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 ad