Growth of C . difficile in caecal emulsions to simulate conditions in vivo did not alter the cell surface hydrophobicity . The phase partition method for charge determination indicated that E . coli and C . difficile had a net negative charge, although this was weaker for C difficile than E . coli . However, although E . coli exhibited a net negative charge as determined by immuno-gold electronmicroscopy (IGEM), in keeping with the results of the phase partition method, C . difficile was shown to be predominantly positively charged by IGEM, and by movement in a charged field as determined by paper electrophoresis and a novel method based on light microscope observation . A cell-wall deficient mutant of C . difficile was weakly positively charged, showing that most of the charge resides in the cell wall.

J Med Microbiol, 1996 Feb, 44(2), 111 - 4
Enhancement of Clostridium difficile toxin production in biotin-limited conditions; Yamakawa K et al.; The effect of biotin on toxin production by Clostridium difficile was examined in a defined medium . When toxin production by strain KZ 1647, which was isolated from a healthy adult, was examined in relation to its biotin requirement, it was found that with decreasing concentrations of biotin, bacterial growth was decreased, but production of both toxins A and B were remarkably increased, particularly with 0.05 nM biotin . The time course of production of both toxins in biotin-limited conditions was similar to that in biotin-enriched conditions . The biotin effect on toxin production was also observed in 15 other strains, suggesting that the effect occurs frequently amongst toxigenic C . difficile strains . The biotin effect is discussed in relation to the pathogenesis of C . difficile colitis.

Bone Marrow Transplant, 1996 Feb, 17(2), 285 - 6
Herpes simplex virus (HSV) colitis in a bone marrow transplant recipient; Naik HR et al.; Herpes simplex virus (HSV) infections are common in bone marrow transplantation patients . Unusual sites may be involved, however colonic disease with HSV is rare . We report a successfully treated case of colitis due to HSV, cytomegalovirus, Clostridium difficile and graft-versus-host disease in an allogeneic marrow recipient.

J Antibiot (Tokyo), 1996 Feb, 49(2), 150 - 4
Antibiotics A21459 A and B, new inhibitors of bacterial protein synthesis . II . Structure elucidation; Ferrari P et al.; The structures of the antibiotics, active against a few Gram-negative bacteria and Clostridium difficile, were determined on the basis of physicochemical analyses on the intact molecules and on the acid hydrolysate of A21459 A . FAB-MS and 1H and 13C NMR investigations identified the amino acid units and determined their sequence . Antibiotics A21459 A and B are homodetic cyclic peptides constituted by eight amino acid units . They are glycine, methoxytryptophan, tryptophan, cysteine, alanine, sarcosine, dehydroalanine, and alpha-aminobutyric acid for A21459 A (alanine for A21459 B) . Cysteine and alanine condensed to form a thiazole moiety, according to the biosynthesis of thiazole containing antibiotics.

EMBO J, 1996 Feb 1, 15(3), 510 - 9
Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes; Lang P et al.; We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function . We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188 . This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity . However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol . Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation . Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.

Appl Environ Microbiol, 1996 Feb, 62(2), 662 - 7
Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms; Moriishi K et al.; The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA . Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D . The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817 . The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity) . When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C . botulinum type C strains 6812 and 6814 . These results suggest that some strains of C . botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.

J Neurochem, 1996 Feb, 66(2), 549 - 58
Lysophosphatidic acid-induced neurite retraction in PC12 cells: neurite-protective effects of cyclic AMP signaling; Tigyi G et al.; Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells . LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric Gi proteins . However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction . In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction . The neurite-protective effect of forskolin was blocked by Rp-adenosine 3',5'-phosphorothioate . Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in A126-1B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase . Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA . ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA-induced neurite retraction . LPA activates Gq- and Gi-linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway . The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.

J Neurochem, 1996 Feb, 66(2), 537 - 48
Lysophosphatidic acid-induced neurite retraction in PC12 cells: control by phosphoinositide-Ca2+ signaling and Rho; Tigyi G et al.; The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells . Neurite retraction was blocked by cytochalasin B and ADP-ribosylation of the small-molecular-weight G protein Rho by the Clostridium botulinum C-3 toxin . LPA induced a transient rise in the level of inositol 1,4,5-trisphosphate, and retraction was blocked by inhibitors of phospholipase beta . Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response . The activation of the phosphoinositide (PIP)-Ca2+ second messenger system played a permissive role in the morphoregulatory response . Blockers of protein kinase C--chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long-term phorbol ester treatment--all diminished neurite retraction by interfering with LPA-induced Ca2+ mobilization, which was required for the withdrawal of neurites . A brief 15-min treatment with 4 beta-phorbol 12-myristate 13-acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor . Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction . Although activation of the PIP-Ca2+ second messenger system appears necessary for the Rho-mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA-induced morphoregulatory response.

J Bacteriol, 1996 Feb, 178(4), 1200 - 3
Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein, CipA; Lytle B et al.; The Clostridium thermocellum cellulosomal scaffolding protein, CipA, acts as an anchor on the cellulose surface for the various catalytic subunits of the cellulosome, a large extracellular cellulase complex . CipA contains nine repeated domains that serve as receptors for the cellulosomal catalytic subunits, each of which carries a conserved, duplicated ligand sequence (DS) . Four representative CipA receptor domains with sequence dissimilarity were cloned and expressed in Escherichia coli . The interaction of these cloned receptor domains with the duplicated ligand sequence of CelS (expressed as a thioredoxin fusion protein, TRX-DSCelS), was studied by nondenaturing polyacrylamide gel electrophoresis . TRX-DSCelS formed a stable complex with each of the four receptor domains, indicating that CelS, the most abundant cellulosomal catalytic subunit, binds nonselectively to all of the CipA receptors . Conversely, the duplicated sequence of CipA (in the form of TRX-DSCipA), which is homologous to that of CelS, did not bind to any of the receptors under the experimental conditions.

J Bacteriol, 1996 Feb, 178(3), 871 - 80
Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri; Sohling B et al.; A region of genomic DNA from Clostridium kluyveri was cloned in Escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene . The gene region (6,575 bp) contained several open reading frames which encoded the coenzyme A (CoA)- and NADP+-dependent succinate-semialdehyde dehydrogenase (sucD), the 4-hydroxybutyrate dehydrogenase (4hbD), and a succinyl-CoA;CoA transferase (cat1), as analyzed by heterologous expression in E . coli . An open reading frame encoding a putative membrane protein (orfY) and the 5' region of a gene encoding a sigma 54-homologous sigma factor (sigL) were identified as well . Transcription was investigated by Northern (RNA) blot analysis . Protein sequence comparisons of SucD and 4HbD revealed similarities to the adhE (aad) gene products from E . coli and Clostridium acetobutylicum and to enzymes of the novel class (III) of alcohol dehydrogenases . A comparison of CoA-dependent aldehyde dehydrogenases is presented.

J Bacteriol, 1996 Feb, 178(3), 735 - 44
Repression of the Escherichia coli modABCD (molybdate transport) operon by ModE; Grunden AM et al.; The modABC gene products constitute the molybdate-specific transport system in Escherichia coli . Another operon coding for two proteins which diverges from the modABCD operon has been identified . The first gene of this operon codes for a 262-amino-acid protein, designated ModE (28 kDa), and the second genes codes for a 490-amino-acid protein . ModF (54 kDa) . The role of ModF has not yet been determined; however, mutations in modE depressed modABCD transcription even in the presence of molybdate, suggesting that ModE is a repressor . ModE, in the presence of 1 mM molybdate, repressed the production of plasmid-encoded ModA and ModB' proteins in an in vitro transcription-translation system . DNA mobility shift experiments confirmed that ModE binds to an oligonucleotide derived from the operator region of the modABCD operon . Further experimentation indicated that ModE binding to target DNA minimally requires an 8-bp inverted-repeat sequence, TAAC GITA . A highly conserved amino acid sequence, TSARNOXXG (amino acids 125 to 133), was identified in ModE and homologs from Azotobacter vinelandii, Haemophilus influenzae, Rhodobacter capsulatus, and Clostridium pasterianum . Mutants with mutations in either T or G of this amino acid sequence were isolated as "superrepressor" mutants . These mutant proteins repressed modABCD transcription even in the absence of molybdate, which implies that this stretch of amino acids is essential for the binding of molybdate by the ModE protein . These results show that molybdate transport in E . coli is regulated by ModE, which acts as a repressor when bound to molybdate.

Rev Prat, 1996 Jan 15, 46(2), 206 - 12
{Diarrhea in immune deficiency status}; Bouchaud O; With a prevalence of approximatively 50%, diarrhoea is a frequent event in immune deficiency of any cause . Because this condition is permanent in AIDS, the main characteristic of diarrhoea is chronicity . Non infectious causes are more common in conditions other than AIDS with, for exemple, intestinal injuries related to graft versus host disease or to chemotherapy toxicity . Among infectious causes, enteric parasitic diseases such as cryptosporidiosis or microsporidiosis are more commonly observed in the immunodeficiency related to HIV while viral infections due to cytomegalovirus or adenovirus seem possible in any case of severe troubles of the immunity . Gram-negative infections and Clostridium difficile colitis have to be diagnosed because efficient treatment is available . In these patients usually in bad general condition, because diarrhoea is often of unknown aetiology or non curable, the diagnostic and therapeutic strategy has to be pragmatic in order to control the symptoms without an excess of invasive procedures.

Rev Prat, 1996 Jan 15, 46(2), 184 - 8
{Infectious diarrhea in the aged}; Jeandel C et al.; Infectious diarrhoea in the elderly is associated with high morbidity and mortality and need early diagnosis and treatment . Polypathology, malnutrition, polytherapy, length of stay in the hospital and residence in nursing-home contribute to the increasing incidence and gravity of these diseases with aging . Viral gastroenteritis is responsible for epidemic in nursing-home residents . Bacterial gastrointestinal infections are primarily caused by enterotoxigenic agents inducing sporadic or epidemic infections food poisoning . Older adults are more particularly exposed to antibiotic-associated diarrhoea . Clostridium difficle has been increasingly recognized as a cause of pseudomembranous colitis in elderly and is associated with increased mortality . The approach to an old patient with suspected infectious diarrhoea should be to eliminate the likely noninfectious causes, to detect immediate complications due to hydroelectrolytic loss, to initiate early appropriated oral rehydration therapy so as to prevent dehydratation, to avoid the use of antimotility drugs, and to evaluate and to take over nutritional consequences of diarrhoea.

Rev Prat, 1996 Jan 15, 46(2), 171 - 6
{Diarrhea caused by antibiotic therapy}; Beaugerie L; Diarrhoea, or any change in bowel habits, occurs in up to 30% of the individuals treated by antimicrobial agents . Most cases of such diarrhoea are benign and secondary to a transient dysfunction of normal colonic flora induced by the antibiotic treatment . In some cases, the antibiotic-induced alteration of the normal gut flora leads to the establishment of pathogens, of which Clostridium difficile is the most important . C . difficile intestinal infection results in a wide spectrum of diseases, ranging from benign diarrhoea without colitis to life-threatening cases of relapsing pseudomembranous colitis.

J Am Vet Med Assoc, 1996 Jan 15, 208(2), 243 - 7
Hepatic abscesses in dogs: 14 cases (1982-1994); Farrar ET et al.; OBJECTIVES--To determine typical clinical signs and clinicopathologic findings in dogs with hepatic abscesses, to assess outcome of treatment, and to evaluate the role that abdominal ultrasonography has in the diagnosis of hepatic abscesses in dogs and in monitoring response to treatment . DESIGN--Retrospective case series . ANIMALS--14 dogs with hepatic abscesses . RESULTS--Anorexia and lethargy were the most common historical complaints, followed by vomiting and diarrhea . Physical abnormalities included fever, dehydration, signs of abdominal pain, hepatomegaly, and mucosal bleeding . Hematologic abnormalities included leukocytosis with neutrophilia, mild to moderate thrombocytopenia, and mild anemia . Serum biochemical abnormalities included high alkaline phosphatase and alanine aminotransferase activities and high bilirubin concentration; hypoalbuminemia and prolonged coagulation values were also reported . Abdominal radiography revealed hepatomegaly, poor abdominal detail, a hepatic mass, or splenomegaly in 9 dogs . Thoracic radiography revealed alveolar consolidation or mixed bronchial/interstitial pulmonary patterns in 6 dogs . Hypoechoic, heteroechoic, or hyperechoic masses were identified in all dogs in which ultrasonography was performed . Escherichia coli, Clostridium sp, Klebsiella pneumoniae, Enterococcus sp, Staphylococcus epidermidis, and S intermedius were the most common bacteria isolated from hepatic abscesses . Concurrent infections were identified in the biliary tract, spleen, blood, endocardium, lung, prostate gland, peritoneum, lymph nodes, salivary gland, or brain of several dogs . Seven dogs died or were euthanatized before definitive treatment could be initiated . One dog was successfully treated with antibiotics and was alive 12 months after medical treatment . Six dogs were treated surgically (ie, full or partial liver lobectomy, drainage, abdominal lavage) and medically (ie, antibiotic administration) . Five of these dogs survived and were alive 12 months after surgery . Ultrasonography was used to monitor response to treatment in several dogs . CLINICAL IMPLICATIONS--Hepatic abscesses are rare in dogs, but the clinical signs and clinicopathologic findings are similar to other inflammatory hepatic disease . Ultrasonography revealed abnormalities in all animals in which imaging studies were performed, and was successfully used to monitor response to treatment in several dogs . Medical and surgical treatments were used successfully to treat hepatic abscesses in dogs.

FEBS Lett, 1996 Jan 15, 378(3), 253 - 7
The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a; Nishiki T et al.; 125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II . Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a . Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes . The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II . These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

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