Clostridia

Curr Treat Options Neurol, 2003 Jan, 5(1), 23 - 31
Botulism; Davis LE; Botulinum toxin is the most potent toxin known to humans and as little as 100 ng can be lethal . The toxin blocks peripheral cholinergic neurotransmission at the neuromuscular junction and cholinergic autonomic nervous system by introducing an endopeptadase enzyme into the presynaptic side of the synapse . The endopeptadase cleaves acetylcholine vesicle docking proteins that are required for the synapse to release acetylcholine into the synaptic cleft . Botulism occurs from consumption or inhalation of preformed botulinum toxin or growth of Clostridium botulinum bacteria in the infant gastrointestinal tract or within a wound . Growth of C . botulinum in the immature gut or wound will release botulinum toxin that reaches the circulation . All forms of botulism cause progressive weakness, bulbar signs (blurred vision, diplopia, mydriasis, dysphagia, and dysarthria), and respiratory failure with normal sensation and mentation . Treatment is aimed at 1) maintaining respiration via intubation and mechanical ventilation, 2) stopping progression of weakness by administration of botulinum antitoxin (equine trivalent botulinum antitoxin for adults and botulism immune-globulin intravenous-human for infant botulism), and 3) preventing complications from weeks of paralysis with good supportive care . The source of the botulinum toxin should be identified to prevent additional cases . Patients can recover normal muscle strength within weeks to months, but usually complain of fatigue for years.

Am J Clin Pathol, 2003 Jan, 119(1), 45 - 9
Clinical usefulness of components of the Triage immunoassay, enzyme immunoassay for toxins A and B, and cytotoxin B tissue culture assay for the diagnosis of Clostridium difficile diarrhea; Massey V et al.; We studied 557 nonduplicate fresh stool specimens from adult patients clinically suspected of having Clostridium difficile-associated diarrhea . All samples were tested in parallel with an in-house cytotoxin B tissue culture assay (CTA), the C DIFFICILE TOX A/B II test (TA/B; TechLab, Blacksburg, VA), and the Triage Micro C DIFFICILE Panel (Biosite Diagnostics, San Diego, CA) . The Triage device detects toxin A (TA) and glutamate dehydrogenase (GDH) simultaneously . Of the specimens, 350 were negative and 95 were positive for all markers . Another 112 specimens yielded discrepant results . The CTA found 143 positive specimens . Results of the components of the Triage and TA/B were compared separately with those of CTA . GDH was the most sensitive but least specific marker, whereas TA and TA/B were less sensitive but highly specific . Because of these attributes and a quick turnaround time, GDH would be the best screening test for C difficile-associated diarrhea . CTA detected the highest number of cases of C difficile-associated diarrhea and would be most useful as a confirmatory test for GDH-positive and TA-negative specimens.

Clin Microbiol Infect, 2002 Dec, 8(12), 814 - 22
In vitro anti-anaerobic activity of the cephalosporin derivative RWJ 54428, compared to seven other compounds; Hoellman DB et al.; Agar dilution MIC was used to test the activity of RWJ 54428, a new cephalosporin derivative, compared to imipenem, meropenem, ceftriaxone, piperacillin, piperacillin-tazobactam, clindamycin and metronidazole against 363 anaerobes isolated from clinical specimens . RWJ 54428 had low MICs against most beta-lactamase-negative Gram-negative rods, and all Gram-positive strains except Clostridium difficile . Imipenem and meropenem had the lowest MICs (MIC50s of 0.125 mg/L and MIC90s of 1.0 mg/L) . Piperacillin-tazobactam, clindamycin and metronidazole were active against most strains, and ceftriaxone was active mainly against beta-lactamase-negative organisms.

J Clin Microbiol, 2003 Jan, 41(1), 509 - 11
Clostridium difficile brain empyema after prolonged intestinal carriage; Gravisse J et al.; Clostridium difficile, the most common cause of antibiotic-associated diarrhea, is occasionally isolated from extraintestinal sites and is usually found as part of a polymicrobial flora . We report a case of brain empyema that occurred after the recurrent intestinal carriage of a nontoxigenic strain of C . difficile . Brain abscess cultures contained both toxigenic and nontoxigenic isolates . Pulsed-field gel electrophoresis showed that nontoxigenic isolates from the intestine and from the brain were identical.

Bioorg Med Chem, 2003 Feb 6, 11(3), 421 - 6
A quantitative structure-activity relationship study on some matrix metalloproteinase and collagenase inhibitors; Kumar D et al.; A quantitative structure-activity relationship (QSAR) study is made on some hydroxamic acid-based inhibitors of matrix metalloproteinases (MMPs) and a bacterial collagenase, namely Clostridium histolyticum collagenase (ChC), that also belongs to an MMP family, M-31, using Kier's valence molecular connectivity index (1)chi(v) of the substituents and electrotopological state (E-state) indices of some atoms . The results indicate that out of the four MMPs (MMP-1, MMP-2, MMP-8, and MMP-9) studied, MMP-2 and MMP-9 can be structurally quite similar, but widely differing from MMP-1 and MMP-8 and ChC . For MMP-2 and MMP-9, the inhibition activity of compounds is shown to depend on both (1)chi(v )and E-state indices, while for MMP-1 and MMP-8 it is shown to depend only on E-state indices and for ChC only on (1)chi(v) . However, in all the cases, an aromatic group like C(6)F(5) or 3-CF(3)-C(6)H(4) attached to SO(2) moiety in the compounds is indicated to be equally beneficial, due to probably the involvement of fluorine atom(s) in charge-charge interactions with the Zn(2+) ion of the enzymes or in the formation of the hydrogen bonds with some sites of the receptors.

J Bacteriol, 2003 Jan, 185(2), 504 - 12
Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis; Arai T et al.; Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety . To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized . Isothermal titration calorimetric studies showed that the association constants (K(a)) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10(4) and 6.4 x 10(4) M(-1), respectively, and that the binding of CBM30 to these ligands is enthalpically driven . Qualitative analyses showed that CBM30 had strong affinity for cellulose and beta-1,3-1,4-mixed glucan such as barley beta-glucan and lichenan . Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley beta-glucan and lichenan . By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates . These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

J Bacteriol, 2003 Jan, 185(2), 391 - 8
CelI, a noncellulosomal family 9 enzyme from Clostridium thermocellum, is a processive endoglucanase that degrades crystalline cellulose; Gilad R et al.; The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G . P . Hazlewood, K . Davidson, J . I . Laurie, N . S . Huskisson, and H . J . Gilbert, J . Gen . Microbiol . 139:307-316, 1993) . We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein . The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b . The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70 degrees C, respectively, using carboxymethylcellulose as a substrate . Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase . A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates . A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined . This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2141 - 6
Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen . nov., comb . nov; Duncan SH et al.; Two newly isolated strains of obligately anaerobic bacteria from human faeces are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon . These strains, along with Fusobacterium prausnitzii ATCC 27768(T) and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products . A new finding is that all four strains produce D-lactate, but not L-lactate . The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid . The DNA G+C content of the four strains is 47-57 mol% . Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group) . It is proposed that a new genus, Faecalibacterium gen . nov . be created; this genus should include Faecalibacterium prausnitzii gen . nov., comb . nov . ATCC 27768(T) (= NCIMB 13872(T)) (formerly Fusobacterium prausnitzii) as the type species together with ATCC 27766 and the newly isolated strains A2-165 and L2-6.

Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 706 - 11
The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis; Frisch C et al.; Clostridium difficile toxin A, the chief pathogenicity factor of the antibiotic-associated pseudomembranous colitis, is an intracellular acting cytotoxin that reaches its targets, the Rho GTPases, after receptor-mediated endocytosis . The C-terminal part, constructed of repetitive peptide elements, is thought to bind to a lot of carbohydrate containing receptor molecules to induce clustering and endocytosis . To study which part of the receptor-binding domain is in charge of addressing toxin A into the target cells, we studied the functional, i.e., endocytosis-inducing, binding of toxin A . By a competition assay between various receptor-binding fragments of toxin A and the holotoxin A we found that the complete receptor-binding domain, encompassing the entire repetitive elements, but not parts of it, is necessary for binding-induced endocytosis . The receptor binding domain itself shows weaker competition with holotoxin A than the fragment consisting of receptor-binding domain plus intermediary part of the toxin . All toxin A fragments that compete with holotoxin A are capable of inducing their own endocytosis . Thus, the entire receptor-binding domain, covering the C-terminal third of the toxin A molecule, is responsible for cell uptake of toxin A and the intermediary part contributes to the correct folding and assembly of the repetitive domains.

Pediatr Infect Dis J, 2002 Dec, 21(12), 1173 - 4
Recurrent crepitant cellulitis caused by Clostridium perfringens; Bryant P et al.; A previously healthy 13-year-old boy developed extensive subcutaneous emphysema of the lower limb after a penetrating injury to the knee . Clostridium perfringens was isolated from the wound . Despite surgical debridement and appropriate antibiotics, the emphysema recurred, and prolonged antibiotic treatment was required . This case highlights the distinction between gas gangrene and the lesser known entity of clostridial crepitant cellulitis.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 73 - 5
{Study on the effect of a biostimulant on the growth and toxigenic function of Clostridium tetani strain Copenhagen-471}; Garib FIu et al.; Bakstim, a new biostimulating preparation obtained from the organs of the immune system of animals, was developed . The impact of Bakstim on the growth and toxigenic function of C . tetani production strain Copenhagen-471 was evaluated . The addition of the preparation to Gluzman commercial medium for obtaining tetanus toxoid led to an increase in the yield of bacterial biomass from 1.9 to 4-fold and an increase in the toxoid production from 2 to 2.8-fold . The optimum concentration of this biostimulant ensuring the maximum yield of tetanus toxin from the production culture was determined (1,000 mg/l) . Bakstim will supposedly be used as additive to nutrient media for the production of tetanus toxoid.

Int J Food Microbiol, 2003 Jan 26, 82(1), 81 - 6
Evaluation of different methods for the detection of Clostridium perfringens phosphatases; Eisgruber H et al.; In order to detect phosphatase activity a total of 137 isolates from 12 Clostridium (C.) species were examined via fluorescence on SCA-agar with methylumbelliferyl phosphate (SCA-MUP), via APIZYM and RAPID ID 32 A as well as using a phosphatase reagent containing 1-naphthyl phosphate . Fluorescence on SCA-MUP showed the presence of acid or alkaline phosphatase in almost all isolates examined . Likewise, acid or alkaline phosphatase could be detected via APIZYM and RAPID ID 32 A in most strains and species . Opposed to this, the majority of the Clostridium bifermentans isolates showed a positive reaction exclusively on SCA-MUP . On the other hand, the phosphatase reagent for the detection of acid phosphatase lead to unambiguously positive results only when examining Clostridium perfringens isolates . Therefore, the SCA-MUP-agar, in contrast to the phosphatase reagent, was proven to be unsuitable for the identification of C . perfringens via detection of acid phosphatase . Using the phosphatase reagent the activity of this enzyme was detected in 95.1% of the C . perfringens isolates included in the study . In addition, the phosphatase reagent showed identical reactions after a 24 h incubation at 37 degrees C and when used on cultures incubated for 6 h at 44 degrees C in the case of 98.5% of the C . perfringens isolates.

Ann Pharmacother, 2003 Jan, 37(1), 127 - 31
Management of botulism; Robinson RF et al.; OBJECTIVE: To provide a concise review of the presentation and treatment of botulism . DATA SOURCES: Searches of MEDLINE (1966-November 2001), tertiary references, and public and government Internet sites were conducted . STUDY SELECTION: All articles and additional references from those articles were thoroughly evaluated . DATA SYNTHESIS: Clostridium botulinum toxin blocks acetylcholine release in a dose-dependent fashion, resulting in acute symmetric diplopia, dysarthria, dysphonia, dysphagia, and possible neurologic sequelae despite the route of exposure (i.e., food-borne, wound, intestinal, inhalation) . Disease secondary to genetically engineered C . botulinum may differ from that of inadvertent exposure . Present treatment is primarily supportive care, respiratory support, rapid decontamination, and antitoxin administration (i.e., trivalent, pentavalent, heptavalent antitoxin) . Early initiation of antitoxin limits the extent of paralysis, but does not reverse it . CONCLUSIONS: Supportive care and the use of antitoxin have been effective in the treatment of botulism from food-borne, intestinal, and wound exposure . However, the effectiveness of antitoxin in the treatment of inhaled C . botulinum has not been proven.

Folia Microbiol (Praha), 2002, 47(5), 559 - 64
Identification and characterization of Clostridium paraputrificum, a chitinolytic bacterium of human digestive tract; Simunek J et al.; A strictly anaerobic, mesophilic and chitinolytic bacterial strain was isolated from human feces . Based on morphological and physiological properties and 16S rRNA sequence analysis the strain was identified as Clostridium paraputrificum . The strain utilized chitin and N-acetyl-D-glucosamine, grew on glucose and hydrolyzed starch . Cultivation of the strain with colloidal chitin as the growth substrate resulted in the production of gas (hydrogen and carbon dioxide) and formation of acetate and lactate (21.6 and 18.9 mmol/L, respectively) and only small quantities of propionate and butyrate (1.7 and 2.6 mmol/L, respectively) . In the course of a 10-d cultivation with chitin, the endochitinase activity was detected after 1 d and gradually increased, reaching maximum after 3 d (251 nkat/L N-acetyl-D-glucosamine) . The beta-N-acetylglucosaminidase activity appeared just at the beginning of the cultivation, increased to day 2 and then remained nearly constant . More than 90% of chitin added was degraded within 2 d of cultivation . On the zymogram of the extracellular chitinolytic complex were visible at least 6 isoenzymes with molar mass 43.5-65.0 kDa . The temperature optimum of endochitinase and beta-N-acetylglucosaminidase activities was 50 degrees C; the optimum activity of both enzymes was found at pH 4-6.

J Agric Food Chem, 2003 Jan 1, 51(1), 76 - 81
Bioactivity of Backhousia citriodora: antibacterial and antifungal activity; Wilkinson JM et al.; Backhousia citriodora products are used as bushfoods and flavorings and by the aromatherapy industry . The antimicrobial activity of 4 samples of B . citriodora oil, leaf paste, commercial tea (0.2 and 0.02 g/mL), and hydrosol (aqueous distillate) were tested against 13 bacteria and 8 fungi . Little or no activity was found to be associated with the leaf tea and hydrosol, respectively . Leaf paste displayed antimicrobial activity against 7 bacteria including Clostridium perfringens, Pseudomonas aeruginosa, and a hospital isolate of methicillin resistant Staphylococcus aureus (MRSA) . The 4 essential oils were found to be effective antibacterial and antifungal agents; however, variation was apparent between oils that did not correlate with citral content . The antimicrobial activity of B . citriodoraessential oils was found to be greater than that of citral alone and often superior to Melaleuca alternifolia essential oil . B . citriodora has significant antimicrobial activity that has potential as an antiseptic or surface disinfectant or for inclusion in foods as a natural antimicrobial agent.

J Gen Appl Microbiol, 1998 Oct, 44(5), 327 - 335
Glucoamylase from Thermoanaerobacterium thermosaccharolyticum: Sequence studies and analysis of the macromolecular architecture of the enzyme; Ducki A et al.; A chromosomal DNA fragment with a length of 2,025 bp, carrying the structural gene coding for glucoamylase in Thermoanaerobacterium thermosaccharolyticum, was cloned and sequenced . It coded for 695 amino acids, representing a polypeptide with a predicted molecular mass of 77.5 kDa . The deduced amino acid sequence exhibited high homologies with the glucoamylase sequence of another bacterial glucoamylase (Clostridium sp . G0005) and with fungal glucoamylases . The catalytic domain (amino acids 271 to 695) of the T . thermosaccharolyticum enzyme shared a high degree of similarity (five conserved regions) with the catalytic domain of Aspergillus awamori glucoamylase . By comparing the secondary structure of the sequence of the catalytic domain of the T . thermosaccharolyticum enzyme with that of glucoamylase from A . awamori, and on the basis of X-ray crystallographic data available for the A . awamori enzyme, it turned out that, most probably, both enzymes have a catalytic domain organized into an "(alpha/alpha)(6)-barrel" and an overall size and shape that is very similar . These findings confirm and extend our working model for the macromolecular architecture of the T . thermosaccharolyticum glucoamylase obtained, in earlier experiments, by electron microscopy of negatively stained isolated enzyme molecules . Antibodies for an enzyme-specific peptide located near the active site were successfully applied for inhibition studies of enzyme activity and for electron microscopic epitope mapping . A study comparing the site of attachment of this kind of antibody to the T . thermosaccharolyticum glucoamylase molecule with the expected attachment site as deduced from the A . awamori enzyme structure confirmed the close similarity of both glucoamylases regarding the macromolecular architecture of that part of the enzyme carrying the catalytic center, though helices H9, H10, and H11 in peripheral parts of the A . awamori enzyme are missing in the T . thermosaccharolyticum enzyme.

J Gen Appl Microbiol, 1999 Aug, 45(4), 163 - 168
Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin; Prabakaran S et al.; A 36-kDa trypsin inhibitor was purified from Clostridium botulinum type E culture supernatant by multiple molecular sieve and ion exchange chromatographic steps . The sequence of the amino-terminal 13 amino acid residues of this single-chain protein is Asn.Gln.Glu.Val.Phe.Asn.Met.Pro.Lys.Phe.Ser.Thr.Ala- . This novel protein that also inhibits chymotrypsin is produced by an organism that does not appear to produce any protease.

J Gen Appl Microbiol, 1997 Oct, 43(5), 249 - 255
Isolation and characterization of anaerobic indole- and skatole-degrading bacteria from composting animal wastes; Kohda C et al.; Four species of indole-degrading Clostridium and 3 species of skatole-degrading Clostridium were isolated from piggery or chicken manure composting processes . Since type strains of respective isolates did not degrade these compounds, the degradability of the compounds was a novel characteristic . All isolates were mesophilic . The maximum growth allowance concentrations of these isolates were 300 to 800 mg/l in indole and 100 to 300 mg/l in skatole . All isolates showed better growth and utilization of indolic compounds in nutrient-rich medium than in minimal medium . Skatole-degrading isolates degraded some substituted indoles tested, 3-indoleacetic acid, indole and oxindole, but did not degrade 1-methylindole, 2-methylindole, isatin or anthranilic acid . On the other hand, indole-degrading isolates degraded only oxindole . The growth of Clostridium malenominatum A-3 was inhibited by a low concentration (0.005%) of indole or skatole, even when 200-fold excess glucose was present in the medium . When 0.03% indole or skatole was added to the medium, C . malenominatum A-3 showed a lag phase for about 10 and 70 h, respectively . When 0.01% of these compounds was added to the medium, the uptake of glucose was inhibited . C . malenominatum A-3 degraded these compounds under nutrient-rich and minimal conditions.

Antimicrob Agents Chemother, 2003 Jan, 47(1), 337 - 41
In vitro activities of daptomycin, vancomycin, quinupristin- dalfopristin, linezolid, and five other antimicrobials against 307 gram-positive anaerobic and 31 Corynebacterium clinical isolates; Goldstein EJ et al.; The activities of daptomycin, a cyclic lipopeptide, and eight other agents were determined against 338 strains of gram-positive anaerobic bacteria and corynebacteria by the NCCLS reference agar dilution method with supplemented brucella agar for the anaerobes and Mueller-Hinton agar for the corynebacteria . The daptomycin MICs determined on Ca(2+)-supplemented (50 mg/liter) brucella agar plates were one- to fourfold lower than those determined in unsupplemented media . Daptomycin was highly active (MICs, <or=2 microg/ml) against many strains including 36 of 37 peptostreptococci, 37 of 48 isolates of the Eubacterium group, and all strains of Propionibacterium spp., Clostridium perfringens, Clostridium difficile, and other Clostridium spp . It was fourfold or greater more active than vancomycin against Clostridium innocuum and 16 of 34 strains of vancomycin-resistant lactobacilli . Three strains of C . difficile for which quinupristin-dalfopristin and linezolid MICs were >8 microg/ml were inhibited by <1 microg of daptomycin per ml . Daptomycin MICs were >or=4 microg/ml for most strains of Clostridium clostridioforme, Clostridium paraputrificum, Clostridium tertium, and Clostridium ramosum; the isolates were generally more resistant to other antimicrobials . Daptomycin was two- to fourfold less active against Actinomyces spp . than vancomycin, quinupristin-dalfopristin, or linezolid . Twenty-nine of 31 strains of Corynebacterium spp., including Corynebacterium jeikeium, Corynebacterium amycolatum, and Corynebacterium pseudodiphtheriticum, were inhibited by <or=0.25 microg of daptomycin per ml . For two strains of "Corynebacterium aquaticum," 8 microg of daptomycin per ml was required for inhibition . Daptomycin demonstrated very good activities against a broad range of gram-positive organisms including vancomycin-resistant C . innocuum and lactobacillus strains and quinupristin-dalfopristin- and linezolid-resistant C . difficile strains.

J Mol Biol, 2003 Jan 17, 325(3), 471 - 83
Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin; Tsuge H et al.; Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals . It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib) . Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton . Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues . The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region . However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT . The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380 . Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity . At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase . Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction .

J Biol Chem, 2003 Mar 7, 278(10), 7956 - 63 Epub 2002 Dec 19.
R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile; Chaves-Olarte E et al.; Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease . Several toxin A-negative strains from C . difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect . Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures . Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect . Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins . Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1 . Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation . Implications for the pathophysiology of C . difficile-induced diarrhea are discussed.

J Hosp Infect, 2003 Jan, 53(1), 1 - 5
General outbreaks of infectious intestinal disease (IID) in hospitals, England and Wales, 1992-2000; Meakins SM et al.; Between 1992 and 2000, 26.6% (1,396/5,257) of all general outbreaks of infectious intestinal disease (IID) reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC) occurred in hospitals . Over 29,000 patients and staff were affected and the mortality risk was higher than for outbreaks in other settings {relative risk 2.00 (95% CI: 1.52-2.63) P<0.001} . Person-to-person spread was the predominant mode of transmission . The mortality risk was highest in foodborne disease outbreaks {relative risk 3.22 (95% CI: 1.41-7.36); P=0.003} . Most outbreaks occurred between November and April . The pathogens most frequently reported were Norwalk-like virus (NLV) (54%) and Clostridium difficile (12.6%) . These findings emphasize the public health importance of outbreaks of IID in hospitals, especially during the winter when pressures on hospitals are at their height.

J Cell Physiol, 2003 Feb, 194(2), 127 - 38
Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts; Slice LW et al.; Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566) . However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated . In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q) . Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11) . GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB) . Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter . Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells . Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction . Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin . These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts .

J Biol Chem, 2003 Feb 21, 278(8), 5956 - 62 Epub 2002 Dec 18.
Down-regulation of Rac-1 GTPase by Estrogen; Laufs U et al.; Rac1 GTPase is essential for the activation of the NAD(P)H oxidase complex and, thereby, regulates the release of reactive oxygen species (ROS) in the vessel wall . 17 beta-estradiol (E2) inhibits vascular ROS production . To elucidate the underlying molecular mechanisms we investigated the potential regulation of Rac1 by E2 in vascular smooth muscle cells . Treatment of vascular smooth muscle cells with angiotensin II as well as overexpression of the constitutively active mutant RacL61 increased ROS release as assessed by dichlorofluorescein fluorescence, whereas inhibition of Rac1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative RacN17 inhibited ROS production . Treatment with E2 (100 nm) completely prevented angiotensin II-induced NAD(P)H oxidase activity and ROS production . E2 time and concentration dependently decreased angiotensin II-induced and basal Rac1 mRNA and protein expression as well as Rac1 activity . Down-regulation of Rac1 expression by E2 was mediated by inhibition of gene transcription (nuclear run-on assays), but E2 had no effect on Rac1 mRNA stability . Regulation of Rac1 was mediated by estrogen receptors since co-incubation with ICI 182.780 prevented down-regulation of Rac1 . To test these observations in vivo, ovariectomized spontaneously hypertensive rats were treated with E2 or vehicle . Real-time PCR and Western blotting showed reduction of aortic Rac1 mRNA and protein by 32 and 58%, respectively . Furthermore, down-regulation of Rac1 by E2 was observed in human mononuclear cells of women with elevated E2 levels after controlled ovarian hyperstimulation . Rac1 GTPase gene-transcription and activity is regulated by 17 beta-estradiol, which may be an important molecular mechanism contributing to the cardiovascular effects of estrogens.

Nippon Yakurigaku Zasshi, 2002 Nov, 120(1), 116P - 118P
{Isolation and determination of an antidote for botulinum neurotoxin from black tea extract}; Sawamura S et al.; The botulinum neurotoxin produced by Clostridium botulinum exhibits the strongest neurotoxicity, and causes botulism in mammals . We have found an inactivator for clostridial neurotoxins in black tea extract (thearubigin fraction) as a natural foodstuff . In this study, we have isolated and identified the inactivators . The activity against the neuromuscular blocking action of botulinus neurotoxin type A was examined in mouse phrenic nerve diaphram preparation . The purification procedure of the inactivators was as follows . Tea was extracted with aqueous acetone, and then filtrated and lyophilized . It was also extracted with n-hexane, chloroform, ethyl acetate, n-butylalchol and water, so the activity of the antidote was recognized to be in the n-butylalchol layer (named the thearubigin fraction) . A two-step reversed phase HPLC was developed for the thearubigin fraction . Three flavonoids were found to have the major activity . The structural elucidation of the compounds by means of NMR spectrascopy revealed, kaempfenol-3-O-{glc-(6-1)-rha-(3-1)-glc};keampfetrin, kaempferol-3-O-{glc-(6-1)-rha};nicotiflorin and quercetin glycoside.

Am J Physiol Cell Physiol, 2003 Apr, 284(4), C1073 - 82 Epub 2002 Dec 18.
Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction; Liu TS et al.; We determined whether the cytoprotective heat shock protein HSP72 protects against the injurious effects of Clostridium difficile toxin A (TxA) on intestinal epithelial cells . Colonic epithelial Caco-2/bbe (C2) cells were stably transfected with HSP72 antisense (C2AS) or vector only (C2VC), resulting in low and high HSP72 expression, respectively . Measurements of epithelial barrier integrity, mitochondrial function, and apoptosis activation were assessed after TxA exposure . HSP72 and RhoA interactions were evaluated with immunoprecipitations . In C2AS cells, TxA was associated with a greater decrease in transepithelial resistance (TER), an increase in {(3)H}mannitol flux, and increased dissociation of perijunctional actin . Although HSP72 binds RhoA, it failed to prevent RhoA glucosylation . TxA caused a more rapid decrease in ATP, release of cytochrome c, and activation of caspase-9 in C2AS cells . To determine whether ATP depletion decreases TER, we treated cells with antimycin A, which caused a decline in TER . We conclude that HSP72 may protect intestinal epithelial cells from TxA-mediated damage through several mechanisms, including actin stabilization, mitochondrial protection, and inhibition of apoptosis activation, but not by prevention of RhoA glucosylation.

Can J Microbiol, 2002 Oct, 48(10), 911 - 21
Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep; Min BR et al.; Condensed tannins in forage legumes improve the nutrition of sheep by reducing ruminal degradation of plant protein and increasing crude protein flow to the intestine . However, the effects of condensed tannins in forage legumes on rumen bacterial populations in vivo are poorly understood . The aim of this study was to investigate the specific effects of condensed tannins from Lotus corniculatus on four proteolytic rumen bacteria in sheep during and after transition from a ryegrass (Lolium perenne)-white clover (Trifolium repens) diet (i.e., low condensed tannins) to a Lotus corniculatus diet (i.e., higher condensed tannins) . The bacterial populations were quantified using a competitive polymerase chain reaction . Lotus corniculatus was fed with or without ruminal infusions of polyethylene glycol (PEG), which binds to and inactivates condensed tannins, enabling the effect of condensed tannins on bacterial populations to be examined . When sheep fed on ryegrass-white clover, populations of Clostridium proteoclasticum B316T, Butyrivibrio fibrisolvens C211a, Eubacterium sp . C12b, and Streptococcus bovis B315 were 1.5 x 10(8), 1.1 x 10(6), 4.6 x 10(8), and 7.1 x 10(6) mL(-1), respectively . When the diet was changed to Lotus corniculatus, the average populations (after 8-120 h) of C . proteoclasticum, B . fibrisolvens, Eubacterium sp., and S . bovis decreased (P < 0.001) to 2.4 x 10(7), 1.1 x 10(5), 1.1 x 10(8), and 2.5 x 10(5) mL(-1), respectively . When PEG was infused into the rumen of sheep fed Lotus corniculatus, the populations of C . proteoclasticum, B . fibrisolvens, Eubacterium sp., and S . bovis were higher (P < 0.01-0.001) than in sheep fed Lotus corniculatus without the PEG infusion, with average populations (after 8-120 h) of 4.9 x 10(7), 3.8 x 10(5), 1.9 x 10(8), and 1.0 x 10(6), respectively . Sheep fed the Lotus corniculatus diet had lower rumen proteinase activity, ammonia, and soluble nitrogen (P < 0.05-0.001) than sheep that were fed Lotus corniculatus plus PEG . The Lotus corniculatus diet reduced rumen nitrogen digestibility (P < 0.05) and ammonia pool size and increased the flow of undegraded feed nitrogen to the abomasum . The nitrogen intake, rumen non-ammonia nitrogen pool size, rumen microbial non-ammonia nitrogen pool size, and abomasal microbial non-ammonia nitrogen fluxes were similar both in sheep fed only Lotus corniculatus and in sheep fed Lotus corniculatus plus PEG, but nonmicrobial non-ammonia nitrogen flux to the abomasum was higher (P < 0.01) for the sheep fed only Lotus corniculatus . Although condensed tannins in Lotus corniculatus reduced the populations of some proteolytic bacteria, total ruminal microbial protein and microbial protein outflow to the abomasum were unchanged, suggesting a species-specific effect of condensed tannins on bacteria in the rumen.

Lett Appl Microbiol, 2003, 36(1), 41 - 5
Variability in spore germination response by strains of proteolytic Clostridium botulinum types A, B and F; Alberto F et al.; AIMS: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum . METHODS AND RESULTS: An automated turbidometric method was used to follow the fall in optical density . Spores of proteolytic Cl . botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions . Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl . botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B . cereus . CONCLUSIONS: Unlike spores of nonproteolytic Cl . botulinum, all proteolytic Cl . botulinum germinate in hydrophobic l-amino acids without l-lactate . However, a great variability of response to germinant is evidenced between the species . SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of a model strain to study germination of Cl . botulinum spores should consider the variability in sensitivity to germinants shown in this work . In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.

J Physiol, 2002 Dec 15, 545(Pt 3), 879 - 86
Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs; Chowdhury HH et al.; We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events . To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin . The average amplitude of exocytic events was not significantly different in control and in CST-treated cells . However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls . The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls . In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1 . In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1) . To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied . The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different . The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis . While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton.

Life Sci, 2003 Jan 3, 72(7), 843 - 50
Inhibition of collagenase and metalloproteinases by aloins and aloe gel; Barrantes E et al.; The effects of Aloe barbadensis gel and aloe gel constituents on the activity of microbial and human metalloproteinases have been investigated . Clostridium histolyticum collagenase (ChC) results dose-dependently inhibited by aloe gel and the activity-guided fractionation led to an active fraction enriched in phenolics and aloins . Aloins have been shown to be able to bind and to inhibit ChC reversibly and non-competitively . Aloe gel and aloins are also effective inhibitors of stimulated granulocyte matrix metalloproteinases (MMPs) . The remarkable structural resemblances between aloins and the pharmacophore structure of inhibitory tetracyclines, suggest that the inhibitory effects of aloins are via an interaction between the carbonyl group at C(9) and an adjacent hydroxyl group of anthrone (C(1) or C(8)) at the secondary binding site of enzyme, destabilizing the structure of granulocyte MMPs.

J Biol Chem, 2003 Feb 14, 278(7), 4882 - 91 Epub 2002 Dec 10.
Cooperation of Gq, Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid; Yuan J et al.; To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control . In RK and Galpha(12/13) KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm . In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells . However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q) . LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B . Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha(12/13) KO cells . Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue . Our results indicate that G(q) is necessary but not sufficient to mediate LPA-induced PKD activation . In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells . We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.

Microbiol Immunol, 2002, 46(10), 647 - 55
Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin; Nagahama M et al.; Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC) . In this study we examined the role of the C-domain (251-370 residues; CP251- 370) in biological activities of the toxin . The N-domain (1-250 residues; CP1- 250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them . A hybrid protein (BC-CP251-370) consisting of BcPLC and CP251- 370 bound to the red cells and lysed them . Incubation of CP1-250 with CP251-370 completely complemented hemolytic and PLC activities . CP251-370 also conferred hemolytic activity on BcPLC . CP251-340 (251-340 residues) significantly stimulated PLC activity of CP1-250), but did not confer hemolytic activity on CP1-250 . Kinetic analysis suggested that CP251-370 increased affinity toward the substrate of CP1-250 . The results suggested that CP251-370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1-250 . Acrylodan-labeled CP251-370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251-370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251-370 to a hydrophobic environment . These observations suggest that interaction of CP251-370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1-250.

Mar Pollut Bull, 2002 Oct, 44(10), 1063 - 70
The effect of the new Massachusetts Bay sewage outfall on the concentrations of metals and bacterial spores in nearby bottom and suspended sediments; Bothner MH et al.; Since the new outfall for Boston's treated sewage effluent began operation on September 6, 2000, no change has been observed in concentrations of silver or Clostridium perfringens spores (an ecologically benign tracer of sewage), in bottom sediments at a site 2.5 km west of the outfall . In suspended sediment samples collected with a time-series sediment trap located 1.3 km south of the outfall, silver and C . perfringens spores increased by 38% and 103%, respectively, in post-outfall samples while chromium, copper, and zinc showed no change . All metal concentrations in sediments are <50% of warning levels established by the Massachusetts Water Resources Authority . An 11-year data set of bottom sediment characteristics collected three times per year prior to outfall startup provides perspective for the interpretation of post-outfall data . A greater than twofold increase in concentrations of sewage tracers (silver and C . perfringens) was observed in muddy sediments following the exceptional storm of December 11-16, 1992 that presumably moved contaminated inshore sediment offshore.

Clin Infect Dis, 2002 Dec 15, 35(12), 1457 - 62 Epub 2002 Dec 02.
Clostridium difficile-associated diarrhea: epidemiological data from Western Australia associated with a modified antibiotic policy; Thomas C et al.; The incidence of Clostridium difficile-associated diarrhea (CDAD) has increased dramatically in hospitals worldwide during the past 2 decades . In Western Australia, this increase was most obvious during the 1980s, when there was also an increase in the use of third-generation cephalosporin antibiotics . A study of the epidemiology of CDAD and the use of third-generation cephalosporins during 1993-2000 was undertaken . From 1993 through 1998, the incidence of CDAD remained relatively stable (2-3 cases per 1000 discharges annually) . Then, a significant decrease in the incidence occurred, from 2.09 cases per 1000 discharges (95% confidence interval {CI}, 1.71-2.47) in 1998 to 0.87 cases per 1000 discharges (95% CI, 0.63-1.11) in 1999 (P<.0001); this decrease persisted into 2000 . A decrease in third-generation cephalosporin use occurred during the period of the study because of changes in the prescribing policy . These findings suggest that a reduction in the use of third-generation cephalosporins can reduce the occurrence of CDAD.

Pediatr Surg Int, 2002 Oct, 18(7), 586 - 90 Epub 2002 Sep 21.
Probiotics up-regulate MUC-2 mucin gene expression in a Caco-2 cell-culture model; Mattar AF et al.; Enteral probiotics such as Lactobacillus casei GG (LGG) have been used in the treatment of a variety of intestinal disorders in infants and children, including diarrhea, malabsorption, and Clostridium difficile colitis . Previous studies have identified the gene locus for mucin (MUC-2) and its expression in Caco-2 cells . Others have demonstrated that mucin, located on the surface of the intestinal epithelium, inhibits bacterial translocation (BT) . We previously demonstrated that both mucin and the probiotic bacterium LGG have an inhibitory effect on BT in both an in-vitro Caco-2 cell model and a neonatal rabbit model . We hypothesized that the decline in BT by LGG is mediated by up-regulation of epithelial MUC-2 . Human enterocyte Caco-2 cells were grown to confluence and incubated at 37 degrees C with either medium (control group) or 10(4) or 10(8) LGG for 180 min . Non-adherent LGG was washed away . Caco-2 cells were then lysed, purified, and quantified for MUC-2 protein and mRNA . The addition of LGG to the enterocyte monolayer surface resulted in significantly ( P < 0.05) increased MUC-2 expression compared to the untreated monolayers . Protein densities for MUC-2 significantly ( P < 0.05) increased with LGG . Density (expressed as ratio to control group) was 8.6 +/- 1.3 in the low-dose group (10(4) LGG) and 15.6 +/- 2.3 in the high-dose group (10(8) LGG) . LGG may thus bind to specific receptor sites on the enterocyte and stimulate the up-regulation of MUC-2, resulting in increased inhibition of BT.

Gynecol Oncol, 2002 Dec, 87(3), 252 - 9
Lysophosphatidic acid induces focal adhesion assembly through Rho/Rho-associated kinase pathway in human ovarian cancer cells; Sawada K et al.; OBJECTIVE: The level of lysophosphatidic acid (LPA) is elevated in patients with ovarian cancer, and LPA has been reported to have a pivotal role in cancer dissemination . In the current study, the effect of LPA on the motility of ovarian cancer cells was investigated . METHODS: We analyzed the effects of LPA on the migration activity, the focal adhesion formation, and the tyrosine phosphorylation of focal adhesion proteins in human ovarian cancer cell lines Caov-3 and OVCAR-3 . Inhibitors of the small GTPase Rho, one of its downstream effectors (Rho-associated kinase (ROCK)), myosin light chain kinase (MLCK), and myosin light chain (MLC) phosphatase were used to examine the mechanism of LPA-induced cellular effects . RESULTS: LPA enhanced the migration of ovarian cancer cells and facilitated their invasion . Rho and ROCK played essential roles in the migratory process, as evidenced by the inhibition of migration and focal adhesion formation of cancer cells by Clostridium botulinum C3 exoenzyme (C3), an inhibitor of Rho, or Y-27632, an inhibitor of ROCK . LPA also evoked the formation of focal adhesions and tyrosine phosphorylation of focal adhesion kinase and paxillin, all of which were inhibited by C3 or Y-27632 . CONCLUSION: These results suggest that LPA induced the migration of ovarian cancer cells, at least in part, through accelerated formation of focal adhesions mediated by Rho/ROCK-induced actomyosin contractility . This study may provide the basis for new therapies to control the metastasis of ovarian cancer.

Appl Microbiol Biotechnol, 2002 Dec, 60(4), 420 - 7 Epub 2002 Oct 18.
A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose; Li H et al.; A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli . The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da . Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain . Nag3A was purified from recombinant E . coli and characterized . The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide {4-MU-(GlcNAc)}, but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine . The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively . SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.

J Clin Endocrinol Metab, 2002 Dec, 87(12), 5808 - 16
Small guanosine triphospatase RhoA and Rho-associated kinase as regulators of trophoblast migration; Shiokawa S et al.; The small guanosine triphosphatase Rho controls cell adhesion and motility through reorganization of the actin cyto-skeleton and regulation of actomyosin contractility . Among the putative target molecules of Rho, a Rho-associated coiled coil-forming protein kinase (ROCK) is thought to participate in Rho-mediated cell adhesion and motility . In the present study, we explored the expression and function of RhoA and ROCK in human trophoblast cells . The colocalization of RhoA, cytokeratin 8/18, and cytokeratin 7 in some cells located in the decidual stromal region indicated that extravillous trophoblast cells expressed RhoA . In double staining for RhoA and ROCK in human chorionic villi, RhoA staining was strongly positive in the cytoplasm of cytotrophoblasts, whereas ROCK stained in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts . Both RhoA and ROCK were stained in cytoplasma of cultured human cytotrophoblast . Cultured human trophoblast cells contained actin stress fibers that were lost after treatment with C3, an exoenzyme produced by Clostridium botulinum . Y-27632, a selective ROCK inhibitor, suppressed RhoA-induced formation of actin stress fibers and formation of focal contact in trophoblast cells . The trophoblast reacquired actin stress fibers and focal contact after withdrawal of Y-27632 . Cultured human cytotrophoblast cells from 7-9 wk of gestation migrated into a fibronectin-coated membrane . Both C3 exoenzyme and Y-27632 inhibited cytotrophoblast migration in a dose-dependent manner . In conclusion, cyto-trophoblasts express RhoA and ROCK in their cytoplasm, and RhoA-ROCK is involved in their assembly of actin stress fibers . Suppression of RhoA-ROCK reduces trophoblast migration . These findings suggest that RhoA-ROCK signaling is a key regulator of trophoblast cell migration.

Postgrad Med, 2002 Nov, 112(5), 53 - 4, 57-8, 65 passim
Recurrent Clostridium difficile colitis . Tackling a tenacious nosocomial infection; Joyce AM et al.; C difficile infection recurs in about 20% of previously treated hospitalized patients . The elderly and patients with underlying colonic disease who have recently used antibiotics are at high risk . Signs and symptoms include diarrhea, abdominal pain, and leukocytosis . Diagnosis is dependent on a high degree of clinical suspicion and ELISA testing of a stool sample for toxins . Recurrence is thought to be due to the persistence of C difficile spores . Treatment can be difficult . Oral vancomycin or metronidazole for 10 to 14 days may be helpful as first-line therapy . Tapering the dose over 1 month helps destroy the spores while enabling the normal colonic flora to regrow . Probiotics, such as lactobacillus GG and S boulardii, are being developed to help restore normal colonic flora . Immunization may help prevent C difficile infection in the first place.

Am J Infect Control, 2002 Dec, 30(8), 449 - 57
Combined application of simulated reuse and quantitative carrier tests to assess high-level disinfection: experiments with an accelerated hydrogen peroxide-based formulation; Sattar SA et al.; BACKGROUND: Heat-sensitive medical devices require chemical disinfection between patients, and certain formulations for this purpose can be reused for several days . Because dilution, evaporation, and breakdown or neutralization of active ingredients can occur during reuse, it is vital to ensure that the solution retains its broad-spectrum germicidal activity even at the end of the recommended reuse period . OBJECTIVE: The purpose of this study was to combine the US Environmental Protection Agency's and the Food and Drug Administration's recommended simulated reuse method with recently developed quantitative carrier tests (QCT) to assess the broad-spectrum germicidal activity of a 7% solution of accelerated hydrogen peroxide (pH 2.9) stressed for 14 days.Materials And Methods: On alternate days baths with 3 lots of the test formulation were stressed by the addition of bacteria (Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas aeruginosa ) on glass beads and spores (Bacillus subtilis and Clostridium sporogenes ) on metallic penicylinders . In addition, one set of respiratory therapy equipment was subjected to 3 daily cycles of disinfection in each bath . The pH and H(2)O(2) levels in the test samples were measured, and they were also subjected to QCTs for their sporicidal, bactericidal, virucidal, fungicidal, and mycobactericidal activities . RESULTS: After 14 days of reuse, the pH of the test solutions remained essentially unchanged . Although the level of H(2)O(2) dropped from a high of 7.66% to as low as 6.40%, all lots showed the required level of broad-spectrum germicidal activity after 14 days of stress . CONCLUSIONS: The stress test and QCT were successfully combined in demonstrating the broad-spectrum germicidal activity of a high-level disinfectant subjected to 14 days of simulated reuse.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 12 - 8 Epub 2002 Jul 13.
Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions; Zheng H et al.; The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants { Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf} were investigated to examine the role of both monopolar and dipolar interactions . The reduction potentials of all variants decreased as the polarity of the solvent decreased . The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies . The entropic contribution {delta( Tdelta S degrees )} to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution {delta(-delta H degrees )} was larger for the other mutants . The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states . Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term {delta( Tdelta S degrees )} and enthalpic term {delta(-delta H degrees )} with opposite signs, was observed . A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed.

Vet Microbiol, 2003 Feb 2, 91(2-3), 239 - 48
PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues; Uzal FA et al.; The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants . Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively . The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C . chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii . Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR . In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C . chauvoei or C . septicum (four animals each) were also tested by the PCR using the three sets of primers . Purified DNA template of all C . chauvoei strains produced PCR amplicons of the expected size for all three primer pairs . However, when biomass from pure cultures of C . chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results . No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates . Therefore, the PCR primer sets appear to be very specific for identifying C . chauvoei in both cultures and tissues.

Int J Food Microbiol, 2003 Mar 15, 81(2), 137 - 45
Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage; Noonpakdee W et al.; A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production . One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus . Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10) . The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases . The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin . The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer . Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27 . The ability of the bacteriocin produced by Lc . lactis WNC 20 may be useful in improving the food safety of the fermented product.

Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 155 - 61
Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification; Perpetuo EA et al.; Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge . The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin . With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx . The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx . Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract . Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10 . The results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx . It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).

J Indian Med Assoc, 2002 Oct, 100(10), 607 - 8, 610-2, 614
Botulinum toxin: a dreaded toxin for use in human being; Ghosh B et al.; Botulinum toxin is a dreaded biological toxin elaborated by Clostridium botulinum . The action of this toxin is to cause paralysis of both voluntary and involuntary muscles . The unique property of paralysing capability of muscles has been used for the benefit of human beings . Dr Allan Scot, an ophthalmologist, first used the toxin in a patient with squint in 1981 and since then the botulinum toxin is being used in various disorders characterised by muscle overactivity such as spasticity in both children and adult, dystonic conditions such as blepharospasm, cervical dystonia, spasmodic dysphonia, writer's cramp, etc, hemifacial spasm and headache . Its main action is at the terminal nerve endings of myoneural junction and it prevents release of acetylcholine from vesicles thus causing chemical denervation . Its action persists for 3 to 4 months on an average . Its side effects such as drooping, diplopia, dysphagia, depending on the sites of injection, are few and usually transient . Generalised anaphylaxis is almost unknown . Now botulinum toxin is being used in non-neurological conditions where muscles are under spasmodic state such as achalasia cardia, anal fissure, spasm of urethral sphincter, etc . Because of wider safety range and fewer complications, botulinum toxin has been an important therapeutic armamentarium in different branches of medicine and surgery.

Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 696 - 703
Clostridium difficile in long-term-care facilities for the elderly; Simor AE et al.; Antimicrobial agents are among the most frequently prescribed medications in long-term-care facilities (LTCFs) . Therefore, it is not surprising that Clostridium difficile colonization and C . difficile-associated diarrhea (CDAD) occur commonly in elderly LTCF residents . C . difficile has been identified as the most common cause of non-epidemic acute diarrheal illness in nursing homes, and outbreaks of CDAD in LTCFs have also been recognized . This position paper reviews the epidemiology and clinical features of CDAD in elderly residents of LTCFs and, using available evidence, provides recommendations for the management of C . difficile in this setting.

Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 660 - 4
The role of Clostridium difficile and viruses as causes of nosocomial diarrhea in children; Langley JM et al.; OBJECTIVE: We report surveillance of nosocomial diarrhea in children at our institution during the past decade and note different epidemiology of diarrhea due to viruses and Clostridium difficile . DESIGN: A prospective cohort study . SETTING: A university-affiliated pediatric hospital with 180 beds serving an urban area and providing referral care for the Maritime Provinces of Canada . PARTICIPANTS: Children younger than 18 years . METHODS: Surveillance was conducted from 1991 to 1999 using personal contact with personnel and review of microbiology and medical records . Nosocomial diarrhea was defined as loose stools occurring more than 48 hours after admission, with at least two loose stools in 12 hours and no likely non-infectious cause . RESULTS: Nosocomial diarrhea was the third most common nosocomial infection (217 of 1,466; 15%), after bloodstream and respiratory infections, with from 0.5 to 1 episode per 1,000 patient-days . Of 217 nosocomial diarrhea episodes, 122 (56%) had identified pathogens: C . difficile (39 of 122; 32%), rotavirus (38 of 122; 31%), adenovirus (36 of 122; 30%), and other viral (9 of 122; 7%) . The median age was 1.3 years (range, 11 days to 17.9 years), 0.80 year for children with viral diarrhea, 3.9 years for children with C . difficile, and 1.5 years for children with diarrhea without a causative organism identified (P< .0001) . Most children with nosocomial diarrhea were incontinent (diapered) at the time of their first episode (138 of 185; 75%), but preexisting incontinence was more common in those with viral diarrhea (93%) compared with those with no organism identified (71%) or those with C . difficile-associated diarrhea (CDAD) (49%) (P <.0001) . CONCLUSIONS: C . difficile is the single most common cause of nosocomial diarrhea in our tertiary-care center, although all viral pathogens account for 69% of cases . Diapered status appears to be a risk factor for CDAD in children, and CDAD occurs more often in older children than viral nosocomial diarrhea . Further characterization of risk factors for, and morbidity associated with, nosocomial CDAD in children is warranted.

Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 653 - 9
Underlying disease severity as a major risk factor for nosocomial Clostridium difficile diarrhea; Kyne L et al.; OBJECTIVE: To determine the diagnostic accuracy of an index of underlying disease severity (Horn's index) in identifying patients with a high probability of having nosocomial Clostridium difficile diarrhea as a complication of antimicrobial therapy . DESIGN: A prospective cohort study of 252 adult patients admitted to the hospital and receiving antibiotics . Risk facctors for C . difficile diarrhea were first determined retrospectively in a different cohort of 300 hospitalized patients (primary cohort) and then prospectively in this cohort of 252 hospitalized patients receiving antibiotics (secondary cohort) . At the time of hospital admission, disease was rated by clinicians as mild (1), moderate (2), severe (3), or extremely severe (4) using a modified Horn's index . Multivariable logistic regression analysis was used to determine the odds ratio (OR) for C . difficile diarrhea associated with increasing levels of disease severity . SETTING: An urban teaching hospital affiliated with a medical school in Boston, Massachusetts . RESULTS: The incidence of nosocomial C . difficile diarrhea was 8.7% in the primary cohort and 11% in the secondary cohort In the prospective cohort study (secondary cohort), the OR for C . difficile diarrhea associated with extremely severe disease was 17.6 (95% confidence interval, 5.8 to 53.5) . The sensitivity, specificity, and positive and negative predictive values of a Horn's index score of 3 or more (severe to extremely severe disease) as a predictor of nosocomial C . difficile diarrhea were 79%, 73%, 27%, and 96%, respectively . CONCLUSIONS: These findings provide a means of early stratification of hospitalized patients receiving antibiotics according to their risk for nosocomial C . difficile diarrhea . Patients with severe to extremely severe disease at the time of admission may benefit from careful monitoring of antibiotic prescribing and early attention to infection control issues . In the future, these "high-risk" patients may benefit from prophylaxis studies of novel agents being developed to prevent C . difficile diarrhea.

Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 648 - 52
Predominance of a single restriction endonuclease analysis group with intrahospital subgroup diversity among Clostridium difficile isolates at two Chicago hospitals; Mekonen ET et al.; OBJECTIVE: To determine the epidemiology and relatedness of Clostridium difficile isolates in two geographically separated hospitals in a large metropolitan area, each with unique patients and personneL DESIGN: Observational descriptive molecular epidemiology of clinical C . difficile isolates . SETTING: Two tertiary-care hospitals in Chicago . METHODS: Consecutive C . difficile isolates from the clinical laboratory of a Veterans Affairs hospital during a 13-month period were typed by restriction endonuclease analysis (REA) . During an overlapping 3-month period, stool specimens that tested positive for C . difficile toxin from patients at a nearby county hospital were cultured and the recovered isolates typed by the same method . RESULTS: Nineteen (68%) of 28 nosocomial isolates at the smaller, Veterans Affairs hospital belonged to REA group K . Within this group of closely related strains, 9 distinct REA types were recognized . Twenty-one (72%) of 29 nosocomial isolates at the larger, county hospital also belonged to group K . However, the predominant REA types within group K differed markedly at each institution . CONCLUSIONS: These findings demonstrate a high degree of similarity among nosocomial C . difficile strains from different hospitals in the same city and suggest the possibility of an extended outbreak of a prototype group K strain with subsequent genetic drift at the two different institutions.

Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 641 - 7
Difference in the incidence of Clostridium difficile among patients infected with human immunodeficiency virus admitted to a public hospital and a private hospital; Pulvirenti JJ et al.; OBJECTIVE: To compare the occurrence of Clostridium difficile among inpatients infected with human immunodeficiency virus (HIV) in two different hospitals . DESIGN: Prospective, observational study . SETTING: Specialized HIV inpatient units . PATlENTS: HIV-infected inpatients at Cook County Hospital (CCH) and Rush Presbyterian St . Luke's Medical Center (RPSLMC) . INTERVENTIONS: A clinical and epidemiologic assessment of patient risk factors for C . difficile was performed . C . difficile isolates found on stool, rectal, and environmental cultures were typed by pulsed-field gel electrophoresis . RESULTS: Twenty-seven percent of patients admitted to CCH versus 4% of patients admitted to RPSLMC had positive cultures for C . difficile (P = .001) . At CCH, 14.7% of environmental cultures were positive versus 2.9% at RPSLMC (P = .002) . Risk factors for C . difficile acquisition included hospitalization at CCH, more severe HIV, use of acyclovir and H2-blockers, and longer hospital stay . Patients admitted to CCH were taking more antibiotics, had longer hospital stays, and more frequently had a history of C . difficile infection . During the study, two strains (CD1A and CD4) extensively contaminated the CCH environment . However, only CD1A caused an outbreak . CONCLUSIONS: The C . difficile acquisition rate at CCH was sevenfold higher than that at RPSLMC, and CCH had a more contaminated environment . Differences in patient acquisition rates likely reflect a greater prevalence of traditional C . difficile risk factors and a concurrent outbreak at CCH . Although two strains heavily contaminated the environment at CCH, only one caused an outbreak, suggesting that factors other than the environment are important in initiating C . difficile outbreaks.

Appl Environ Microbiol, 2002 Dec, 68(12), 6425 - 8
Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens; Knarreborg A et al.; A quantitative assay based on high-performance liquid chromatography analysis of bile salts and bacterial protein determination was established for investigating bile salt hydrolase (BSH) activity in bacteria isolated from the small intestine of chickens . Bacteria were isolated using various media and were subsequently grouped according to cell morphology, fermentation profile, and 16S ribosomal DNA sequence . Representative isolates from each bacterial group were assayed for BSH activity . The isolates differed in BSH activity with respect to the state of growth and preculturing with and without taurochenodeoxycholate . The highest levels of BSH activity were found with Enterococcus faecium and Clostridium perfringens.

Appl Environ Microbiol, 2002 Dec, 68(12), 6399 - 402
Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation; Kosugi A et al.; The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain . The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates . rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan . Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction . These results indicate that XynA is a key enzymatic subunit for xylan degradation in C . cellulovorans.

Appl Environ Microbiol, 2002 Dec, 68(12), 6256 - 62
Growth of iron(III)-reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms; Kostka JE et al.; Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe . While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor . Here we have demonstrated that a pure culture of Shewanella oneidensis strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor . Pure cultures of S . oneidensis were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors {smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen} in the same minimal medium . Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon) . Cell counts showed that similar growth of S . oneidensis (10(8) cells ml(-1)) occurred with smectitic Fe(III) and on other Fe forms {amorphous Fe(III) oxyhydroxide, and Fe citrate} or oxygen as the electron acceptor . In contrast, cell yields of S . oneidensis measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions . Over a range of particle loadings (0.5 to 4 g liter(-1)), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced . From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the Bacteria (Clostridium and Desulfitobacterium) and the delta-Proteobacteria (members of the Geobacteraceae) . Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe(III) oxide minerals and is dependent upon the mineral surface area available . Iron(III) bound in clay minerals should be considered an important electron acceptor supporting the growth of bacteria in soils or sedimentary environments.

Appl Environ Microbiol, 2002 Dec, 68(12), 5918 - 24
Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages; Knarreborg A et al.; The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed(-1) and salinomycin at 40 mg kg of feed(-1)) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture . The bacterial origin of fragments in DGGE profiles was identified by sequencing . Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation . An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated . Lactobacilli and C . perfringens were the bacterial groups most strongly affected by the dietary treatments . Moreover, different strains (clonal variants of the alpha-toxin gene) of C . perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.

Appl Environ Microbiol, 2002 Dec, 68(12), 5891 - 903
Macedocin, a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198; Georgalaki MD et al.; Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin . Macedocin has a molecular mass of 2,794.76 +/- 0.42 Da, as determined by electrospray mass spectrometry . Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes . Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum . It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp . sakei LMG 13558(T), while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production . Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121 degrees C with 1 atm of overpressure . Inhibition of macedocin by proteolytic enzymes is variable.

Appl Environ Microbiol, 2002 Dec, 68(12), 5870 - 6
Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France; Fach P et al.; The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France . A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C . botulinum spores per 25 g and 100% of samples inoculated with more than 30 C . botulinum spores per 25 g . The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did . The prevalence of C . botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C . botulinum most-probable-number counts, respectively, and is in the low range of C . botulinum contamination reported elsewhere . The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E . Type F was not detected . The high prevalence of C . botulinum type B in fish samples is relatively unusual compared with the high prevalence of C . botulinum type E reported in many worldwide and northern European surveys . However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 625 - 30
Bacterial spores in silage and raw milk; te Giffel MC et al.; Spore-forming bacteria can survive food-processing treatments . In the dairy industry, Bacillus and Clostridium species determine the shelf-life of a variety of heat-treated milk products, mainly if the level of post-process contamination is low . In order to minimize problems caused by bacterial spores in foods and food production processes a chain management approach, from raw materials, ingredients and environmental sources to final product storage conditions, is most effective . Silage is considered to be a significant source of contamination of raw milk with spores . PCR-RAPD fingerprinting and heat resistance studies of populations of aerobic spore-formers isolated from grass and maize silage and from raw milk confirmed this assumption . Prevention of outgrowth of aerobic spores in silage will contribute to reduction of the total spore load of raw milk . Therefore, it is important that the silage fermentation process is controlled . Application of cultures of lactic acid bacteria or chemical additives can aid silage fermentation and improve aerobic stability.

J Med Microbiol, 2002 Nov, 51(11), 1001 - 8
An investigation into the microflora of heroin; McLauchlin J et al.; In 2000, an unusual increase of morbidity and mortality among illegal injecting drug users in the UK and Ireland was reported and Clostridium novyi was identified as the likely source of the serious infection, although infections due to C . botulinum and Bacillus cereus were also reported . Because heroin was a possibile source of infection, this study investigated the microflora of heroin samples seized in England during 2000 and 2002 . Two methods were developed for the examination of the microflora of heroin . The first consisted of suspension of the drug in maximum recovery diluent (MRD) which was inoculated directly into Clostridium Botulinum Isolation Cooked Meat Broth (CBI) . The second method rendered the heroin soluble in citric acid, concentrated particulate material (and bacterial cells) by filtration and removed heroin residues by washing with citric acid and phosphate-buffered saline before placing the filter in CBI broth . Duplicate CBI broths from both methods were incubated without heating and after heating at 60 degrees C for 30 min . Subcultures were made after incubation for 7 and 14 days on to eight different solid media . The methods were evaluated with heroin samples spiked with either C . botulinum or C . novyi spore suspensions; recovery of 10 spores in the original sample was demonstrated . Fifty-eight heroin samples were tested by citric acid solubilisation and 34 by the MRD suspension technique . Fifteen different gram-positive species of four genera were recognised . No fungi were isolated . Aerobic endospore-forming bacteria (Bacillus spp . and Paenibacillus macerans) were the predominant microflora isolated and at least one species was isolated from each sample . B . cereus was the most common species and was isolated from 95% of all samples, with B . licheniformis isolated from 40% . Between one and five samples yielded cultures of B . coagulans, B . laterosporus, B . pumilus, B . subtilis and P . macerans . Staphylococcus spp . were isolated from 23 (40%) samples; S . warneri and S . epidermidis were the most common and were cultured from 13 (22%) and 6 (10%) samples respectively . One or two samples yielded cultures of S . aureus, S . capitis and S . haemolyticus . The remainder of the flora detected comprised two samples contaminated with C . perfringens and two samples with either C . sordellii or C . tertium . Multiple bacterial species were isolated from 43 (74%) samples, a single species from the remaining 15 . In 13 samples B . cereus alone was isolated, in one B . subtilis alone and in one sample B . pumilus alone . C . botulinum and C . novyi were not isolated from any of the heroin samples . Recommendations for the optimal examination of the microflora of heroin are given.

J Med Microbiol, 2002 Nov, 51(11), 990 - 1000
Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C . perfringens and Bacillus cereus isolated from injecting drug users during 2000; McLauchlin J et al.; As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C . perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis . Among the 43 C . novyi isolates, 23 different AFLP profiles were detected . The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user . Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988) . The 40 C . perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin . Sixteen AFLP types of C . perfringens were distinguished and there was little evidence for commonality among the isolates . The AFLP types of C . perfringens from heroin differed and were unique . Six isolates of B . cereus were from four drug users and two samples of heroin . Four different AFLP patterns were distinguished . Three AFLP types were isolated from four drug users . B . cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user . The AFLP type of the isolate from a second and unrelated heroin sample was unique . The AFLP results showed no or very limited evidence for commonality between the different isolates of B . cereus and C . perfringens . In marked contrast, the C . novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C . novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

J Med Microbiol, 2002 Nov, 51(11), 985 - 9
Isolation and identification of Clostridium spp . from infections associated with the injection of drugs: experiences of a microbiological investigation team; Brazier JS et al.; Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease . C . novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly . The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification . Additional phenotypic tests that are useful in recognising the main pathogenic species are described . Differentiation of C . novyi type A from C . botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture.

J Med Microbiol, 2002 Nov, 51(11), 978 - 84
An outbreak of serious illness and death among injecting drug users in England during 2000; Jones JA et al.; An outbreak of serious illness and death occurred in injecting drug users during 2000 in Scotland, Ireland and England . National and international collaboration was necessary for the investigation and management of this outbreak . In England and Wales active case-finding was initiated, coupled with standardised data collection and microbiological investigation of cases . Twenty-six definite or probable cases were identified in England between 1 April and 31 Aug . 2000; 17 of these occurred in the North . The overall case fatality was 50% (13/26) . The principal apparent risk factor was a history of intramuscular or subcutaneous injection of heroin and the limited duration of the outbreak suggested that the problem might have been related to a particular supply of heroin . Clostridium novyi was isolated from two English cases . Taken in conjunction with contemporaneous microbiological and epidemiological results from Scottish and Irish cases, the probable aetiology for this outbreak was infection with C . novyi associated with both a particular supply of heroin and the method of preparation and injection used . A 'toolkit' was distributed in Sept . 2000 to all Consultants for Communicable Disease Control in England and Wales to assist them with the ongoing surveillance, investigation and management of this condition . Lessons learned have been used to produce guidance for the investigation and management of outbreaks of unexplained serious illness of possible infective aetiology.

J Med Microbiol, 2002 Nov, 51(11), 971 - 7
Lethal outbreak of infection with Clostridium novyi type A and other spore-forming organisms in Scottish injecting drug users; McGuigan CC et al.; This report describes the investigation and management of an unprecedented outbreak of severe illness among injecting drug users (IDUs) in Scotland during April to August 2000 . IDUs with severe soft tissue inflammation were prospectively sought among acute hospitals and a mortuary in Scotland . Cases were categorised as definite or probable: probable cases had severe injection site inflammation or multi-system failure; definite cases had both . Information about clinical course, mortality, post-mortem findings and laboratory data was gathered by standardised case-note review and interview . Sixty cases were identified--23 definite and 37 probable . Most had familial or social links with each other and 50 were from Glasgow . Median age was 30 years; 31 were female . The majority, especially definite cases, injected heroin/citric acid extravascularly . Of definite cases, 20 died (87% case-fatality rate; 13 after intensive care), 15 had necrotising fasciitis, 22 had injection site oedema and 13 had pleural effusion . Median white cell count was 60 x 10(9)/L . Of 37 probable cases, three died (8% case-fatality rate) . Overall, the most frequently isolated pathogen was Clostridium novyi type A (13 cases: 8 in definite cases) . The findings are consistent with an infection resulting from injection into soft tissue of acidified heroin contaminated with spore-forming bacteria . Toxin production led to a severe local reaction and, in many, multi-system failure.

J Infect Dis, 2002 Dec 15, 186(12), 1781 - 9 Epub 2002 Nov 22.
Colonization for the prevention of Clostridium difficile disease in hamsters; Sambol SP et al.; Studies suggest that asymptomatic colonization with Clostridium difficile (CD) decreases the risk of CD-associated disease (CDAD) in humans . A hamster model was used to test the efficacy of colonization with 3 nontoxigenic CD strains for preventing CDAD after exposure to toxigenic CD . Groups of 10 hamsters were given 10(6) nontoxigenic CD spores 2 days after receiving a single dose of clindamycin . Five days later, the hamsters were given 100 spores of 1 of 3 toxigenic CD strains previously shown to cause mortality within 48 h . Each nontoxigenic strain prevented disease in 87%-97% of hamsters that were challenged with toxigenic strains . Failure to prevent CDAD was associated with failure of colonization with nontoxigenic CD . Colonization with nontoxigenic CD strains is highly effective in preventing CDAD in hamsters challenged with toxigenic CD strains, which suggests that use of a probiotic strategy for CDAD prevention in humans receiving antibiotics might be beneficial.

J Biol Chem, 2003 Feb 7, 278(6), 4112 - 20 Epub 2002 Nov 22.
Recruitment of murine neutrophils in vivo through endogenous sialidase activity; Cross AS et al.; Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface . This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis . We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo . A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activities in vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8 . We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitment in vivo . The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes . Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response.

J Bacteriol, 2002 Dec, 184(24), 6859 - 65
Characterization of two noncellulosomal subunits, ArfA and BgaA, from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation; Kosugi A et al.; Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases . To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C . cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated . The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci . The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively . Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside . On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside . However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase . Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction . When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C . cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone . These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.

FEMS Microbiol Lett, 2002 Nov 19, 217(1), 15 - 22
Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum; Sabathe F et al.; A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824 . Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp . Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes . As C . acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution . Biochemical evidence for the expression of a cellulosomal protein complex was investigated . The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C . cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present . In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C . acetobutylicum.

Inorg Chem, 2002 Dec 2, 41(24), 6358 - 71
Mössbauer study of reduced rubredoxin as purified and in whole cells . Structural correlation analysis of spin Hamiltonian parameters; Vrajmasu VV et al.; The {Fe(II)(Cys)(4)}(2-) site of rubredoxin from Clostridium pasteurianum (Rd(red)) has been studied by Mossbauer spectroscopy in both purified protein and whole cells of Escherichia coli overproducing it . Excellent fits were obtained to an S = 2 spin Hamiltonian for D = 5.7(3) cm(-1), E/D = 0.25(2), delta = 0.70(3) mm/s, DeltaE(Q) = -3.25(2) mm/s, eta = 0.75(5), A(x) = -20.1(7) MHz, A(y) = -11.3(2) MHz, and A(z) = -33.4(14) MHz . These parameters were analyzed with crystal-field theory for the (5)D manifold of iron(II), revealing a d(z(2)) orbital ground state that is admixed by approximately 0.21 d(x(2) - y(2)) . The spin-Hamiltonian parameters are consistent within the (5)D theory, apart from the zero-field splitting parameter, D . This problem was solved by extending the crystal-field treatment with spin-orbit coupling to spin-triplet d-d excited states of the iron . Theoretical estimates are given for the spin-triplet (D(T)) and spin-quintet contributions (D(Q)) to D based on excitation energies derived from time-dependent density functional theory, TD-DFT . The computational results were interpreted in terms of crystal-field theory, yielding the Racah parameters B = 682 cm(-1) and C = 2583 cm(-1) . The theoretical analysis gives the relative magnitudes D(Q):D(T):D(ss) = 51%: 42%:7% (D(ss) originates from spin-spin interaction) . The DFT analysis corroborates the pivotal role of the torsion angles (omega(i)) of the C-S(i) bonds in shaping the electronic structure of the iron(II) site . Rd(red) in overexpressing whole cells accounts for 60% of the Mossbauer absorption . The Rd(red) spectra from whole cells are virtually identical to those of the purified protein . By using the theoretical omega dependence of the spin Hamiltonian parameters, the torsions for Rd(red) in whole cells and purified protein samples are estimated to be the same within 2 degrees . These findings establish Mossbauer spectroscopy as a structural tool for investigating iron sites in whole cells.

Naunyn Schmiedebergs Arch Pharmacol, 2002 Dec, 366(6), 501 - 12 Epub 2002 Sep 24.
The uptake machinery of clostridial actin ADP-ribosylating toxins--a cell delivery system for fusion proteins and polypeptide drugs; Barth H et al.; Several bacterial protein toxins, including Clostridium botulinum C2 toxin, Clostridum perfringens iota toxin, Clostridium difficile ADP-ribosyltransferase, and the Bacillus-produced vegetative insecticidal proteins, target the cytoskeleton by ADP-ribosylation of actin . All these toxins are binary in structure and consist of an enzyme component, possessing ADP-ribosyltransferase activity and a separated binding and translocation component, which is involved in the delivery of the enzyme component into the cell . The toxins are not only important virulence factors but also cell biological tools to study the function of the actin cytoskeleton . Moreover, the binary toxins turned out to be effective transporter systems for the delivery of specific fusion toxins (e.g., Rho-ADP-ribosylating C3 exoenzyme) into cells . The present review describes the biological functions of the toxins, focuses on recent studies on the uptake and delivery mechanism and discusses the usage as a drug delivery system.

Ann Med Interne (Paris), 2002 Sep, 153(5), 291 - 9
{Clostridium difficile infection in a Department of Internal Medicine . A consecutive series of 45 patients}; Bligny D et al.; OBJECTIVES AND METHODS: A retrospective study of 45 patients with Clostridium difficile infection over a 4-year period in a department of Internal Medicine . RESULTS: Mean age was 79 years; sex-ratio (F/M)=1.5; 38% of the patients had neurological or severe psychiatric disorders; 20% had a neoplastic disease . Ninety-three percent of cases had received one or more antibiotics before onset of diarrhea, prescribed mainly for a pulmonary infection . Amoxicillin clavulanic acid and cephalosporins were the most frequently used treatments, respectively in 48% and 40% of cases . For 25 patients (56%) Clostridium difficile-associated diarrhea was considered as a nosocomial infection, and as community-acquired diarrhea in 20 cases (44%) . Treatment included isolation of the patient as soon as bacteriological diagnosis was known and specific therapy was instituted by metronidazole or vancomycin for a mean of 18 days . The addition of Saccharomyces boulardii was used in of cases . The clinical course was rapidly favorable for 80% of patients . Five patients died with complications of severe colitis in 2 cases . Mean hospital stay was 49 days (annual mean of the department=10 days) . CONCLUSION: Clostridium difficile diarrhea concerns above all elderly patients with one or more underlying pathologies . Amoxicillin clavulanic acid and third-generation cephalosporins are the most frequently prescribed antibiotics in these cases and have the highest correlation with this infectious complication . This medical problem requires greater knowledge as it causes significant morbidity and increases the risk of prolonged hospital stays.

J Am Chem Soc, 2002 Nov 27, 124(47), 14039 - 48
Rotation of the exo-methylene group of (R)-3-methylitaconate catalyzed by coenzyme B(12)-dependent 2-methyleneglutarate mutase from Eubacterium barkeri; Pierik AJ et al.; 2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate . Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered . In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4 . In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4 . In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4 . In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl{2'-(2)H(1)}itaconate and (Z)-3-{2'-(2)H(1),methyl-(2)H(3)}methylitaconate . The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene{2'-(2)H(1)}glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR . In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl{2'-(2)H(1)}itaconate over its equilibrium value was observed ("E-overshoot") . The E-overshoot was only 3-4% with (Z)-3-{2'-(2)H(1),methyl-(2)H(3)}methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical . The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical . It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway.

J La State Med Soc, 2002 Sep-Oct, 154(5), 251 - 5
Clostridial enteritis necroticans versus secondary clostridial infection superimposed upon ischemic bowel disease; Zhao W et al.; Clostridial enteritis necroticans, or pig-bel, as seen in Papua New Guinea, is a necrotizing, segmental gangrene of the small intestine occurring in members of a malnourished population, who become ill after consuming large quantities of pork contaminated with Type C . Clostridium perfringens . We report a case of possible Clostridial enteritis necroticans with concomitant ischemic intestinal disease secondary to superior mesenteric arterial thrombosis occurring in a 53-year-old woman with a long history of diabetes mellitus, hypertension, and peripheral vascular disease . The differential diagnosis and the pathogenesis of C . perfringens enteritis necroticans are discussed.

Clin Infect Dis, 2002 Dec 1, 35(11), 1441 - 3 Epub 2002 Nov 13.
Toxic shock syndrome due to Clostridium sordellii: a dramatic postpartum and postabortion disease; Sinave C et al.; We describe a young woman who developed Clostridium sordellii toxic shock syndrome after having had an abortion medically induced by mifepristone (RU-486; Mifeprex {Danco Laboratories}) 7 days before admission to our hospital . Although the patient was aggressively treated, death occurred <3 days after admission . It is hoped that very early recognition of this disease will decrease the mortality associated with this rarely reported ailment that occurs among young, otherwise healthy women.

New Microbiol, 2002 Oct, 25(4), 413 - 20
Fate of bacterial indicators, viruses and protozoan parasites in a wastewater multi-component treatment system; Bonadonna L et al.; The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture . The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages . Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment . All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8 . The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores . Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated . The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment . The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.

J Med Microbiol, 2002 Oct, 51(10), 891 - 4
Laboratory diagnosis of Clostridium perfringens antibiotic-associated diarrhoea; Asha NJ et al.; Clostridium perfringens has been reported as the cause of up to 15% of cases of antibiotic-associated diarrhoea (AAD) and may be diagnosed by detection of enterotoxin (CPEnt) in faeces . The performance of a commercial ELISA method for CPEnt, with culture and PCR methods to confirm the presence of enterotoxigenic C . perfringens, was evaluated in 200 consecutive specimens from patients with clinical details suggestive of AAD: 8% of the specimens were positive for CPEnt, 16% were positive for C . difficile cytotoxin and 2% gave positive test results for both C . perfringens and C . difficile toxins . Culture and PCR results confirmed the majority of ELISA results, although 2 (12.5%) reactive specimens were only weakly positive . C . perfringens is a potentially important cause of infective AAD and can be detected with the C . perfringens enterotoxin ELISA kit, although weak positive results should be considered with caution.

J Med Microbiol, 2002 Oct, 51(10), 813 - 20
Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component; Mahmut N et al.; Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox) . The amino acid sequences of type C and D nonTox components are almost identical . In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine . This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins . The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin . This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both . The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera . When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice . These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine . The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

Med Klin (Munich), 2002 Nov 15, 97(11), 650 - 8
{Late infectious complications after high-dose therapy and autologous blood stem cell transplantation}; Metzner B et al.; AIM: Only a few data on frequency and character of late infectious complications after high-dose therapy (HDT) and autologous blood stem cell transplantation (ASCT) have been published . This prospective study was carried out to identify potential predictive factors for late infections (occurring after discharge following HDT) and to clarify the usefulness of prophylactic measures . PATIENTS AND METHODS: Clinical data of 192 consecutive patients treated with HDT and ASCT in a single hospital were analyzed on late infectious complications . After discharge following HDT, the 166 evaluable patients (84 with hematologic malignancies, 82 with solid tumors) had been examined and interviewed on infections every 4-12 weeks after ASCT . For Pneumocystis carinii prophylaxis, inhalation with pentamidine or oral cotrimoxazole was used for 3-4 months following ASCT . RESULTS: In the first 6 months following ASCT (after discharge) we saw on average one infectious episode per patient (median, range 0-6), usually light infections (mostly banal upper airway infections) . 17 patients had to be treated in hospital for infectious (15 of whom with hematologic malignancies), three of whom (only with hematologic malignancies) died in spite of intensive care as a result of pneumonias due to opportunistic causative agents (mainly Pneumocystis carinii {PcP}) . In the second half of the year after ASCT, five patients (with hematologic malignancies) had to be hospitalized due to infections . No further infection-related death occurred . Early documented infections (pneumonia, bacteremia or Clostridium difficile colitis) were associated with an increased risk for late serious infections . Zoster occurred in 18% of patients within 12 months, more frequently after increased pretreatment (25% vs . 11% after less pretreatment), most frequently in patients with relapsed lymphomas (32%) . CONCLUSIONS: Significant late infectious complications after ASCT are uncommon . Patients with hematologic malignancies have a significantly increased risk of more serious infections and should be observed carefully . For risk patients with hematologic malignancies and possibly solid tumors, a strict PcP prophylaxis is required . Patients with relapsed lymphomas could possibly be treated preventively against zoster with low-dose aciclovir to reduce the extent of zoster disease . Each patient should be informed carefully that early signs of zoster require an effective zoster treatment as soon as possible.

Curr Infect Dis Rep, 2002 Dec, 4(6), 477 - 483
Clostridium difficile-associated Enteric Disease; Bartlett JG; Clostridium difficile was identified as the putative agent of antibiotic-associated pseudomembranous colitis in 1978 and is now recognized as the major identifiable cause of antibiotic-associated diarrhea . This microbe causes a spectrum of enteric disease ranging from nuisance diarrhea to life-threatening colitis . Risk factors include increasing age, exposure to antibiotics, colonization or acquisition of toxin-producing strains of C . difficile, and lack of circulating antibody to C . difficile toxin A . Detection is relatively simple by stool assay for C . difficile toxin--usually an enzyme immunoassay that will detect toxin A and B . Most nonsevere cases will respond with discontinuation of the implicated antibiotic . More severe cases require metronidazole and supportive care . The major complications include ileus, toxic megacolon, relapsing disease after antibiotic treatment, and nosocomial epidemics.

Pharmacotherapy, 2002 Nov, 22(11), 1479 - 83
Association between honey consumption and infant botulism; Tanzi MG et al.; Infant botulism, a disease that results in a blockade of voluntary motor and autonomic functions, was first recognized in the United States in the late 1970s . Since then, more than 1000 cases in this country have been reported to the Centers for Disease Control and Prevention (CDC) . Numerous studies have shown that the ingestion of honey is linked with infant botulism . In addition, honey samples across the United States have tested positive for Clostridium botulinum spores and toxins . Such substantial evidence led the CDC to recommend that honey not be given to infants younger than 12 months old . It is important that clinicians be familiar with this risk and should not recommend honey-containing products or supplements or the use of honey as a flavoring agent for infants in this age group.

J Mol Microbiol Biotechnol, 2002 Sep, 4(5), 503 - 13
Regulatory relationship of two-component and ABC transport systems and clustering of their genes in the Bacillus/Clostridium group, suggest a functional link between them; Joseph P et al.; On the Bacillus subtilis chromosome there are five examples of genes encoding two-component systems with response regulators of the OmpR family adjacent to genes encoding sub-family 9 ABC transport systems . Three of these (yts, yvc, yxd) are very similar in gene organization and in sequence . We demonstrate that the TCS and ABC transporter genes do not belong to the same transcriptional unit . The ABC transport and TCS systems are functionally linked, each response regulator controlling the expression of its cognate ABC transporter genes but not its own . Analysis of 48 bacterial genomes revealed that such family clusters only exist in the Bacillus/Clostridium group . Evolutionary analyses indicated that almost all clustered OmpR response regulators constitute two groups ("GI" and "GIIl") whereas almost all clustered sub-family 9 nucleotide-binding domains belong to two other groups ("9A" and "9B") . Interestingly, there is a mutually exclusive clustering between genes encoding "GI" or a "GII" response regulators and genes encoding "9A" or a "9B" nucleotide binding proteins . We propose that a two-component system and its cognate ABC transporter genes have evolved as a unit in Bacillus/Clostridium, both systems participating in a common physiological process.

Rev Esp Enferm Dig, 2002 Jun, 94(6), 361 - 6
Clostridium septicum infection associated with perforation of colon diverticulum; San Ildefonso A et al.; Non-traumatic or spontaneous gas gangrene by Clostridium septicum is a rare infection in humans, characterised by extensive destruction of muscle tissue, a mortality rate of 100% if left untreated (1), and often associated with haematological or colorectal diseases . An associated malignancy was found in 80% of patients with Clostridium septicum infection: 34% had a colorectal carcinoma and 40% had a haematological malignancy (2); the infection usually lies in an often-ulcerated intestinal lesion (3) . We report here a case of spontaneous gas gangrene by Clostridium septicum in the right thigh due to perforation of a diverticulum in the sigmoid colon.

Aesthetic Plast Surg, 2002 Sep-Oct, 26(5), 356 - 9
Long-term prospective follow-up of botulinum toxin treatment for facial rhytides; Bulstrode NW et al.; Some wrinkles and unsightly facial expressions are due to hyperactivity of the underlying facial musculature . Clostridium botulinum type A exotoxin reversibly paralyzes selected muscles and is a safe, helpful adjunct to many other treatments for facial rejuvenation . Fifty-two patients were treated and all data recorded in a prospective fashion . Only areas requested by the patient were treated . The dosage and dilution given in each area were carefully noted and all patients had pretreatment and posttreatment photographs . The effect of botulinum toxin injections on the horizontal brow rhytides was recorded by measuring the distance from the frontal hairline to the superior edge of the eyebrow in the mid-pupillary line . Patients were followed for one to three years (mean 16.3 months) . One patient was not responsive to botulinum toxin in spite of repeated injections . Three further patients required touch-up injections two weeks after the initial treatment due to a weak initial response . Repeat injections were required every three to six months (mean 4.05) to maintain the desired improvement . Asymmetry of the brow was seen in two patients and corrected with further administration of botulinum toxin . Twenty-five patients had their forehead rhytides injected and the appropriate measurements taken . Brow ptosis occurred in 22 of the 25 patients and varied 1-6 mm with a mean value of 2.3 mm . This difference was statistically significant (paired t-test p <0.001) . Two patients reported dryness and flakiness of the frontal area after injections . No cases of eyelid ptosis or hypersensitivity were seen . Botulinum toxin injections are safe and all undesired effects are reversible . Great care has to be taken not to aggravate the degree of brow ptosis . Injection of the forehead depressors minimizes the risk of brow ptosis . Careful planning of injection sites and doses avoids a mask-like upper face . The use of botulinum toxin provides a useful adjunct to laser and surgical procedures for facial rejuvenation.

Pediatr Allergy Immunol, 2002 Oct, 13(5), 357 - 60
Clostridium difficile, atopy and wheeze during the first year of life; Woodcock A et al.; Differences have been suggested to occur in the composition of intestinal microflora from allergic and non-allergic children . In this study we used a semi-quantitative enzyme-linked immunosorbent assay (ELISA) for the measurement of Clostridium difficile-specific immunoglobulin G (IgG) (CDIgG) . CDIgG was excellent in differentiating between adults with or without Cl . difficile colitis (absorbance levels, positive vs . negative controls: geometric mean (GM) 0.301, 95% CI: 0.289-0.314 vs . GM 0.167, 95% CI: 0.155-0.181; mean difference 1.8-fold, 95% CI: 1.65-1.95; p < 0.0001) . We used this technique to investigate whether there are any differences between atopic wheezy infants and non-atopic non-wheezy controls . In a prospective cohort study (n = 390) 10 patients were identified at 1 year of age (atopic, history of recurrent wheeze) and matched (gender, month of birth, exposure to Der p 1, Fel d 1 and Can f 1) with a control group of infants (non-atopic, no history of wheeze) . The patients had significantly higher Cl . difficile-specific IgG absorbance levels (GM 0.298, 95% CI: 0.249-0.358) compared with controls (GM 0.235, 95% CI: 0.201-0.274; mean difference 1.27-fold, 95% CI: 1.07-1.50; p = 0.01) . These results suggest that there may be differences in the composition of intestinal microflora between allergic and non-allergic infants at 1 year of age, with allergic children having higher Cl . difficile IgG antibody levels.

Microbiology, 2002 Nov, 148(Pt 11), 3651 - 60
Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria; Salzman NH et al.; Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers . Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences . Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences . A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga-Flavobacter-Bacteroides phylum, which the authors named 'mouse intestinal bacteria' . 16S rRNA probes were developed for this new OTU . Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale-Clostridium coccoides group and three clustered within the Bacteroides group . One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides . Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated . Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).

J Biol Chem, 2003 Feb 21, 278(8), 6120 - 7 Epub 2002 Nov 08.
Recognition and hydrolysis of noncrystalline cellulose; Boraston AB et al.; Cellulase Cel5A from alkalophilic Bacillus sp . 1139 contains a family 17 carbohydrate-binding module (BspCBM17) and a family 28 CBM (BspCBM28) in tandem . The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved . BspCBM28 was obtained as a discrete polypeptide by engineering the cel5A gene . BspCBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans, CcCBM17, was used to compare the binding characteristics of the two families of CBM . Both CcCBM17 and BspCBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site (K(a) approximately 1 x 10(6) M(-1)) and a low affinity site (K(a) approximately 2 x 10(4) M(-1)) . They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose . A polypeptide, BspCBM17/CBM28, comprising the tandem CBMs from Cel5A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of CcCBM17 and BspCBM28, indicating cooperativity between the linked CBMs . Cel5A mutants were constructed that were defective in one or both of the CBMs . The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose . This suggests that either the CBMs can modify the action of the catalytic module of Cel5A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.

J Assoc Physicians India, 2002 Aug, 50, 1013 - 6
Prevalence of pathogens in diabetic foot infection in South Indian type 2 diabetic patients; Viswanathan V et al.; AIM: To determine the prevalence of pathogens in diabetic foot infections, in relation to parameters like Wagner's grading, duration of diabetes and healing time . MATERIAL AND METHODS: A group of 654 (M:F, 433:221) type 2 diabetic patients with foot ulcers were studied . Specimens like pus, wound exudate and tissue were processed for smear for Gram's staining, aerobic and anaerobic culture, and biochemical identifications . RESULTS: In 654 diabetic patients, 728 pathogens were isolated . Aerobic pathogens were isolated in 437 (66.8%) patients and anaerobic pathogens were isolated in 217 (33.2%) . As Wagner's grading increased, the prevalence of anaerobic pathogens also increased . Neuropathy was common in diabetic patients infected with both aerobic and anaerobic pathogens . Ulcers infected with anaerobic pathogens showed a longer healing time than ulcers infected with aerobic pathogens . There was no significant difference in peripheral vascular disease (PVD) in patients selected for the study . Among aerobic pathogens, Enterobacteriaceae family (48%), Staphylococcus species (spp) (18.2%), Streptococcus spp (16.8%) and Pseudomonas spp (17%) were seen frequently . Among anaerobes Peptostreptococcus spp and Clostridium spp formed 69.4% . Gram-negative anaerobes like Bacteroides spp and Fusobacterium spp were present in 30.6% . Healing time was longer when strict aerobic pathogen Pseudomonas spp and strict anaerobic pathogens were present (136.1 +/- 28.6 and 136.4 +/- 34.7 days, respectively) . CONCLUSIONS: Diabetic foot infection is polymicrobial in nature . The healing time of wound infected with anaerobic pathogens was higher than those infected with aerobic pathogens . Anaerobic pathogens increased with the Wagner's grading . Presence of neuropathy increased the risk of foot infection.

Eur J Nutr, 2002 Nov, 41 Suppl 1, I26 - 31
Effects of lactulose on the intestinal microflora of periparturient sows and their piglets; Krueger M et al.; The periparturient period of animals (and humans) is very stressful and influenced by the microecosystem of the gastrointestinal tract (GIT) . Performance and productivity of animal husbandry depend on the health of animal mothers and their offspring . We investigated the influence of prebiotic amounts of lactulose in sows and their piglets . Two experimental trial sows received daily 30 ml lactulose, 71 field trial sows received daily 45 ml lactulose during their periparturient period (10 days before until 10 days after parturison) . The weaners of trial sows received 15 ml lactulose per 1 kg baby food 10 days before and 10 days after weaning.The effect of lactulose was recorded by performance parameters like number of piglet born alive, losses until weaning, body mass of piglets, daily weight gain of weaners until 35 days after weaning . The effect of lactulose on GIT microflora was estimated by bacterial counts of faeces of sows (total aerobic bacteria, Gram-negative bacteria, Clostridium (C.) perfringens) . In order to show a previously unknown effect of lactulose we investigated the levels of antibodies to phospholipase C (PLC) of C . perfringens in plasma of experimental sows and in colostral and ripe milk of field sows . Lactulose influenced the performance parameters of sows in a non-significant way . In case of weaners we recorded significant daily weight gains . Lactulose significantly influenced total aerobic bacterial counts, C . perfringens counts in faeces of sows 20 days after parturison . Under experimental conditions it was shown that trial sows and their piglets had higher IgG-antibody levels to C . perfringens PLCs than the control animals . Similar results were found under field conditions . Trial sows had significant higher IgG-anti LPS (J5) antibodies in milk 10 days after birth.

Trends Biochem Sci, 2002 Nov, 27(11), 552 - 8
Botulinum and tetanus neurotoxins: structure, function and therapeutic utility; Turton K et al.; The toxic products of the anaerobic bacteria Clostridium botulinum, Clostridium butyricum, Clostridium barati and Clostridium tetani are the causative agents of botulism and tetanus . The ability of botulinum neurotoxins to disrupt neurotransmission, often for prolonged periods, has been exploited for use in several medical applications and the toxins, as licensed pharmaceutical products, now represent the therapeutics of choice for the treatment for several neuromuscular conditions . Research into the structures and activities of botulinum and tetanus toxins has revealed features of these proteins that might be useful in the design of improved vaccines, effective inhibitors and novel biopharmaceuticals . Here, we discuss the relationships between structure, mechanism of action and therapeutic use.

J Biol Chem, 2003 Jan 10, 278(2), 1067 - 74 Epub 2002 Oct 31.
A thermally sensitive loop in clostridial glutamate dehydrogenase detected by limited proteolysis; Aghajanian S et al.; The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C . Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C . Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site . SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa . Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change . Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH . Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme . Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments . However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure . Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states . N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.

Plant J, 2002 Nov, 32(3), 391 - 400
NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS; Kirby J et al.; GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues . A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant . A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase . Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity . For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes . NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer . Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

Dig Liver Dis, 2002 Sep, 34 Suppl 2, S44 - 7
New therapeutic approach in the management of intestinal disease: probiotics in intestinal disease in paediatric age; Cucchiara S et al.; Current evidence supports the view that oral administration of probiotics may be of therapeutic usefulness in several clinical disorders by reestablishing normal flora in the gastrointestinal tract . These entities include inflammatory and infectious diseases of the gut as well as extraintestinal disorders (such as atopic eczema) in which a defective intestinal permeability plays a role . The probiotic effects are attributed to restoration to normal of increased intestinal permeability, unbalanced gut microecology, improved immunological gut barrier function, downregulation of the intestinal inflammatory responses with reduced generation of proinflammatory cytokines . Entities for which the impact of probiotic administration can be considered as proven are Rotavirus diarrhoea, Clostridium difficile diarrhoea, post-antibiotic diarrhoea, allergic diseases . On the other hand, entities for which administration of probiotics is considered under investigation are inflammatory bowel disease, necrotizing enterocolitis, cystic fibrosis, small bowel bacterial contamination, functional gastrointestinal disorders . The value of probiotics as therapy for a variety of gastrointestinal disorders in childhood still needs to be investigated in detail, through well controlled and rigorous studies, including a placebo group and strict criteria of randomisation . Much work needs to be done in this area by clearly defining indications, delivery system, costs, safety long-term effects.

Appl Environ Microbiol, 2002 Nov, 68(11), 5311 - 7
The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains; Zimmer M et al.; Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry . As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C . perfringens bacteriophage phi3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene . Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin . A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology . Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage lambda S holin in lambdadeltaSthf . The endolysin gene ply3626 was cloned in Escherichia coli . However, protein synthesis occurred only when bacteria were supplemented with rare tRNA(Arg) and tRNA(Ile) genes . Formation of inclusion bodies could be avoided by drastically lowering the expression level . Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography . Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme . All 48 tested strains of C . perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected . This highly specific activity towards C . perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.

Mol Microbiol, 2002 Oct, 46(2), 439 - 52
Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier; Purdy D et al.; Progress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems . We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT-based mobilization from Escherichia coli donors . Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C . difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems . The characterized replicon is the first C . difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times . Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M . CdiCD6II, with equivalent specificities to Sau96I/M . Sau96I (5'-GGNMCC-3') and MboI/M . MboI (5'-GMATC-3') respectively . A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5'-CATCG-3' between the fourth and fifth nucleotide to give a blunt-ended fragment . This is the first time that an enzyme with this specificity has been reported . The sequential addition of this site to vectors showed that each site caused between a five- and 16-fold reduction in transfer efficiency . The transfer efficiencies achieved with both strains equated to between 1.0 x 10-6 and 5.5 x 10-5 transconjugants per donor.

J Infect Dis, 2002 Nov 15, 186(10), 1438 - 47 Epub 2002 Oct 29.
Mechanism of Clostridium difficile toxin A-induced apoptosis in T84 cells; Brito GA et al.; This study is an investigation into the mechanism of Clostridium difficile toxin A-induced apoptosis in human intestinal epithelial cells . Toxin A induced apoptosis of T84 cells in a dose- and time-dependent fashion . Toxin A-induced apoptosis was completely inhibited by blocking toxin enzymatic activity on Rho GTPases with uridine 5'-diphosphate-2',3'-dialdehyde by a nonspecific caspase inhibitor and was partially inhibited by caspase-1, -3, -6, -8, and -9 inhibitors . Caspases 3, 6, 8, and 9 and Bid activation were detected . Toxin A also induced changes in mitochondrial membrane potential and cytochrome c release at 18-24 h, a time course similar to caspase-9 activation . In conclusion, toxin A induces apoptosis by a mechanism dependent on inactivation of Rho, activation of caspases 3, 6, 8, and 9 and Bid, and mitochondrial damage followed by cytochrome c release . Toxin A proapoptotic activity may contribute to the mucosal disruption seen in toxin A-induced enteritis.

Am J Clin Nutr, 2002 Nov, 76(5), 1117 - 25
Selective growth of mucolytic bacteria including Clostridium perfringens in a neonatal piglet model of total parenteral nutrition; Deplancke B et al.; BACKGROUND: Compromised barrier function and intestinal inflammation are common complications of total parenteral nutrition (TPN) . OBJECTIVE: We tested the hypothesis that the lack of enteral nutrients in TPN might select commensal or pathogenic bacteria that use mucus as a substrate, thereby weakening the protection provided by the intestinal mucus layer . DESIGN: Ileal microbiota profiles of piglets fed by total enteral nutrition (TEN; n = 6) or TPN (n = 5) were compared with the use of 16S ribosomal DNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis and with a PCR-based method developed to specifically measure Clostridium perfringens concentrations . Ileal bacteria from TEN and TPN piglets were also examined for their ability to grow on mucin or sulfated monosaccharides . RESULTS: Bacterial community structure was equally complex in the ileum of TEN and TPN piglets, but profiles clustered according to mode of nutrition . Sixty-two percent of total mucus-associated bacteria (100 colonies tested) in TPN compared with 33% of mucus-associated bacteria (100 colonies tested) in TEN ileal samples grew on mucin . Bacteria capable of using sulfated monosaccharides were also enriched in TPN samples . C . perfringens, an opportunistic pathogen, was specifically enriched in the TPN ileum (P < 0.05) . These results were corroborated by cultivation-based studies that showed rapid growth of C . perfringens on mucin-based substrates . CONCLUSIONS: Mucolytic potential is widespread among intestinal bacteria . Mucolytic bacteria in general and C . perfringens in particular were selected when enteral nutrients were withheld in this TPN piglet model . Similar enrichment processes may occur in humans nourished by TPN and may thereby contribute to intestinal dysfunction.

Ann Pharmacother, 2002 Nov, 36(11), 1767 - 75
Treatment of Clostridium difficile-associated diarrhea; Malnick SD et al.; OBJECTIVE: To review the literature related to the treatment and infection control of Clostridium difficile-associated diarrhea (CDAD) . DATA SOURCES: A MEDLINE search (1966-August 2001) of the English literature was conducted . DATA SYNTHESIS: C . difficile is a leading cause of antibiotic-related diarrhea . The clinical spectrum extends from simple diarrhea to fulminant colitis . Cessation of antibiotic therapy alone is sufficient for mild cases; however, the majority of cases require oral metronidazole as the drug of choice . Vancomycin orally is reserved for patients who have failed to respond to metronidazole, are pregnant, or are severely ill . There is an important role for infection control interventions . CONCLUSIONS: CDAD is a common infection . Appropriate antibiotic treatment and infection control policies can prevent the spread and reduce the morbidity associated with this disease.

Mol Hum Reprod, 2002 Nov, 8(11), 1014 - 22
Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: rho protein regulation; Heneweer C et al.; Embryo implantation involves adhesion of trophoblast cells to the epithelial lining of the endometrium . Using an in-vitro model to simulate this initial interaction, we previously reported that attachment of human trophoblast-like JAR spheroids to human uterine epithelial RL95-2 cells provokes a Ca(2+) influx in RL95-2 cells depending on apically localized integrin receptors . Here, we demonstrate that adhesiveness of RL95-2 cells for JAR spheroids, measured by a centrifugal force-based adhesion assay, is dependent on Rho GTPases, most likely RhoA . Cellular expression and distribution of RhoA were studied by fluorescence confocal microscopy, focusing on the localization of RhoA and F-actin within the adhesion sites between JAR and RL95-2 cells . Contact areas contained high amounts of RhoA and F-actin fibres near the plasma membrane . To determine whether Rho GTPases may influence JAR cell binding, we treated RL95-2 cells with Clostridium difficile toxin A, which specifically inactivates Rho GTPases . Toxin A treatment changed the subcellular distribution of endogenous RhoA in RL95-2 cells and altered RhoA and F-actin colocalization . Adhesion of JAR spheroids to RL95-2 cells treated with toxin A was largely suppressed . These data indicate that Rho GTPases, most likely RhoA, play an important role in uterine epithelial RL95-2 cells for trophoblast binding, and suggest that RhoA may be involved in local signalling cascades during early embryo implantation in vivo.

Pediatr Rehabil, 2002 Jan-Mar, 5(1), 11 - 9
Joint and bone infections due to anaerobic bacteria in children; Brook I; The current review describes the microbiology, diagnosis and management of septic arthritis and osteomyelitis due to anaerobic bacteria in children . Staphylococcus aureus, Haemophilus influenzae type-b, and Group A streptococcus, Streptococcus pneumoniae, Kingela kingae, Neisseria meningiditis and Salmonella spp are the predominant aerobic bacteria that cause arthritis in children . Gonococcal arthritis can occur in sexually active adolescents . The predominant aerobes causing osteomyelitis in children are S . aureus, H . influenzae type-b, Gram-negative enteric bacteria, beta-hemolytic streptococci, S . pneumoniae, K . kingae, Bartonella henselae and Borrelia burgdorferi . Anaerobes have rarely been reported as a cause of these infections in children . The main anaerobes in arthritis include anaerobic Gram negative bacilli including Bacteroides fragilis group, Fusobacterium spp., Clostridium spp . and Peptostreptococcus spp . Most of the cases of anaerobic arthritis, in contrast to anaerobic osteomyelitis, involved a single isolate . Most of the cases of anaerobic arthritis are secondary to hematogenous spread . Many patients with osteomyelitis due to anaerobic bacteria have evidence of anaerobic infection elsewhere in the body, which is the source of the organisms involved in osteomyelitis . Treatment of arthritis and osteomyelitis involving anaerobic bacteria includes symptomatic therapy, immobilization in some cases, adequate drainage of purulent material and antibiotic therapy effective to these organisms.

C R Biol, 2002 Aug, 325(8), 863 - 78; discussion 879-83
{Botulism: the agent, mode of action of the botulinum neurotoxins, forms of acquisition, treatment and prevention}; Marvaud JC et al.; The botulinum neurotoxins are produced by anaerobic, spore-forming bacteria belonging to the Clostridium genus . They are synthesised as a single chain protein (150 kDa), which is not or weakly active . The active form results from a proteolysis cleaving the precursor in a light chain (about 50 kDa) and a heavy chain (about 100 kDa), which are linked by a disulfide bridge . The heavy chain is involved in the recognition of a specific neuronal surface receptor and mediates the internalization of the light chain into the cytosol . The light chain is responsible for the intracellular activity . It catalyses the proteolysis of SNARE proteins, which are involved in the exocytosis of synaptic vesicles containing acetylcholine . Hence, the release of acetylcholine at the neuromuscular junction is blocked, leading to a flaccid paralysis . Human botulism, usually type A, B or E, is associated with intoxination, ingestion of preformed toxin in food, with digestive toxi-infection, mainly in newborns (infant botulism), or with wound contamination (wound botulism) . The treatment of botulism is usually symptomatic . The specific treatment is based on the serotherapy or on the use of purified specific antibodies . The vaccination against botulism is efficient . However, since the botulinum neurotoxins are widely used for the treatment of numerous dystonias, a generalised vaccination is not conceivable.

Mol Biol (Mosk), 2002 Sep-Oct, 36(5), 868 - 76
{A thermostable Clostridium thermocellum lichenase-based reporter system for studying the gene expression regulation in prokaryotic and eukaryotic cells}; Goldenkova IV et al.; A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells . The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter . The gene product was active and did not undergo modification in heterologous hosts . Simple and sensitive methods were used to detect and to quantitate the lichenase activity . Inducible licBM2 expression was demonstrated with E . coli and yeast cells, allowing the system to be employed in dynamic studies.

Arch Intern Med, 2002 Oct 28, 162(19), 2223 - 8
Health and economic outcomes of vancomycin-resistant enterococci; Carmeli Y et al.; BACKGROUND: The health and economic impact of vancomycin-resistant enterococci has not been quantified . METHODS: A retrospective matched cohort study was conducted comparing the outcomes of patients with vancomycin-resistant enterococci (cases) with those of control subjects matched for length of hospital stay until inclusion in the cohort, hospital location, and calendar date . The propensity to be a vancomycin-resistant enterococci case was modeled based on patient characteristics, and included in multivariable models to adjust for confounding . Analyses included the following: (1) conditional logistic regression for mortality, surgery, intensive care unit admission, and discharge to long-term care; (2) linear regression for the logarithm of cost; and (3) accelerated failure time model for length of stay . RESULTS: A total of 233 cases were compared with 647 controls . Groups were similar in age (mean, 62 years), sex (female, 47%), and length of stay before inclusion in the cohort (mean, 8.1 days), but differed in primary diagnosis and comorbidities, past infection or colonization with methicillin sodium-resistant Staphylococcus aureus or Clostridium difficile, and treatment with cephalosporins or metronidazole . These variables were included in the propensity score, which had good to excellent prediction . Outcomes for cases vs controls and adjusted risks (relative risks {RRs}) were as follows: (1) case fatality rate, 17% vs 6% (RR, 2.13; P =.04); (2) length of stay after inclusion in the cohort, 15.1 vs 8.5 days (RR, 1.73; P<.001); (3) hospital costs, $52 449 vs $31 915 (RR, 1.40; P<.001); (4) surgery after inclusion in the cohort, 18% vs 10% (RR, 2.74; P =.001); (5) intensive care unit admission after inclusion in the cohort, 25% vs 14% (RR, 3.47; P<.001); and (6) transfer to an institution, 51% vs 35% (RR, 2.01; P =.001) . CONCLUSION: Compared with a matched hospital population, a population with vancomycin-resistant enterococci was associated with severe adverse outcomes: increased mortality, morbidity, and costs.

Arch Intern Med, 2002 Oct 28, 162(19), 2177 - 84
The spectrum of pseudomembranous enterocolitis and antibiotic-associated diarrhea; Hurley BW et al.; Pseudomembranous (entero)colitis is primarily caused by Clostridium difficile infection . The most common predisposing factor is prior use of antibiotics, including vancomycin and metronidazole, which themselves are therapy for C difficile colitis . Other risk factors have also been described . The presence of C difficile in the gastrointestinal tract leads to a spectrum of manifestations from the asymptomatic carrier state to fulminant colitis . Successful treatment of C difficile colitis requires prompt treatment with appropriate antibiotics, withdrawal of the suspected predisposing antibiotics, and, in rare cases, total colectomy . Preventive measures of adequate infection control and judicious use of antibiotics are necessary means in attempting to control the spread of C difficile infection . Attempts at making an effective human vaccine are currently under way.

Lett Appl Microbiol, 2002, 35(5), 446 - 50
PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats; Boerema JA et al.; AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition . METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl . putrefaciens 16S rDNA gene fragments in meat . The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms . The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples . Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl . algidicarnis and/or Cl . putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours . CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs . SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.

Lett Appl Microbiol, 2002, 35(5), 433 - 8
Safety evaluation of sous vide-processed ready meals; Nissen H et al.; AIMS: To assess survival, growth and toxin production of spore-forming bacteria in sous vide products exposed to a relatively high heat treatment . METHODS AND RESULTS: During a three-year period, 2,168 sous vide-processed, commercially available ready-made meals with a shelf life of 3-5 weeks were examined . The products were stored at 4 degrees C for the first 1/3 and at 7 degrees C for the remaining 2/3 of their shelf life period . Three-fourths of the samples had less than 10 bacteria per gram the day after production, and none had more than 1,000 . Similar numbers were found at the end of the shelf life when stored as described above . At abuse temperature (20 degrees C), the number of bacteria increased to 10(6)-10(7) cfu g(-1) 7 d after production . A total of 350 isolates of Bacillus spp . were collected, but no Clostridium strains were detected . Only 11 of the 113 tested strains were able to grow at 7 degrees C in broth, and none of the psychrotrophic strains were able to produce substantial amounts of toxins causing food poisoning . CONCLUSION: The health risk of these products is small as long as the temperature during storage is low . For microbial testing of the end products, traditional plating will suffice.

Lett Appl Microbiol, 2002, 35(5), 366 - 9
Recognition of anaerobic bacterial isolates in vitro using electronic nose technology; Pavlou A et al.; AIMS: Use of an electronic nose (e.nose) system to differentiation between anaerobic bacteria grown in vitro on agar media . METHODS AND RESULTS: Cultures of Clostridium spp . (14 strains) and Bacteroides fragilis (12 strains) were grown on blood agar plates and incubated in sampling bags for 30 min before head space analysis of the volatiles . Qualitative analyses of the volatile production patterns was carried out using an e.nose system with 14 conducting polymer sensors . Using data analysis techniques such as principal components analysis (PCA), genetic algorithms and neural networks it was possible to differentiate between agar blanks and individual species which accounted for all the data . A total of eight unknowns were correctly discriminated into the bacterial groups . CONCLUSIONS: This is the first report of in vitro complex volatile pattern recognition and differentiation of anaerobic pathogens . SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest the potential for application of e.nose technology in early diagnosis of microbial pathogens of medical importance.

Environ Sci Technol, 2002 Oct 15, 36(20), 4370 - 81
Reactivity of partially reduced arylhydroxylamine and nitrosoarene metabolites of 2,4,6-trinitrotoluene (TNT) toward biomass and humic acids; Ahmad F et al.; Sequential anaerobic/aerobic treatment of 2,4,6-trinitrotoluene (TNT) generally results in the incorporation of residues into biomass and natural organic matter fractions of a system . To better understand the potential contribution of hydroxylamine and nitroso moieties in these reactions, studies were conducted using model systems taking advantage of the biocatalytic-activity of Clostridium acetobutylicum that does not produce aminated TNT derivatives . To evaluate binding to biomass only, systems containing cell-free extracts of C . acetobutylicum and molecular hydrogen as a reductant were employed . At the end of treatment, mass balance studies showed that 10% of the total 14C was associated with an insoluble protein-containing precipitate that could not be extracted with organic solvents . Model reactions were conducted between a mixture of 2,4-dihydroxylamino-6-nitrotoluene (DHA6NT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) and 1-thioglycerol to test the involvement of the nitroso-thiol reaction in binding to biomass . It was demonstrated that DHA6NT formed a new and relatively polar product with 1-thioglycerol only in the presence of oxygen . The oxygen requirement confirmed that the nitroso functionality was responsible for the binding reaction . The reactivity of arylhydroxylamino and nitrosoarene functionalities toward International Humic Substance Society (IHSS) peat humic acid was evaluated under anaerobic and aerobic conditions, respectively . 4HADNT showed no appreciable reactivity toward peat humic acid . Conversely, the nitrosoarene compound, nitrosobenzene, showed rapid reactivity with peat humic acid (50% removal in 48 h) . When tested with two other humic acids (selected on the basis of their protein content), it became apparent that the proteinaceous fraction was responsible at least in part for the nitrosoarene's removal from solution . Furthermore, the pretreatment of the humic acids with a selective thiol derivatizing agent had a considerable effect on their ability to react with nitrosobenzene . Finally, molecular modeling tools were used to compare the electrophilic characteristics of potential nitroso intermediates forming from the oxidation of arylhydroxylamino metabolites of TNT . Molecular modeling analysis demonstrated that the more reduced TNT derivative containing nitroso groups were more likely to react with nucleophiles in humic substances than the less reduced nitroso intermediates.

Science, 2002 Oct 18, 298(5593), 567 - 72
A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase; Doukov TI et al.; A metallocofactor containing iron, sulfur, copper, and nickel has been discovered in the enzyme carbon monoxide dehydrogenase/acetyl-CoA (coenzyme A) synthase from Moorella thermoacetica (f . Clostridium thermoaceticum) . Our structure at 2.2 angstrom resolution reveals that the cofactor responsible for the assembly of acetyl-CoA contains a {Fe4S4} cubane bridged to a copper-nickel binuclear site . The presence of these three metals together in one cluster was unanticipated and suggests a newly discovered role for copper in biology . The different active sites of this bifunctional enzyme complex are connected via a channel, 138 angstroms long, that provides a conduit for carbon monoxide generated at the C-cluster on one subunit to be incorporated into acetyl-CoA at the A-cluster on the other subunit.

Microb Pathog, 2002 Oct, 33(4), 177 - 84
Characterization of Clostridium butyricum neurotoxin associated with food-borne botulism; Tsukamoto K et al.; The neurotoxin of Clostridium butyricum strain LCL155 (BuNT/LCL155) associated with type E food-borne botulism showed antigenic and biological properties different from those of C . botulinum type E (BoNT/E) andC . butyricum strain BL5262 (BuNT/BL5262) . The specific toxicity of BuNT/LCL155 was found to be about 10% those of BoNT/E and BuNT/BL5262 . Immunological analysis with monoclonal antibodies against BoNT/E showed that the heavy chain of BuNT/LCL155 differs partially from those of BoNT/E and BuNT/BL5262 . Binding experiments with rat brain synaptic membrane revealed that BuNT/LCL155 possesses a binding activity lower than either BoNT/E or BuNT/BL5262 . There was no difference in the catalytic activity of the three neurotoxins, which had been determined with a recombinant of the intracellular target protein SNAP-25 . These data suggest that the BuNT/LCL155 shares the receptor-recognition site structurally different from BoNT/E and BuNT/BL5262, perhaps causing a decreased specific toxicity.

Int J Antimicrob Agents, 2002 Oct, 20(4), 270 - 4
In vitro activity of 15 antimicrobial agents against clinical isolates of Clostridium difficile in Kuwait; Jamal WY et al.; A total of 73 clinical isolates of Clostridium difficile isolated from stool/rectal swabs of patients admitted to the intensive care units at Mubarak Hospital, Ibn Sina Hospital Burn unit and Haematology wards at the Kuwait Cancer Control Centre, were investigated for their susceptibility to 15 antibiotics using the Etest . Amoxycillin-clavulanic acid, ampicillin, meropenem, metronidazole, penicillin, piperacillin, piperacillin/tazobactam, teicoplanin and vancomycin had excellent activities with MIC(90)s of 0.38, 0.5, 1, 0.19, 1.5, 2, 3, 0.25 and 0.75 mg/l, respectively . Of the 73 C . difficile isolates, 86% were resistant to imipenem (MIC(90) >32 mg/l) and almost 97% were resistant to trovafloxacin (MIC(90)>256 mg/l) . Forty eight percent of the isolates were resistant to clindamycin . A total of 18 isolates were highly clindamycin-resistant with an MIC of >256 mg/l; 10 of these were toxin producers . Multiple antibiotic resistance (two or more antibiotics) was noted in 63 isolates . These were more common among the toxigenic strains than the non-toxigenic strains by a ratio of 2.5:1.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3418 - 21
gyrA and gyrB mutations are implicated in cross-resistance to Ciprofloxacin and moxifloxacin in Clostridium difficile; Dridi L et al.; A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 micro g of ciprofloxacin per ml . The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997 . Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable . Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished . All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71-->Val in one, Thr82-->Ile in six, and Ala118-->Thr in one) or in gyrB (six mutations, amino acid changes Asp426-->Asn in five and Arg447-->Leu in one) . These changes are similar to those already described in other species except for Asp71-->Val, which is novel, and Ala118-->Thr, which is exceptional . Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful . The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 micro g/ml . In conclusion, decreased susceptibility to fluoroquinolones in C . difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.

Plasmid, 2002 Sep, 48(2), 98 - 103
Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure; Danielsen M; The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced . The sequence revealed a composite structure containing DNA from up to four different sources . The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus . Within the tetracycline resistance region a Lactobacillus IS-element was found . The remaining part of the plasmid contained three open reading frames with unknown functions . The composite structure with several truncated genes suggests a recent assembly of the plasmid . This is the first sequence of an antibiotic resistance plasmid isolated from L . plantarum.

Int J Food Microbiol, 2002 Nov 15, 79(1-2), 3 - 16
Preservation and fermentation: past, present and future; Ross RP et al.; Preservation of food and beverages resulting from fermentation has been an effective form of extending the shelf-life of foods for millennia . Traditionally, foods were preserved through naturally occurring fermentations, however, modern large scale production generally now exploits the use of defined strain starter systems to ensure consistency and quality in the final product . This review will mainly focus on the use of lactic acid bacteria (LAB) for food improvement, given their extensive application in a wide range of fermented foods . These microorganisms can produce a wide variety of antagonistic primary and secondary metabolites including organic acids, diacetyl, CO2 and even antibiotics such as reuterocyclin produced by Lactobacillus reuteri . In addition, members of the group can also produce a wide range of bacteriocins, some of which have activity against food pathogens such as Listeria monocytogenes and Clostridium botulinum . Indeed, the bacteriocin nisin has been used as an effective biopreservative in some dairy products for decades, while a number of more recently discovered bacteriocins, such as lacticin 3147, demonstrate increasing potential in a number of food applications . Both of these lactococcal bacteriocins belong to the lantibiotic family of posttranslationally modified bacteriocins that contain lanthionine, beta-methyllanthionine and dehydrated amino acids . The exploitation of such naturally produced antagonists holds tremendous potential for extension of shelf-life and improvement of safety of a variety of foods.

J Assoc Res Otolaryngol, 2002 Sep, 3(3), 289 - 301 Epub 2002 Feb 27.
Structural microdomains in the lateral plasma membrane of cochlear outer hair cells; Zhang M et al.; The basal and lateral regions of the plasma membrane of cochlear outer hair cells are structurally and functionally distinct . The lateral region contains thousands of motor proteins but few voltage-gated channels . The basal region, conversely, contains a high number of voltage-gated channels but is devoid of motor proteins . It has been suggested that the cortical cytoskeleton is responsible for maintaining this regional distinction . Toward elucidating the structure of the outer hair cell's electromotile mechanism, we investigated the physical organization of the lateral plasma membrane in living guinea pig outer hair cells by analyzing the distribution pattern of the anionic long-chain carbocyanine SP-DiIC18(3) within this area, before and after electrical stimulation and with an intact and a disrupted cytoskeleton . We observed punctate, intensely fluorescent patches as well as areas of weaker fluorescence, with clear local maxima and minima, upon labeling the cells with this membrane-soluble probe . This discrete distribution of SP-DilC18(3) suggests that the lateral plasma membrane of guinea pig outer hair cells may be composed of small structural domains (microdomains) . Disrupting the cytoskeleton with either trypsin or toxin B from Clostridium difficile did not change this pattern of distribution, thus indicating that this treatment did not facilitate the lateral diffusion of the probes . Electrical stimulation using whole-cell patch-clamp techniques, on the other hand, induced two responses: fast motility and reversible displacement of the fluorescent probes . Both responses were inhibited by internal perfusion with salicylate, while disruption of the cytoskeleton did not inhibit OHC fast motility but affected the electrically induced redistribution of fluorescent probes . Together, these results suggest that the lateral plasma membrane of guinea pig outer hair cells contains structural microdomains and that the cytoskeleton does not appear to be playing a major role in maintaining the lateral separation of these distinct molecular regions.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14476 - 81 Epub 2002 Oct 15.
Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton; Shupliakov O et al.; Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles . In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone . Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon . In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster . The filaments proliferated after stimulation and extended toward the vesicle cluster . Synaptic vesicles were tethered along the filaments . Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited . Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses . Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.

J Food Prot, 2002 Oct, 65(10), 1667 - 9
Inhibition of growth, enterotoxin production, and spore formation of Clostridium perfringens by extracts of medicinal plants; Garcia S et al.; The extracts of 14 plants used in the traditional medicine of Mexico were evaluated for their effects on the growth, spore formation, and enterotoxin production of Clostridium perfringens type A . The extracts of Psidium guajava L., Haemotoxylon brasiletto, and Euphobia prostata were the most effective inhibitors of growth, spore formation, and enterotoxin production . No enterotoxins were detected when extracts were added to the media at less than the MIC for growth.

J Food Prot, 2002 Oct, 65(10), 1580 - 5
Effective use of nisin to control Bacillus and Clostridium spoilage of a pasteurized mashed potato product; Thomas LV et al.; Heat-resistant spore-forming bacteria such as Bacillus and Clostridium can survive and grow in cooked potato products . This situation represents both a public health problem and an economic problem . The natural food preservative nisin is used in heat-treated foods to prevent the growth of such bacteria . A cocktail of Clostridium sporogenes and Clostridium tyrobutyricum spores was inoculated into cooked mashed potatoes, which were vacuum packed, pasteurized, and incubated at 8 and 25 degrees C . The shelf life of the mashed potatoes at 25 degrees C was extended by at least 58 days with the addition 6.25 microg of nisin per g . At 8 degrees C, in control samples not containing nisin, the natural contaminant Bacillus grew, but the inoculated Clostridium strains did not until the temperature was raised to 20 degrees C after 39 days . No bacterial growth occurred in nisin-containing samples . The shelf life of the mashed potatoes was extended by at least 30 days with 6.25 microg of nisin per g . In trials involving a cocktail of Bacillus cereus and Bacillus subtilis strains, 6.25 microg of nisin per g extended the shelf life of mashed potato samples that were not vacuum packed by at least 27 days at 8 degrees C . At 25 degrees C, 25 microg of nisin per g extended shelf life by a similar period . Shelf life extension was also observed at lower nisin levels . Microbiological analysis of the mashed potato ingredients showed that a high spore level was associated with the onion powder . It is emphasized that the preservative and the ingredients must be well mixed to ensure good nisin efficacy . Nisin remained at effective levels after pasteurization, and good retention was observed throughout the shelf life of the mashed potatoes.

Bone Marrow Transplant, 2002 Oct, 30(8), 517 - 9
Rarity of toxigenic Clostridium difficile infections after hematopoietic stem cell transplantation: implications for symptomatic management of diarrhea; Tomblyn M et al.; Diarrhea is a common complication of high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) . The frequent and prolonged use of multiple antibiotics in this setting can predispose to infection with toxigenic Clostridium difficile and the development of pseudomembranous colitis . Anti-motility agents are usually not administered in this setting until C . difficile infection has been excluded . The objective of this study was to determine the incidence of C . difficile toxin (CDT) positivity at the time of initial diarrhea in HSCT recipients, and to see if the practice of ensuring negative CDT assays prior to initiating symptomatic management of diarrhea needs modification . One hundred and nineteen patients with malignant diseases undergoing autologous or allogeneic HSCT were studied to determine the incidence of diarrhea and CDT positivity with initial diarrhea . One hundred and nine (91%) had diarrhea . Of these, only seven (6%) were CDT+ at the time of initial diarrhea . The median interval between onset of diarrhea and starting symptomatic anti-diarrheal therapy was 1 day . There were no significant differences between the patients with CDT+ diarrhea and the others in terms of timing or severity of diarrhea, number or duration of antibiotic usage, or leukocyte count . The infection resolved in all patients with metronidazole therapy . Our data suggest that the incidence of CDT+ diarrhea is low in HSCT recipients . Concern about C . difficile infection should not delay symptomatic therapy of initial diarrhea in HSCT recipients.

Infect Immun, 2002 Nov, 70(11), 5924 - 30
Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans; Palaniappan RU et al.; A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection . LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats . A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis . Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions . No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30 degrees C or when cultures were shifted to 37 degrees C . Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression . These findings suggest that LigA is specifically induced only in vivo . Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA . LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection . Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.

J Biol Chem, 2002 Dec 13, 277(50), 48949 - 59 Epub 2002 Oct 09.
Comparative genomics of thiamin biosynthesis in procaryotes . New genes and regulatory mechanisms; Rodionov DA et al.; Vitamin B(1) in its active form thiamin pyrophosphate is an essential coenzyme that is synthesized by coupling of pyrimidine (hydroxymethylpyrimidine; HMP) and thiazole (hydroxyethylthiazole) moieties in bacteria . Using comparative analysis of genes, operons, and regulatory elements, we describe the thiamin biosynthetic pathway in available bacterial genomes . The previously detected thiamin-regulatory element, thi box (Miranda-Rios, J., Navarro, M., and Soberon, M . (2001) Proc . Natl . Acad . Sci . U . S . A . 98, 9736-9741), was extended, resulting in a new, highly conserved RNA secondary structure, the THI element, which is widely distributed in eubacteria and also occurs in some archaea . Search for THI elements and analysis of operon structures identified a large number of new candidate thiamin-regulated genes, mostly transporters, in various prokaryotic organisms . In particular, we assign the thiamin transporter function to yuaJ in the Bacillus/Clostridium group and the HMP transporter function to an ABC transporter thiXYZ in some proteobacteria and firmicutes . By analogy to the model of regulation of the riboflavin biosynthesis, we suggest thiamin-mediated regulation based on formation of alternative RNA structures involving the THI element . Either transcriptional or translational attenuation mechanism may operate in different taxonomic groups, dependent on the existence of putative hairpins that either act as transcriptional terminators or sequester translation initiation sites . Based on analysis of co-occurrence of the thiamin biosynthetic genes in complete genomes, we predict that eubacteria, archaea, and eukaryota have different pathways for the HMP and hydroxyethylthiazole biosynthesis.

J Bacteriol, 2002 Nov, 184(21), 5971 - 8
Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression; Mani N et al.; TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis . Whether expressed in C . difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes . As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium . That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase . In the presence of excess glucose, expression from the txeR promoter was repressed . The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals . The increased level of TxeR then permits high-level expression of the toxin genes . The study of txeR gene regulation in C . difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E . coli.

J AOAC Int, 2002 Sep-Oct, 85(5), 1025 - 8
Specific detection of Clostridium botulinum types A, B, E, and F using the polymerase chain reaction; Craven KE et al.; Clostridium botulinum organisms generally produce 1 of 4 neurotoxin types (A, B, E, and F) associated with human illness . Neurotoxin type determination is important in identification of the bacterium . A polymerase chain reaction (PCR) method was developed to identify 24 h botulinal cultures as potential types A, B, E, and F neurotoxin producers as well as other clostridial species which also produce neurotoxins . Components of the PCR and amplification conditions were adjusted for optimal amplification of toxin gene target regions to enable simultaneous testing for types A, B, E, and F in separate tubes using a single thermal cycler . Each primer set was specific for its corresponding toxin type . A DNA extraction procedure was also included to remove inhibitory substances that may affect amplification . This procedure is rapid, sensitive, and specific for identification of toxigenic C . botulinum.

Scand J Infect Dis, 2002, 34(9), 694 - 6
Venous sinus thrombosis after Proteus vulgaris meningitis and concomitant Clostridium abscess formation; Bodur H et al.; A 19-y-old woman presented with Proteus vulgaris meningitis as a complication of chronic otitis media . Despite treatment with ceftazidime and amikacin no clinical improvement was observed . Cranial MRI revealed right-sided mastoiditis/otitis media and venous sinus thrombosis . After mastoidectomy, repeat cranial MRI demonstrated abscess formation in the venous sinuses . The abscess was drained . Clostridium spp . was isolated from the abscess culture.

Scand J Gastroenterol, 2002 Sep, 37(9), 1034 - 41
Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls; Kleessen B et al.; BACKGROUND: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD) . The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa . METHODS: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis . RESULTS: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls (P < 0.05) . Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls . Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria, the Enterobacteriaceae, the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups . CONCLUSION: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.

Infect Dis Clin North Am, 2002 Sep, 16(3), 589 - 605
Central nervous system infections in injection drug users; Tunkel AR et al.; Central nervous system infections in injection drug users are often devastating in terms of excess morbidity and mortality . In injection drug users with infective endocarditis, embolization from infected valvular vegetations may cause cerebral infarction, intracranial hemorrhage, and the formation of brain abscess . Focal intracranial infections (i.e., brain abscess and spinal epidural abscess) may occur in the absence of infective endocarditis, resulting from bacteremia that seeds the brain or epidural space . Antimicrobial therapy, combined with surgical intervention, may be essential to improve outcome from these neurologic complications . Toxin-mediated diseases (especially tetanus and wound botulism) are also seen in injection drug users . Inoculation of Clostridium spp at injection sites may lead to toxin generation and disease . Clinicians must maintain a high level of suspicion for these diagnoses in injection drug users.

Curr Protein Pept Sci, 2000 Jul, 1(1), 91 - 103
The C-terminal domain of pancreatic lipase: functional and structural analogies with c2 domains; Chahinian H et al.; The 3D structure of pancreatic lipase (PL) consists of two functional domains . The N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site, which involves a catalytic triad analogous to that present in serine proteases . The beta-sandwich C-terminal domain of PL plays an important part in the binding process between the lipase and colipase, the specific PL cofactor . Recent structure-function studies have suggested that the PL C-terminal domain may have an extra role apart from that of binding colipase . This domain contains an exposed hydrophobic loop (beta5') which was found to be located on the same side as the hydrophobic loops surrounding the active site, and it may be involved in the lipid binding process . Indirect evidence for this new function of the PL C-terminal domain has been provided by studies with monoclonal antibodies directed against the beta5' loop . The catalytic activity of the PL-antibody complexes on water insoluble substrates decreased drastically, whereas their esterase activity on a soluble substrate remained unchanged . During the last few years, a number of protein structures (15-lipoxygenase, alpha-toxin from Clostridium perfringens) have been determined that contain domains with close structural homologies with the beta-sandwich C-terminal domain of PL . Generally speaking, these domains show structural homologies with the C2 domains occurring in a wide range of proteins involved in signal transduction (e.g . phosphoinositide-specific phospholipase C, protein kinase C, cytosolic phospholipase A2), membrane traffic (e.g . synaptotagmin I, rabphilin) and membrane disruption (e.g . perforin) . Here it is proposed to review the structure and function of the C2 domains, based on the recent 3D structures and improved sequence alignments.

Nat Struct Biol, 2002 Nov, 9(11), 823 - 7
Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin; Ramachandran R et al.; Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes . Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition . By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted oligomeric form of the toxin . Only the short hydrophobic loops at the tip of the D4 beta-sandwich are exposed to the bilayer interior, whereas the remainder of D4 projects from the membrane surface and is surrounded by water, making little or no contact with adjacent protein monomers in the oligomer . Thus, a limited interaction of D4 with the bilayer core seems to be sufficient to accomplish cholesterol recognition and initial binding of PFO to the membrane . Furthermore, D4 serves as the fulcrum around which extensive structural changes occur during the formation and insertion of the large transmembrane beta-barrel into the bilayer.

J Mol Biol, 2002 Oct 4, 322(5), 997 - 1011
The FxRxHrS motif: a conserved region essential for DNA binding of the VirR response regulator from Clostridium perfringens; McGowan S et al.; The VirSR two-component signal transduction pathway regulates virulence and toxin production in Clostridium perfringens, the causative agent of gas gangrene . The response regulator, VirR, binds to repeat sequences located upstream of the promoter and is directly responsible for the transcriptional activation of pfoA, the structural gene for the cholesterol-dependent cytolysin, perfringolysin O . Comparative sequence analysis of the 236 amino acid residue VirR protein revealed a two-domain structure: a typical N-terminal response regulator domain and an uncharacterised C-terminal domain . Database searching revealed that over 40 other proteins, many of which appeared to be response regulators or transcriptional activators, had homology with the VirR C-terminal domain (VirRc) . Multiple sequence alignment of this VirRc family revealed a highly conserved region that was designated the FxRxHrS motif . By deletion analysis this motif was shown to be essential for the functional integrity of the VirR protein . Alanine scanning mutagenesis and subsequent phenotypic analysis indicated that conserved residues located within the motif were required for activity . These residues extended from L179 to N194 . More detailed site-directed mutagenesis showed that amino acid residues R186, H188 and S190 were essential for activity since even conservative substitutions in these positions resulted in non-functional proteins . Three of the mutant proteins, R186K, S190A and S190C, were purified and shown by in vitro gel shift analysis to be unable to bind to the specific target DNA with the same efficiency as the wild-type protein . These data reveal for the first time that VirRc functions as a DNA binding domain in which the highly conserved FxRxHrS motif has a functional role . These studies have important implications for this new family of transcriptional factors since they imply that the conserved FxRxHrS motif may be involved in DNA binding in all of these proteins, irrespective of their biological role.

Br Poult Sci, 2002 Sep, 43(4), 569 - 79
The influence of grinding and pelleting of feed on the microbial composition and activity in the digestive tract of broiler chickens; Engberg RM et al.; 1 . The influence of feed grinding (coarsely or finely ground feed) and feed form (mash or pellets) on the intestinal environment was investigated in a growth experiment with broiler chickens taking the intestinal microflora, intestinal viscosity, and the activities of pancreatic digestive enzymes into consideration . 2 . As compared to mash the feeding of pellets was associated with a significantly higher body weight due to increased feed intake and improved feed utilisation . 3 . Pellet-fed birds had significantly decreased gizzard weights, a higher gizzard pH and a lower intestinal pH than mash-fed birds . 4 . Pellet-fed birds had significantly lower relative pancreas weights and lower activities of pancreatic digestive enzymes (amylase, lipase, chymotrypsin), which indicates the existence of a feedback mechanism, which may have been triggered by the intestinal concentration of enzymatically hydrolysed products or of the respective digestive enzymes . 5 . Pellet-fed birds had larger numbers of coliform bacteria and enterococci in the ileum and a reduced number of Clostridium perfringens and lactobacilli in the distal end of the digestive tract (caeca and rectum) . Microbial fermentation in terms of volatile fatty acid (VFA) concentration was found to be significantly higher in the caeca of pellet-fed birds than in mash-fed birds.

Br Poult Sci, 2002 Sep, 43(4), 545 - 50
Effect of administration of Lactobacillus crispatus, Clostridium lactatifermentans and dietary lactose on the development of the normal microflora and volatile fatty acids in the caeca of broiler chicks; van der Wielen PW et al.; 1 . Lactobacillus crispatus and Clostridium lactatifermentans, both isolated from the caeca of chickens, grown together in an in vitro model system are able to ferment lactose to acetate and propionate . In this study, the capabilities of these organisms were studied in vivo . 2 . The effect on concentrations of volatile fatty acids and lactate, together with the development of some bacterial groups in the caeca of chicks, was studied after oral inoculation with L . crispatus and C . lactatifermentans together with dietary lactose . For this purpose, chicks were divided into 4 groups: (i) control group, (ii) dietary lactose, (iii) L . crispatus and C . lactatifermentans, and (iv) dietary lactose together with L . crispitaus and C . lactatifermentans . 3 . In general, concentrations of (undissociated) volatile fatty acids in the caeca were not significantly different in broilers receiving both bacteria and dietary lactose compared with control broilers . Concentrations of lactate in the caeca of 14-d-old broilers treated with any of the three treatments were significantly higher than in the caeca of control broilers . 4 . This indicates that L . crispatus or other lactate-producing organisms were responsible for the elevated concentrations of lactic acid . Clostridium lactatifermentans has probably not colonised the caeca sufficiently to ferment this lactate further to acetate and propionate . 5 . Numbers of Enterobacteriaceae and enterococci in the caeca of broilers receiving both bacteria and dietary lactose were not different from control broilers . 6 . We conclude from these results that under the conditions applied in this study a mixture of L . crispatus and C . lactatifermentans with dietary lactose was able to increase lactate concentrations but was unable to increase concentrates of acetate and propionate in the caeca of broiler chicks.

Hear Res, 2002 Oct, 172(1-2), 81 - 6
Rescue of auditory hair cells from aminoglycoside toxicity by Clostridium difficile toxin B, an inhibitor of the small GTPases Rho/Rac/Cdc42; Bodmer D et al.; The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss . Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies . Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides (J . Neurosci . 20 (2000) 43) . We have studied the upstream events leading to JNK activation in aminoglycoside toxicity in vitro . The small GTPases Rac and Cdc42 are well known upstream activators of JNK in other cell types . Clostridium difficile toxin B monoglucosylates all members of the Rho GTPase subfamily (Rho, Rac and Cdc42 isoforms) and inhibits GTP binding by steric interference (Nature 341 (1989) 209) . Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with C . difficile toxin B for 12 h . They were then treated with toxin B plus gentamicin for 72 h . Significantly less HC death was observed compared to with gentamicin alone . Toxin B alone had no effect on HCs at the highest concentration used . Using antibodies against phospho-c-Jun, we observed background immunoreactivity in control explants, strong staining of outer hair cell nuclei in gentamicin treated explants, and weaker immunostaining in explants treated with gentamicin and C . difficile toxin B . We conclude that Rho family small GTPases play a role in aminoglycoside toxicity signaling as upstream activators of the JNK signaling pathway.

Arch Surg, 2002 Oct, 137(10), 1096 - 100
Clostridium difficile colitis: an increasingly aggressive iatrogenic disease?
Morris AM, Jobe BA, Stoney M, Sheppard BC, Deveney CW, Deveney KE.
HYPOTHESIS: The diagnosis of Clostridium difficile colitis is increasing in frequency, with worsening patient outcomes . DESIGN: Retrospective cohort study . SETTING: University hospital . PATIENTS: One hundred fifty-seven patients diagnosed with C difficile colitis between 1994-2000 . MAIN OUTCOME MEASURES: Resolution of disease, operative intervention, and death . RESULTS: Compared with our previous 10-year experience, overall cases of C difficile colitis have risen by more than 30%, and immunocompromised patients comprise a larger proportion of those affected . One third of patients were receiving posttransplantation medication, chemotherapy, or had human immunodeficiency virus . Of these, 2 (4%) of 51 required surgical intervention and 10 (20%) of 51 died . An additional 18.5% of patients had diabetes, renal failure, or both . Of these, 2 (7%) of 30 required surgery and 4 (13%) of 30 died . Only 9.5% of patients had prophylactic perioperative antibiotics as a sole risk factor; 2 (13%) of 15 required surgery and 3 (20%) of 15 died . The overall mortality rate was 15.3%, increased from 3.5% in our previous series . Neither need for surgery nor mortality differed among these patient groups . CONCLUSIONS: The frequency of C difficile colitis remains high and seems to be associated with increasing mortality . Among patients with positive C difficile toxin assay results, immunocompromise and delayed diagnosis no longer seem to be associated with higher risk for death . All patients taking antibiotics are at risk and require early recognition and aggressive medical intervention.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1469 - 75
Shuttleworthia satelles gen . nov., sp . nov., isolated from the human oral cavity; Downes J et al.; Nine strains of anaerobic, non-spore-forming, gram-positive bacilli, isolated from the human oral cavity and provisionally identified as belonging to the genus Eubacterium, were subjected to a comprehensive range of phenotypic and genetic tests . Biochemically, they were found to comprise a homogeneous group, and phylogenetic analysis of their 16S rRNA sequences indicated that they constitute a unique branch within the Clostridium-Bacillus subphylum of the phylum Firmicutes . All of the isolates displayed an unusual colonial morphology after extended incubation . This resembled a contaminated culture in that small, secondary colonies were seen to arise around and from within the primary colony form, and a third, independent, colony type was also seen . However, inspection of the colonies by Gram-staining and scanning electron microscopy together with protein profile analysis and 16S rRNA gene sequence comparison of the two independent colony types revealed that only a single organism was present . A new genus, Shuttleworthia, and the species Shuttleworthia satelles gen . nov., sp . nov., are proposed . The cells are saccharolytic, and acetate, butyrate and lactate are produced as end products of glucose fermentation . Aesculin is hydrolysed and indole is produced . The G+C content of the DNA of the type strain is 51 mol% . The type strain is strain DSM 14600T (= CCUG 45864T = VPI D143K-13T).

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1461 - 8
Clostridium thiosulfatireducens sp . nov., a proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor; Hernandez-Eugenio G et al.; A strictly anaerobic, gram-positive, sporulating rod (0.5-0.6 x 2.0-4.0 microm), designated strain Lup 21T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating cheese-factory wastewater . Strain Lup 21T was motile by means of peritrichous flagella, had a G+C content of 31.4 mol% and grew optimally at 37 degrees C, pH 7.4, in the absence of NaCl . It is a heterotrophic micro-organism, utilizing proteinaceous compounds (gelatin, peptides, Casamino acids and various single amino acids) but unable to use any of the carbohydrates tested as a carbon and energy source . It reduced thiosulfate and elemental sulfur to sulfide in the presence of Casamino acids as carbon and energy sources . Acetate, butyrate, isobutyrate, isovalerate, CO2 and sulfide were end products from oxidation of gelatin and Casamino acids in the presence of thiosulfate as an electron acceptor . In the absence of thiosulfate, serine, lysine, methionine and histidine were fermented . On the basis of 16S rRNA similarity, strain Lup 21T was related to members of the low-G+C Clostridiales group, Clostridium subterminale DSM 6970T being the closest relative (with a sequence similarity of 99.4%) . DNA-DNA hybridization was 56% with this species . On the basis of phenotypic, genotypic and phylogenetic characteristics, the isolate was designated as a novel species of the genus Clostridium, Clostridium thiosulfatireducens sp . nov . The type strain is strain Lup 21T (= DSM 13105T = CIP 106908T).

Planta Med, 2002 Sep, 68(9), 767 - 9
Free fatty acids inhibit the activity of Clostridium histolyticum collagenase and human neutrophil elastase; Rennert B et al.; We investigated the ability of free fatty acids to inhibit the activity of Clostridium histolyticum collagenase (EC 3.4.24.3) and human neutrophil elastase (EC 3.4.21.37) . We determined the activity of collagenase by degradation of resorufin-labeled casein fluorimetrically . The determination of the elastase activity was performed by a spectrophotometric method using a 4-nitroanilide peptide substrate . We found that most of the tested fatty acids inhibited collagenase at concentrations between 50 microM and 500 microM . For elastase we found an inhibition of the activity at concentrations between 500 nM and 50 microM . The most potent inhibitory fatty acids of both enzymes differed . Thus, as a result for collagenase we can assume that the saturated fatty acids with C(16)-C(19) were the most potent ones . For elastase the inhibition rate of unsaturated acids was much higher than the rate of the saturated ones . The highly active erucic acid with an IC(50) value of 450 nM (elastase) is remarkable.

Invest Ophthalmol Vis Sci, 2002 Oct, 43(10), 3181 - 9
ECM-stimulated signaling and actin reorganization in embryonic corneal epithelia are Rho dependent; Reenstra WR et al.; PURPOSE: The goal of this study was to investigate the role of the small guanosine triphosphatase (GTPase), Rho, in the corneal epithelial response to extracellular matrix (ECM) molecules . The avian corneal epithelial model was used to establish that Rho is required for actin reorganization and tyrosine phosphorylation of integrin-mediated signal pathway proteins . METHODS: Whole embryonic corneal epithelia were isolated without the basal lamina and either transfected with Rho-specific antisense oligonucleotides or treated with Clostridium botulinum C3 exoenzyme and then stimulated with fibronectin (FN) or collagen (COL) . The epithelia were evaluated for actin reorganization and protein production including Rho protein levels and tyrosine phosphorylation with Western blot analysis . RESULTS: After an overnight transient transfection with antisense oligonucleotides, Rho protein levels were decreased more than 80%, and tyrosine phosphorylation of all integrin-mediated signal transduction proteins was decreased compared with control epithelia . Intracellular Rho distribution did not change in the presence of antisense oligonucleotides; however, the amount of immunolabeled Rho decreased . Disrupting the signaling cascade with Rho antisense also blocked FN- and COL-stimulated actin cortical mat reformation . C . botulinum C3 exoenzyme, a pharmacologic agent that specifically causes adenosine diphosphate (ADP) ribosylation and inactivation of Rho, also blocked actin reorganization and tyrosine phosphorylation . In contrast, decreasing Raf protein levels did not change FN-mediated actin reorganization or tyrosine phosphorylation . CONCLUSIONS: Decreasing Rho protein or blocking its function inhibited ECM-stimulated actin reorganization and signal transduction, as measured by tyrosine phosphorylation.

EMBO J, 2002 Oct 1, 21(19), 5047 - 56
Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin; Hong Y et al.; Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes . The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL . These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane . We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants . One mutant unexpectedly expressed GPI-anchored proteins, but nevertheless bound aerolysin poorly and was 10-fold less sensitive than wild-type cells . A cDNA of N-acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant . Therefore, N-glycan is involved in the binding . Removal of mannoses by alpha-mannosidase II was important for the binding of aerolysin . In contrast, the alpha-toxin killed GnTI-deficient and wild-type CHO cells equally, indicating that its binding to GPI-anchored proteins is independent of N-glycan . Because SL bound to wild-type but not to GnTI-deficient cells, and because a hybrid toxin consisting of SL and the alpha-toxin killed wild-type cells 10-fold more efficiently than GnTI- deficient cells, SL with its binding site for N-glycan contributes to the high binding affinity of aerolysin.

J Chromatogr A, 2002 Sep 13, 970(1-2), 95 - 115
Characterisation of botulinum toxins type A and B, by matrix-assisted laser desorption ionisation and electrospray mass spectrometry; van Baar BL et al.; A method earlier developed for the mass spectrometric (MS) identification of tetanus toxin (TTx) was applied to botulinum toxins type A and B (BTxA and BTxB) . Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent . Biologically active BTxA and BTxB are comprised of a protein complex of the respective neurotoxins with specific haemagglutinins (HAs) and non-toxic non-haemagglutinins (NTNHs) . These protein complexes are also observed in mass spectrometric identification . The particular BTxA complex, from Clostridium botulinum strain 62A, almost completely matched database data derived from genetic sequences known for this strain . Although no such database information was available for BTxB, from C . botulinum strain okra, all protein sequences from the complex except that of HA-70 were found to match proteins known from other type B strains . It was found that matrix-assisted laser desorption ionisation MS provides provisional identification from trypsin digest peptide maps and that liquid chromatography electrospray (tandem) mass spectrometry affords unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin or pepsin digestion.

CVI Forum, 1994 Nov, (8), 6 - 9
Neonatal tetanus: the final countdown; Health benefits of legal abortion: an analysis; PIP: The abolition of legal abortion in the US would seriously threaten the health, and even the lives, of women and children . Statistics on the relationship between abortion and health attained before and after abortion was legalized were used to project some of the probable consequences of reversing the US Supreme Court's 1973 Roe v . Wade decision . Abortion has been widely practiced throughout US history, but the actual number of procedures performed before some states legalized abortion is unknown . Few legal procedures were performed for medical reasons, yet many illegal abortions took place . In 1955, a panel of experts could only provide a "best estimate" of between 200,000 and 1,200,000 illegally induced abortions occurring annually in the US . The actual number was most likely closer to the higher figure . The complication rates for illegal abortions, most of which were performed by unskilled practitioners in unsafe settings, were much higher than the rates for legal abortion now . Complications were related to ineffective or unsafe methods, Sepsis, particularly with the bacterium "Clostridium prefringens," which causes gas gangrene, was a major problem that has virtually disappeared . Each year prior to the 1970s, more than 100 women in the US died of abortion complications . Due to the fact that vital statistics reflect an incomplete ascertainment of deaths, the actual number of deaths is probably larger, possibly by as much as 50% . In 1983 more than 1.3 million procedures were performed -- a figure close to the estimated number of illegal abortions performed before 1970 . In comparison, 672,000 hysterectomies and 424,000 tonsillectomy operations were performed the same year . The number of abortion-related deaths in the US decreased between 1972 and 1980, from 90 to 16 . Most of this decrease resulted from the availability and safety of legal abortion . Legal abortion carries an especially low risk of death, particularly when performed in the 1st trimester . For the 1972-80 period, the risk of death was 1.9/100,0000 abortion procedures . Legal abortion also has a lower risk of death than does childbearing . Using even conservative data, the comparison of mortality rates reveals that terminating a pregnancy through legally induced abortion is 7 times safter than carrying the pregnancy to term . Legal abortion also results in low morbidity rates . As legal abortions became widely available, the numbers of women treated for septic abortion complications in emergency rooms and hospitals decreased markedly . If abortions were again made illegal, the number of illegal abortions in the US would probably increase from fewer than 20,000 at present to more than 1,000,000 per year . Because illegal abortion may carry a risk of death as much as 30 times that of legal abortion, deaths to women of reproductive age in the US would be expected to increase .

Contracept Fertil Sex (Paris), 1985 Feb, 13(2), 475 - 82
{A comparative study of the various organisms causing salpingitis and their potential presence in IUD wearers}; Kaufmann E; PIP: A prospective study was undertaken to compare the enbdometrial and vaginal bacteria of a continuous series of 283 patients who had IUDs removed between June 1, 1979 and June 1, 1983, by the same clinician . Measures were taken to avoid contamination of the IUD upon removal by the vaginal bacteria, but as a practical matter it was impossible to avoid all contact . The IUDs involved were Multiload copper 250 or 375, Gyne T and T 200, Nova T, and Dimelys . The number of positive cultures from the IUD and the vagina respectively were 1 and 3 of Trichomonas, 82 and 142 Doederlein, 17 and 47 Candida, 131 and 141 white Staphylococcus, 77 an 71 enterococci, 97 and 132 Coli bacilli, 41 abd 54 Lancedfield streptococci, 15 and 58 Hemophilus vaginalis, 13 and 24 Proteus, 1 and 1 Veillonella alcalescens, 3 and 11 Vibrions, 2 and 3 staphylococci dores, 1 and 2 Klebsiella, 1 and 1 Clostridium, 9 and 6 Peptostreptococcus putridus, 1 and 1 Peptococcus prevoti, 6 and 6 Bacteroides fragilis, 2 and 2 Peptococcus intermedius, 0 and 1 Peptococcus aerogenes, and 1 and 1 Citobacter diverous . In almost all cases the concentrartion of bacteria was much greater in the vagina . The rarity of sterile IUDs was not surprising considering the method or removal and the almost inevitable contact with the vagina . The number of organisms declined significantly with parity, but when non-pathogenic bacteria were excluded, the number of sterile IUDs was greatest among nulliparas and primaparas . The Nova T and Dimelys had less contamination by all types of organisms than did the Gravigarde, Multiload copper, or Gyne T . The greatly reduced quantity of bacteria in the uterine cavity relative to the vagina indicates that the cervix is still an effective filter even when it is traversed by the IUD thread .

Appl Environ Microbiol, 2002 Oct, 68(10), 4925 - 31
Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen; Eschenlauer SC et al.; Excessive NH(3) production in the rumen is a major nutritional inefficiency in ruminant animals . Experiments were undertaken to compare the rates of NH(3) production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH(3) production . Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet . The calculated rate of NH(3) production from Trypticase varied from 1.8 to 19.7 nmol mg of protein(-1) min(-1) depending on the substrate, its concentration, and the method used . Monensin (5 micro M) inhibited NH(3) production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011) . Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 micro M monensin . Many fewer bacteria (0.002% of the total) grew on amino acids alone . Nineteen isolates capable of growth on Trypticase were obtained from four sheep . 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups . All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH(3) at >250 nmol min(-1) mg of protein(-1), depending on the medium, as determined by a batch culture method . All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity . Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp . Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra) . Group VI was 95% similar to Acidaminococcus fermentans . Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars . This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH(3) production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Sep, 22(9), 791 - 3
{PCR cloning of 3'-terminal repeat fragments of Clostridium difficile toxin A and toxin B gene}; Yang XQ et al.; OBJECTIVE: To explore a method to rapidly detect the presence of Clostridium difficile . METHODS: PCR was used to amplify DNA fragments from the highly conserved and repeated domains of the 3'-terminal of Clostridium difficile toxin A gene and toxin B genes . RESULTS: The fragments 960 bp and 1,851 bp in length were respectively amplified from toxin A and toxin B, while the control bacteria presented no distinct results . CONCLUSION: The 960 bp and 1,851 bp fragments are specific for Clostridium difficile and PCR can be used to detect Clostridium difficile from the stool . As the peptides encoded by these conserved domains have high hydrophobicity and strong antigenicity, manufacture of the protein vaccine by gene engineering is possible on the basis of these conserved domains.

J Vet Diagn Invest, 2002 Sep, 14(5), 441 - 4
Enteric coronavirus infection in a juvenile dromedary (Camelus dromedarius); Wunschmann A et al.; A case of an enteric coronavirus infection in a 6-week-old dromedary calf is described . The animal had diarrhea for 5 days and died despite symptomatic treatment . Numerous viral particles, approximately 140 nm in diameter, with club-like projections were detected in the feces by electron microscopy . These characteristics were consistent with a coronavirus . Immunohistochemical reactivity with 2 antigenic group II coronavirus-specific antibodies confirmed the presence of viral antigen in colonic epithelial cells . The death of the animal was attributed to a neutrophilic and emphysematous colitis that likely was caused by an infection with a Clostridium sp.

Vaccine Wkly . 1995 Jan 2;:12-3.
Despite progress, NT elimination efforts need stepping up . International / neonatal tetanus; Mortality from tetanus neonatorum in Punjab (Pakistan); PIP: Researchers conducted a survey study of 59,598 households in 3 major socioeconomic groups (urban slums, rural agricultural areas, and rural cattle and horse raising areas) in the Punjab province of Pakistan to estimate mortality from neonatal tetanus and to develop a strategy for its control . The investigators learned of 13,831 live births . 724 of these died in the 1st month of life with 432 (60%) dying from neonatal tetanus . Village untrained "dai" or trained midwives delivered all infants in all 3 areas . Often these deliverers placed cow dung on the stump of the severed umbilical cord and used a dirty cloth for cleaning the infant . In addition, the trained midwives would use unclean unsterilized tools to assist in delivery . In rural areas, animals sleep inside with the family and they are always with the family . Since Clostridium tetani is found in intestines of animals, especially horses, the neonatal tetanus rates as a percentage of all neonatal deaths for the rural agricultural and rural cattle and horse raising areas were higher (60% and 73%) than for the urban slums (45%) . The village barber circumcises male infants on or before the 7th day of life . Since he stuffs the wound with ash or cow dung or rubs it with a dirty cloth, the chances of infecting the wound with C1 . tetani increases . Therefore the total ratio of male deaths to female deaths was 1.61:1 . Most males died during the last 3 weeks of the 1st month which can be attributed to circumcision . Overall most deaths occurred between 4-19 days . None of the mothers interviewed had been vaccinated with a tetanus toxoid during pregnancy which greatly contributed to the neonatal tetanus mortality rate .

Vet Microbiol, 2002 Oct 22, 89(2-3), 141 - 50
Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection; Scherm B et al.; Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas . The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M . agalactiae, M . arthritidis, M . bovis and M . hyopneumoniae representing almost the entire coding region were amplified by PCR . Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M . hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed . Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical . Identities to Clostridium perfringens, Bacillus subtilis and E . coli were determined between approximately 50 and 60% . The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above . Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated . M . hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection.

J Ind Microbiol Biotechnol, 2002 Sep, 29(3), 117 - 23
Production of butanol from starch-based waste packing peanuts and agricultural waste; Jesse TW et al.; We examined the fermentation of starch-based packing peanuts and agricultural wastes as a source of fermentable carbohydrates using Clostridium beijerinckii BA101 . Using semidefined P2 medium containing packing peanuts and agricultural wastes, instead of glucose as a carbohydrate source, we measured characteristics of the fermentation including solvent production, productivity, and yield . With starch as substrate (control), the culture produced 24.7 g l(-1) acetone-butanol-ethanol (ABE), while with packing peanuts it produced 21.7 g l(-1) total ABE with a productivity of 0.20 g l(-1) h(-1) and a solvent (ABE) yield of 0.37 . Cell growth in starch, packing peanuts, and agricultural wastes medium was different, possibly due to the different nature of these substrates . Using model agricultural waste, 20.3g l(-1) ABE was produced; when using actual waste, 14.8 g l(-1) ABE was produced . The use of inexpensive substrates will increase the economic viability of the conversion of biomass to butanol, and can provide new markets for these waste streams.

Gene, 2002 Jul 24, 295(1), 51 - 9
Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships; Sanchez L et al.; Sequences of putative fructose-1,6-bisphospate aldolases (FBA) in five amitochondriate unicellular eukaryotes, the diplomonads Giardia intestinalis (published earlier) and Spironucleus barkhanus, the pelobiont Mastigamoeba balamuthi,the entamoebid Entamoeba histolytica, and the parabasalid Trichomonas vaginalis all belong to Class II of FBAs and are highly similar to each other (>48% amino acid identity) . The five protist sequences, however, do not form a monophyletic group . Diplomonad FBAs share a most recent common ancestor, while FBAs of the three other protist species are part of a lineage that also includes sequences from a few eubacteria (Clostridium difficile, Treponema pallidum, Chlorobium tepidum) . Both clades are part of the Type B of Class II aldolases, a complex that contains at least three additional lineages (subgroups) of enzymes . Type B enzymes are distant from Type A Class II aldolases, which consists of a number of bacterial and fungal enzymes and also contains the cytosolic FBA of Euglena gracilis . Class II aldolases are not homologous to Class I enzymes, to which animal and plant enzymes belong . The results indicate that amitochondriate protists acquired their FBAs from separate and different sources, involving lateral gene transfer from eubacteria, than did all other eukaryotes studied so far and underscore the complex composition of the glycolytic machinery in unicellular eukaryotes.

Endocrinology, 2002 Oct, 143(10), 3830 - 8
Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor; Pi M et al.; We investigated the role of Galphaq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling . We found that Ca(2+), Mg(2+), or the calcimimetic R isomer of N-(3-{2-chlorophenyl}propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct . Coexpression of either the dominant negative Galphaq(305-359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of Galphaq . The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and GalphaqQL-stimulated SRE activity . Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530-1875) inhibited CasR-mediated activation of SRE . A carboxyl-terminal CasR minigene, CasR(906-980), encoding a filamin binding region, also blocked CasR- and GalphaqQL-stimulated SRE activity . Potential interactions between CasR, RhoGEF Lbc, Rho A, Galphaq, and filamin were demonstrated by reciprocal coimmunoprecipitation studies . Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of Galphaq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.

Braz Dent J, 2002, 13(2), 118 - 22
Activity of endodontic antibacterial agents against selected anaerobic bacteria; Ferreira CM et al.; The antimicrobial activity of substances used as antibacterial agents (solutions of 10% calcium hydroxide, camphorated paramonochlorophenol - PMCC, 2% chlorhexidine digluconate and 10% castor oil plant detergent) on anaerobic bacteria (Fusobacterium nucleatum ATCC 25586, Prevotella nigrescens ATCC 33563, Clostridium perfringens ATCC 13124 and Bacteroidesfragilis ATCC 25285), using a broth dilution technique, was evaluated in vitro . For determination of minimum inhibitory and minimum bactericide concentrations (MIC and MBC), two culture broths, Reinforced Clostridial Medium (RCM) and supplemented Brucella, standardized inoculum and serially diluted solutions were used . All antibacterial agents presented antimicrobial activity that varied for different bacteria . There were no differences in the performance of the two broths . Chlorhexidine digluconate was the most effective, with the lowest MICs, followed by castor oil detergent, PMCC and calcium hydroxide . C . perfringens and B . fragilis were the most resistant bacteria to all agents.

Br J Cancer, 2002 Sep 9, 87(6), 635 - 44
Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters; Fritz G et al.; In the present study, we addressed the question of a putative relevance of Rho proteins in tumour progression by analysing their expression on protein and mRNA level in breast tumours . We show that the level of RhoA, RhoB, Rac1 and Cdc42 protein is largely enhanced in all tumour samples analysed (n=15) as compared to normal tissues originating from the same individual . The same is true for (32)P-ADP-ribosylation of Rho proteins which is catalysed by Clostridium botulinum exoenzyme C3 . Also the amount of Rho-GDI and ERK2 as well as the level of overall (32)P-GTP binding activity was tumour-specific elevated, yet to a lower extent than Rho proteins . Although the amount of Rho proteins was enhanced in tumours, most of them did not show changes in rho mRNA expression as compared to the corresponding normal tissue . Thus, elevated gene expression seems not to be the underlying mechanism of tumour-specific overexpression of Rho proteins . Sequence analysis of RhoA, RhoB, RhoC and Rac1 failed to detect any mutations in both the GTP-binding site and effector binding region . By analysing >50 tumour samples, the amount of RhoA-like proteins (i.e . RhoA, B, C), but not of Rac1, was found to significantly increase with histological grade and proliferation index . Rho protein expression was neither related to p53 nor to HER-2/neu oncogene status . Expression of rho mRNAs did not show a significant increase with histological grade . Overall the data show that (1) Rho proteins are overexpressed in breast tumours (2) overexpression is not regulated on the mRNA level (3) the expression level of RhoA-like proteins correlates with malignancy and (4) Rho proteins are not altered by mutation in breast tumours.

J Biol Chem, 2002 Nov 1, 277(44), 42136 - 43 Epub 2002 Aug 30.
The HIV-1 Nef protein and phagocyte NADPH oxidase activation; Vilhardt F et al.; Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway . Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst . Microglia (cell line and primary culture) were transduced with lentiviral expression vectors . Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin) . These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits . Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway . The following results suggest a key role for Rac in the priming effect of Nef . (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect . (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef . These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes . Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.

Gut, 2002 Oct, 51(4), 522 - 8
Antidiarrhoeal properties of a novel sigma ligand (JO 2871) on toxigenic diarrhoea in mice: mechanisms of action; Theodorou V et al.; BACKGROUND AND AIMS: Sigma ligands display antisecretory activity against various secretagogues, suggesting antidiarrhoeal properties . In this study, we evaluated: (i) the antidiarrhoeal effect of JO 2871, a high affinity sigma ligand, in three models of toxigenic diarrhoea in mice; and (ii) the site and mechanism of action of this compound . METHODS: Faeces were collected after toxin or vehicle administration in male DBA2 or NMRI mice . Diarrhoea was determined by cumulative stool weight (mg) over a 120 minute period . Diarrhoea was induced by intravenous administration of Salmonella enteriditis lipopolysaccharide (LPS), or oral administration of Escherichia coli heat stable (E coli-sta) or Clostridium difficile toxins . Two sigma ligands, igmesine and JO 2871, were administered either orally or intravenously, 60 and 30 minutes before the toxins, respectively . JO 2871 was also given orally 30 minutes after E coli-sta . In addition, JO 2871 was administered intracerebroventricularly five minutes before LPS and E coli-sta . BMY 14802 (1000 microg/kg orally), a sigma receptor antagonist, or cyclosomatostatin (CSS 1 microg/kg intravenously), a somatostatin antagonist, were given five minutes prior to JO 2871 in LPS, E coli-sta, and C difficile toxin treated mice . Gastric emptying and intestinal transit were evaluated after oral JO 2871 and BMY 14802 and intravenous CSS . RESULTS: Stool weight measured 120 minutes after administration of the toxins was significantly increased . Oral JO 2871 and igmesine dose dependently inhibited toxigenic diarrhoea in all models . ED(50) values obtained using JO 2871 (1-20 microg/kg) were more than 40 times lower than those obtained with igmesine . Oral JO 2871 given after E coli-sta also inhibited diarrhoea in a dose dependent manner (ED(50) 50 microg/kg) . Both sigma ligands were active by the intravenous route on LPS and E coli-sta induced stool weight increases . JO 2871 administered intracerebroventricularly failed to block this effect at any dose tested . Both BMY 14802 and CSS reversed the antidiarrhoeal effect of oral JO 2871 . JO 2871, BMY 14802, and CSS did not affect transit parameters . CONCLUSIONS: JO 2871 exerts a potent oral antidiarrhoeal effect, acting peripherally through sigma sites and somatostatin release.

J Appl Microbiol, 2002, 93(4), 697 - 705
Ascopyrone P, a novel antibacterial derived from fungi; Thomas LV et al.; AIMS: To assess the antimicrobial efficacy of ascopyrone P (APP), a secondary metabolite formed by the fungi Anthracobia melaloma, Plicaria anthracina, Plic . leiocarpa and Peziza petersi belonging to the order Pezizales . METHODS AND RESULTS: In vitro testing using a well diffusion procedure showed that APP at a high concentration (approximately 5%) inhibited the growth of Gram-positive and Gram-negative bacteria . Using an automated microbiology reader, growth curve analysis showed that 2000-4000 mg l(-1) APP caused total or significant bacterial inhibition after incubation for 24 h at 30 degrees C . Against certain yeast strains, 1000- 2000 mg l(-1) APP enhanced growth, although at higher concentrations inhibition of some yeasts was observed . Clostridium and fungal strains were not sensitive to 2000 mg l(-1) APP . No significant cidal effect was observed after 2 h against Listeria monocytogenes or Escherichia coli . Results were identical whether the APP samples tested had been produced enzymatically or chemically . CONCLUSIONS: At a level of 2000 mg l(-1), APP demonstrated growth inhibitory activity against a broad range of bacteria, but not yeasts or moulds . SIGNIFICANCE AND IMPACT OF THE STUDY: A possible application for this novel natural antimicrobial is in food preservation, to control the growth of Gram-negative and Gram-positive bacteria in raw and cooked foods . Effective dosage levels would be 500-4000 mg kg(-1), depending on food type . The efficacy, organoleptic and safety aspects of this compound in food still need to be assessed.

Biochemistry, 2002 Sep 24, 41(38), 11390 - 7
Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates; Seah SY et al.; Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B . sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources . This difference was proposed to be the basis for the poor discrimination by the B . sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine . Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively . The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine . This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine . The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate . Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes . As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.

J Food Prot, 2002 Sep, 65(9), 1457 - 62
Optimizing sporulation of Clostridium perfringens; de Jong AE et al.; Many sporulation media have been developed for Clostridium perfringens, but none stimulates sporulation for all strains . The aim of our experiments was to develop a sporulation method using Duncan and Strong (DS) medium, which supports sporulation of a wide variety of strains . Different inoculation levels were tested, and the effects of sporulation-promoting substances and acid shock were evaluated . Furthermore, DS medium was compared with other sporulation media . Highest spore numbers in DS medium were obtained with a 10% 24-h fluid thioglycollate broth inoculum (5.0 x 10(5)/ml) . Addition of theophylline and replacement of starch by raffinose increased spore yields for some strains, but most strains were not affected (average increases in log N/ml of 0.2 and 0.3, respectively) . One strain was enhanced by the addition of bile, but other strains were strongly inhibited (average decrease in log N/ml of 2.5); agar did not influence sporulation . Neither short-time acid exposure nor addition of culture supernatant fluids of well-sporulating strains resulted in higher spore numbers in DS medium . None of the tested methods enhanced sporulation in general; only strain-dependent effects were obtained . Peptone bile theophylline medium was the most promising sporulation medium tested; peptone bile theophylline starch medium yielded highest spore numbers (2.5 x 10(5)/ml), but some strains failed to sporulate . In conclusion, adding theophylline to DS medium may optimize sporulation of C . perfringens, but peptone bile theophylline medium with or without starch is most suitable.

Infect Immun, 2002 Oct, 70(10), 5770 - 8
Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues; Calabi E et al.; Clostridium difficile is the etiological agent of antibiotic-associated diarrhea, a potentially serious condition frequently affecting elderly hospitalized patients . While tissue damage is primarily induced by two toxins, the mechanism of gut colonization, and particularly the role of bacterial adherence to the mucosa, remains to be clarified . Previous studies have shown binding of C . difficile whole cells to cultured cell lines and suggested the existence of multiple adhesins, only one of which has been molecularly characterized . In this paper, we have investigated tissue binding of C . difficile surface layer proteins (SLPs), which are the predominant outer surface components and are encoded by the slpA gene . The adherence of C . difficile to HEp-2 cells was studied by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis, which showed that antibodies to the high-molecular-weight (MW) SLP inhibited adherence . Immunohistochemical analysis of human gastrointestinal tissue sections revealed strong binding both to the surface epithelium lining the digestive cavities and to the subjacent lamina propria, while glands were negative . A similar pattern was observed in the mouse . By using purified recombinant SLPs, we show that binding is largely mediated by the high-MW SLP . By Western blotting analysis, we have identified two potential ligands of the C . difficile SLPs, one of which may be specific to the gut . By using purified extracellular matrix components immobilized on nitrocellulose, we also show SLP binding to collagen I, thrombospondin, and vitronectin, but not to collagen IV, fibronectin, or laminin . These results raise the possibility that the SLPs play a role both in the initial colonization of the gut by C . difficile and in the subsequent inflammatory reaction.

Curr Infect Dis Rep, 2002 Oct, 4(5), 415 - 419
Skin and Soft Tissue Infections in Injection Drug Users; Brown PD et al.; Skin and soft tissue infections (SSTIs) are common among injection drug users (IDUs) . Subcutaneous and intramuscular injection ("skin-popping") and the injection of "speedballs" (a mixture of heroin and cocaine) are important risk factors for SSTIs in this patient population . Female IDUs appear to be at greater risk of SSTIs than male IDUs, probably because of more difficult venous access . There are conflicting data regarding the impact of HIV and human T-cell lymphotrophic virus II infection on the risk of SSTIs in IDUs; however, an expanding body of evidence suggests that immunosuppressive effects of the drugs themselves may play a role . Most information regarding the microbiology of SSTIs in IDUs comes from data on skin and subcutaneous abscesses, where Staphylococcus aureus and organisms that originate from the oral flora predominate . Clonal outbreaks and uncommon infections including tetanus, wound botulism, and a sepsis/myonecrosis syndrome due to Clostridium species have been recently reported in IDUs.

Am J Physiol Lung Cell Mol Physiol, 2002 Oct, 283(4), L830 - 8
Rho protein inactivation induced apoptosis of cultured human endothelial cells; Hippenstiel S et al.; Small GTP-binding Rho GTPases regulate important signaling pathways in endothelial cells, but little is known about their role in endothelial cell apoptosis . Clostridial cytotoxins specifically inactivate GTPases by glucosylation {Clostridium difficile toxin B-10463 (TcdB-10463), C . difficile toxin B-1470 (TcdB-1470)} or ADP ribosylation (C . botulinum C3 toxin) . Exposure of human umbilical cord vein endothelial cells (HUVEC) to TcdB-10463, which inhibits RhoA/Rac1/Cdc42, or to C3 toxin, which inhibits RhoA, -B, -C, resulted in apoptosis, whereas inactivation of Rac1/Cdc42 with TcdB-1470 was without effect, suggesting that Rho inhibition was responsible for endothelial apoptosis . Disruption of endothelial microfilaments as well as inhibition of p160ROCK did not induce endothelial apoptosis . Exposure to TcdB-10463 resulted in activation of caspase-9 and -3 but not caspase-8 in HUVEC . Moreover, Rho inhibition reduced expression of antiapoptotic Bcl-2 and Mcl-1 and increased proapoptotic Bid but had no effect on Bax or FLIP protein levels . Caspase-3 activity and apoptosis induced by TcdB-10463 were abolished by cAMP elevation . In summary, inhibition of Rho in endothelial cells activates caspase-9- and -3-dependent apoptosis, which can be antagonized by cAMP elevation.

Can J Microbiol, 2002 Jul, 48(7), 655 - 74
Molecular characterization of bacterial diversity from British Columbia forest soils subjected to disturbance; Axelrood PE et al.; Bacteria from forest soils were characterized by DNA sequence analysis of cloned 16S rRNA gene fragments (16S clones) . Surface organic matter and mineral soil samples from a British Columbia Ministry of Forests Long-Term Soil Productivity (LTSP) installation were collected during winter and summer from two disturbance treatments: whole-tree harvesting with no soil compaction (plot N) and whole-tree harvesting plus complete surface organic matter removal with heavy soil compaction (plot S) . Phylogenetic analyses revealed that 87% of 580 16S clones were classified as Proteobacteria, Actinobacteria, Acidobacterium, Verrucomicrobia, Bacillus/Clostridium group, Cytophaga-Flexibacter-Bacteroides group, green nonsulfur bacteria, Planctomyces, and candidate divisions TM6 and OP10 . Seventy-five 16S clones could not be classified into known bacterial divisions, and five 16S clones were related to chloroplast DNA . Members of Proteobacteria represented 46% of the clone library . A higher proportion of 16S clones affiliated with y-Proteobacteria were from plot N compared with plot S . 16S rRNA gene fragments amplified with Pseudomonas-specific primers and cloned (Ps clones) were examined from mineral-soil samples from plots N and S from three LTSP installations . A significantly greater proportion of sequenced Ps clones from plot N contained Pseudomonas 16S rRNA gene fragments compared with Ps clones from plot S.

Can J Microbiol, 2002 Jul, 48(7), 643 - 54
Cultivation-dependent characterization of bacterial diversity from British Columbia forest soils subjected to disturbance; Axelrood PE et al.; Bacteria from forest surface organic matter and mineral soil horizons were cultivated using four methods and characterized by fatty acid methyl ester (FAME) analysis . Soil samples from a British Columbia Ministry of Forests Long-Term Soil Productivity (LTSP) installation were collected during winter and summer from two disturbance treatments (whole-tree harvesting with no soil compaction (plot N) and whole-tree harvesting plus complete surface organic matter removal with heavy soil compaction (plot S)) and from an unlogged reference plot (REF) . Seventy-five percent of 1795 bacterial isolates were affiliated with 42 genera representing beta- and gamma-Proteobacteria, Actinobacteria, the Bacillus/Clostridium group, and the Cytophaga-Flexibacter-Bacteroides group . Approximately half of the culture collection represented genetic diversity confined to four bacterial genera: Pseudomonas, Bacillus, Paenibacillus, and Arthrobacter . A significantly higher proportion of bacterial isolates belonging to Actinobacteria, and the member genus Arthrobacter, were isolated from plot S soil samples compared with soil samples from plots N and REF . Twenty-five percent of bacterial isolates were not conclusively identified to genus with FAME analysis . Sherlock Tracker cluster analysis and partial 16S rRNA gene sequence analysis enabled classification of a subset of these isolates.

Nahrung, 2002 Aug, 46(4), 276 - 8
The effect of different cooking procedures on microbiological and chemical quality characteristics of Tekirdağ meatballs; Yilmaz I et al.; In this research, the effects of different cooking processes (grilling, oven, and microwave cooking) on microbial flora and chemical composition of the raw and cooked meatballs as consumed in Tekirdag were investigated . Microbial flora of the raw meatballs was as follows: total bacteria, 6.02 x 10(6) cfu/g; psychrophilic bacteria, 1.3 x 10(5) cfu/g; yeast and mould, 2.4 x 10(5) cfu/g; coliforms, 1.1 x 10(5) cfu/g; Escherichia coli, 1.0 x 10(2) cfu/g; total staphylococcae, 3.3 x 10(2) cfu/g; Staphylococcus aureus, 85 cfu/g . While Salmonella was found in only one sample, none of the samples contained Clostridium perfringens . The cooking processes clearly decreased the microbial flora (2-3 log cycles in grilling (71 degrees C) and oven-cooked (79 degrees C), 3-4 log cycles in microwave (97 degrees C) heating) of the meatballs . However, because of the crust formation and high moisture losses from the meatball surface in microwave heating, some sensorial defects were observed in the final product . Also, fat and moisture losses were higher in microwave cooking compared to the other cooking processes . In conclusion, it is advised to use slightly higher temperatures than used in the grilling or conventinal cooking procedures to increase microbial quality of the meatballs studied in this research.

Prenat Diagn, 2002 Sep, 22(9), 783 - 5
Clostridium welchii infection following amniocentesis: a case report and review of the literature; Hamoda H et al.; We report a case of severe Clostridium welchii infection following amniocentesis with septicaemia, haemolysis, DIC, pulmonary oedema and renal failure . Full recovery occurred following aggressive conservative management using antibiotics, endometrial curettage and intensive monitoring . The patient retained her uterus and had a successful pregnancy two years later although caesarean section for uterine rupture was required . Conservative management with conservation of the uterus and ovaries may be a safe and effective option in the management of severe Clostridium infections, using antibiotics, endometrial curettage and multidisciplinary team input .

J Neurosci, 2002 Sep 15, 22(18), 7968 - 81
Rac GTPase plays an essential role in exocytosis by controlling the fusion competence of release sites; Humeau Y et al.; The role of small GTPases of the Rho family in synaptic functions has been addressed by analyzing the effects of lethal toxin (LT) from Clostridium sordellii strain IP82 (LT82) on neurotransmitter release at evoked identified synapses in the buccal ganglion of Aplysia . LT82 is a large monoglucosyltranferase that uses UDP-glucose as cofactor and glucosylates Rac (a small GTPase related to Rho), and Ras, Ral, and Rap (three GTPases of the Ras family) . Intraneuronal application of LT (50 nm) rapidly inhibits evoked acetylcholine (ACh) release as monitored electrophysiologically . Injection of the catalytic domain of the toxin similarly blocked ACh release, but not when key amino acids needed for glucosylation were mutated . Intraneuronal application of competitive nucleotide sugars that differentially prevent glucosylation of Rac- and Ras-related GTPases, and the use of a toxin variant that affects a different spectrum of small GTPases, established that glucosylation of Rac is responsible for the reduction in ACh release . To determine the quantal release parameters affected by Rac glucosylation, we developed a nonstationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action . The results indicate that neither the quantal size nor the average probability for release were affected by lethal toxin action . ACh release blockage by LT82 was only caused by a reduction in the number of functional release sites . This reveals that after docking of synaptic vesicles, vesicular Rac stimulates a membrane effector (or effectors) essential for the fusion competence of the exocytotic sites.

J Biol Chem, 2002 Nov 15, 277(46), 43659 - 66 Epub 2002 Sep 06.
Clostridium perfringens iota toxin . Mapping of the Ia domain involved in docking with Ib and cellular internalization; Marvaud JC et al.; Clostridium perfringens iota toxin consists of two unlinked proteins . The binding component (Ib) is required to internalize into cells an enzymatic component (Ia) that ADP-ribosylates G-actin . To characterize the Ia domain that interacts with Ib, fusion proteins were constructed between the C . botulinum C3 enzyme, which ADP-ribosylates Rho, and various truncated versions of Ia . These chimeric molecules retained the wild type ADP-ribosyltransferase activity specific for Rho and were recognized by antibodies against C3 enzyme and Ia . Internalization of each chimera into Vero cells was assessed by measuring the disorganization of the actin cytoskeleton and intracellular ADP-ribosylation of Rho . Fusion proteins containing C3 linked to the C terminus of Ia were transported most efficiently into cells like wild type Ia in an Ib-dependent manner that was blocked by bafilomycin A1 . The minimal Ia fragment that promoted translocation of Ia-C3 chimeras into cells consisted of 128 central residues (129-257) . These findings revealed that iota toxin is a suitable system for mediating the entry of heterologous proteins such as C3 into cells.

Biochemistry, 2002 Sep 17, 41(37), 11284 - 93
Detection of multiple active site domain motions in transient-state component time courses of the Clostridium symbiosum L-glutamate dehydrogenase-catalyzed oxidative deamination reaction; Tally JF et al.; We present a multiwavelength, transient-state kinetic study of the oxidative deamination reaction catalyzed by Clostridium symbiosum glutamate dehydrogenase (csGDH) producing the real-time reaction courses of spectroscopically resolved kinetically competent intermediate complexes . The results show striking differences from a corresponding transient-state study of the same reaction by the structurally homologous enzyme from beef liver (blGDH) . In addition to the highly blue-shifted alpha-iminoglutarate and highly red-shifted carbinolamine complexes observed in both reactions, the csGDH reaction appeared to show the release of free NADH at a very early and mechanistically unlikely point in the reaction . Using lactic acid dehydrogenase as a "reporter" for free NADH, we show that the early portion of this signal reflects previously unobserved spectrally unshifted enzyme-bound NADH complexes . We provide experimental evidence to show that such spectrally anomalous complexes must represent forms of the known alpha-imino and alpha-carbinolamine complexes in which the active site cleft is open . This evidence includes isothermal calorimetric measurements and pH-jump experiments that show the existence of differing two-state transitions in blGDH and csGDH and locate active site domain motions at differing points in the transient-state time courses of the two enzyme reactions . We prove the kinetic competence of a new and more highly detailed mechanism for the csGDH reaction that involves the alternation of open and closed enzyme complexes as integral steps . These findings, supported by the available X-ray crystal structure data, suggest the existence of a programmed time course of protein domain motions coordinated with the classically considered chemical time course . This new viewpoint may be presumed to be applicable to enzyme reactions other than those of the alpha-amino acid dehydrogenases.

Bioorg Med Chem Lett, 2002 Oct 7, 12(19), 2667 - 72
Protease inhibitors: synthesis of matrix metalloproteinase and bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions; Scozzafava A et al.; Matrix metalloproteinase (MMP)/bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions are reported . A series of compounds was prepared by reaction of arylsulfonyl isocyanates or arylsulfonyl halides with phenylalanyl-alanine, followed by coupling with 5-amino-2-mercapto-1,3,4-thiadiazole in the presence of carbodiimides . These new compounds were assayed as inhibitors of human MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from the anaerobe Clostridium histolyticum (ChC) . The new derivatives proved to be powerful inhibitors of these metalloproteases, with activities in the low micromolar range for some of the target enzymes, depending on the substitution pattern at the arylsulfonyl(ureido) moieties.

Circ Res, 2002 Sep 6, 91(5), 406 - 13
Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators; Seshiah PN et al.; Angiotensin II (Ang II)-stimulated hypertrophy of vascular smooth muscle cells is mediated by reactive oxygen species (ROS) derived from NAD(P)H oxidases . The upstream signaling mechanisms by which Ang II activates these oxidases are unclear but may include protein kinase C, tyrosine kinases, phosphatidylinositol-3-kinase, and Rac, a small molecular weight G protein . We found that Ang II-stimulated ROS production is biphasic . The first phase occurs rapidly (peak at 30 seconds) and is dependent on protein kinase C activation . The larger second phase of ROS generation (peak at 30 minutes) requires Rac activation, because inhibition of Rac by either Clostridium difficile toxin A or dominant-negative Rac significantly inhibits Ang II-induced ROS production . Phosphatidylinositol-3-kinase inhibitors (wortmannin or LY294002) and the epidermal growth factor (EGF) receptor kinase blocker AG1478 attenuate both Rac activation and ROS generation . The upstream activator of EGF receptor transactivation, c-Src, is also required for ROS generation, because PP1, an Src kinase inhibitor, abrogates the Ang II stimulation of both responses . These results suggest that c-Src, EGF receptor transactivation, phosphatidylinositol-3-kinase, and Rac play important roles in the sustained Ang II-mediated activation of vascular smooth muscle cell NAD(P)H oxidases and provide insight into the integrated signaling mechanisms whereby Ang II stimulation leads to activation of the growth-related NAD(P)H oxidases.

J Biol Chem, 2002 Nov 15, 277(46), 43980 - 6 Epub 2002 Sep 04.
A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport; Gual P et al.; Osmotic shock stimulates the translocation of the glucose transporter Glut 4 to plasma membrane by a tyrosine kinase signaling pathway involving Gab-1 (the Grb2-associated binder-1 protein) . We show here that, in response to osmotic shock, Gab-1 acts as a docking protein for phospholipase Cgamma1, the p85 subunit of the phosphoinositide 3-kinase and Crk-II . It has been shown that the adapter Crk-II is constitutively associated with C3G, a GDP to GTP exchange factor for several small GTP-binding proteins . We found that inhibition of the activity of phosphoinositide 3-kinase or phospholipase C did not prevent the stimulation of glucose transport by osmotic shock, whereas inactivation of Rho proteins by Clostridium difficile toxin B severely inhibited glucose uptake . Among the Rho family members, overexpression of dominant-interfering TC10/T31N mutant inhibited osmotic shock-mediated Glut 4 translocation suggesting that TC10 is required for this process . Further, disruption of cortical actin integrity by latrunculin B or jasplakinolide severely impaired osmotic shock-induced glucose transport . In contrast, osmotic shock increased the amount of cortical actin associated with caveolin-enriched plasma membrane domains . These data provide the first evidence that activation of TC10 and remodeling of cortical actin, which could occur through the TC10 signaling, are required for osmotic shock-mediated Glut 4 translocation and glucose uptake.

Br J Nutr, 2002 Sep, 88 Suppl 1, S51 - 7
Probiotics and intestinal health effects: a clinical perspective; Marteau P et al.; Probiotics are viable non-pathogenic micro-organisms which, when ingested, exert a positive influence on host health or physiology . We have critically analysed the evidence for the efficacy of specific probiotic strains in human gastrointestinal diseases . The best evidence can be obtained with randomised controlled trials which avoid bias . Good evidence has been obtained with several strains in the prevention or treatment of antibiotic-associated disorders, in the treatment (and to a lesser extent prevention) of gastroenteritis and acute diarrhoea and in the alleviation of lactose intolerance . We also analysed the recent randomised controlled trials performed in patients with Clostridium difficile or Helicobacter pylori, inflammatory bowel disease, irritable bowel syndrome, non-ulcer dyspepsia and colon cancer.

Br J Nutr, 2002 Sep, 88 Suppl 1, S5 - 9
Probiotics and inflammatory bowel disease: from fads and fantasy to facts and future; Shanahan F; Probiotic therapy is attracting the renewed interest of clinicians and basic investigators from a variety of traditional research disciplines . While the theoretical rationale for modifying the commensal flora of the gastrointestinal tract in specific circumstances appears sound and requires scientific pursuit, the field of probiotics has been clouded by exaggerated claims from some quarters . In general, many of the claims for therapeutic efficacy have not been well substantiated, but the field is now poised for evaluation within the realm of evidence-based medicine . Alterations in commensal bacterial flora within the gastrointestinal tract are associated with susceptibility to pathogens such as Clostridium difficile and there is persuasive evidence that the normal flora may participate in the pathogenesis of inflammatory bowel disease and other chronic diseases in genetically susceptible individuals . This has prompted various strategies to fortify or otherwise modify the enteric flora by dietary supplements containing probiotic formulations . Detailed comparisons of probiotic performance amongst different bacterial strains have not been performed in vivo in man or under clinical trial conditions, and the level of scientific characterisation of individual organisms has been variable . In addition, it cannot be assumed that the same probiotic is equally suitable for all individuals . Moreover, the heterogeneity of clinical disorders such as Crohn's disease and ulcerative colitis implies that strain-specific properties may be required for subset-specific categories of patients . While cocktails of probiotics offer convenience, therapeutic progress may require clarification of the mechanism of probiotic action and may be delayed until individual bacterial components have been rigorously studied . More importantly, the full potential of therapeutic manipulation of the enteric flora with probiotics or other strategies may not be optimally realised until the composition and metabolic activities of the normal flora are better understood.

Rev Gastroenterol Mex, 2002 Apr-Jun, 67(2), 126 - 33
{Usefulness of botulinum toxin in gastrointestinal disorders}; Zepeda-Gomez S et al.; Botulinum toxin (Botox) produced by Clostridium botulinum is a potent neuromuscular blocker agent that inhibits acetylcholine release from presynaptic nerve endings . This effect was confirmed in the smooth muscle of the gastrointestinal tract and led to clinical trials investigating the efficacy of Botox for treatment of several gastrointestinal disorders . Multiple controlled studies have shown that Botox is effective in short-term management of achalasia . Botox reduces lower esophageal sphincter pressure, improves esophageal clearance, and alleviates symptoms in up to 70% of patients; however, its long-term efficacy decreases to 30% and repeated injections are often necessary . Botox is reserved for older patients and with high surgical risk . The main predictors of a good response are older age and presence of vigorous achalasia . Biliary or pancreatic sphincter of Oddi dysfunction (SOD) has been another indication for Botox administration . Transendoscopic injection of Botox in the papilla of Vater has shown relief of symptoms in more than 50% of cases of SOD . Furthermore, a Botox clinical response in this condition can predict a long-term benefit with endoscopic sphincterotomy . Botox decreases resting anal pressure, has healing rates of approximately 80% at six months after injection in patients with chronic anal fissure, and has a better outcome than topic nitroglycerine . Case reports have shown good results with Botox administration in treatment of diffuse esophageal spasm, anismus, oropharyngeal dysphagia, anterior rectocele, and secondary achalasia . Administration of botulinum toxin has a low rate of adverse reactions and complications.

Biochem Pharmacol, 2002 Sep, 64(5-6), 971 - 7
Reduction of tumor necrosis factor-alpha (TNF-alpha) related nuclear factor-kappaB (NF-kappaB) translocation but not inhibitor kappa-B (Ikappa-B)-degradation by Rho protein inhibition in human endothelial cells; Hippenstiel S et al.; Degradation of inhibitor kappa-B (Ikappa-B) followed by translocation of nuclear factor-kappaB (NF-kappaB) into the nucleus and activation of gene expression is essential in tumor necrosis factor-alpha (TNF-alpha)-signaling . In order to analyze the role of Rho proteins in TNF-alpha-induced NF-kappaB-activation in human umbilical cord vein endothelial cells (HUVEC) we used Clostridium difficile toxin B-10463 (TcdB-10463) which inactivates RhoA/Rac1/Cdc42 by glucosylation and Clostridium botulinum C3-toxin which inhibits RhoA/B/C by ADP-ribosylation . Exposure of HUVEC to 10 ng/mL TcdB-10463 or 2.5 microg/mL C3-toxin inhibited TNF-alpha (100 ng/mL)-induced expression of a NF-kappaB-dependent reporter gene . Moreover, preincubation of HUVEC with 10 ng/mL TcdB-10463 reduced TNF-alpha-related expression of interleukin-8 (IL-8), TNF-receptor associated factor-2 (TRAF2), and human inhibitor of apoptosis protein 1 (hIAP1)-mRNA . Blocking of Rho reduced NF-kappaB DNA-binding as shown by electrophoretic mobility shift assays . TcdB-10463 and C3-toxin blocked TNF-alpha-related nuclear translocation of NF-kappaB although Ikappa-Balpha/beta was still degraded . In contrast, TcdB-10463 had no effect on IL-1beta-related NF-kappaB-translocation and activation in HUVEC . Neither 1 microM Rho kinase inhibitor Y-27632 nor microfilament depolymerization by 50 ng/mL C . botulinum C2-toxin blocked TNF-alpha-induced degradation of Ikappa-B, nuclear NF-kappaB translocation or expression of a NF-kappaB-dependent reporter gene . Therefore, TNF-alpha-related Ikappa-B-degradation is Rho-independent in HUVEC, whereas a Rho protein-dependent signal is necessary to induce nuclear transport of NF-kappaB in these cells pointing to a novel and unique role of Rho in NF-kappaB-translocation.

Kansenshogaku Zasshi, 2002 Jul, 76(7), 562 - 5
{A case of invasive Clostridium perfringens infection complicated intravascular hemolysis}; Fukuhara J et al.; We experience a case of a 83-year-old male who was admitted complaining of chills, cramp, high fever and respiratory distress . His blood revealed marked hemolysis . Gram positive Rods was observed in the hemoliesed blood taken on admission . About 2 hours after admission, he suddenly fell into a critical condition . He died about 6 hours after admission in spite of resuscitation . Clostridium perfringens was detected from the blood and liver obtained by autopsy . We suspected that he died of acute intravascular hemolysis caused by alpha-toxin produced by C . perfringens . In conclusion, for a patient who has a high fever with strong hemolysis such as our case, C . perfringens infection should be considered.

Vet Rec, 2002 Aug 17, 151(7), 210 - 3
Toxin types of Clostridium perfringens isolated from free-ranging, semi-domesticated reindeer in Norway; Aschfalk A et al.; Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence of Clostridium perfringens . The organism was isolated from 98 (59 per cent) of the reindeer . The isolates were classified into C perfringens toxin types by PCR analysis specific for the genes encoding the four major toxins (alpha, beta, epsilon and tau) and were subclassified by the detection of the genes encoding C perfringens beta2-toxin and enterotoxin . All the isolates belonged to C perfringens toxin type A . In addition, 15 of the 98 isolates were PCR-positive for the beta2-toxin gene, and two of the isolates had the the gene encoding for enterotoxin.

Cancer Res, 2002 Sep 1, 62(17), 4879 - 83
The c-kit tyrosine kinase inhibitor STI571 for colorectal cancer therapy; Attoub S et al.; The c-kit tyrosine kinase inhibitor STI571 exhibits a substantial therapeutic activity in patients with chronic myeloid leukemia and gastrointestinal stromal tumors respectively associated with constitutive activation of the BCR-ABL and c-kit tyrosine kinases . Human colorectal tumors also express the c-kit proto-oncogene . The present study focuses on the anticancer activity of STI571 in human colorectal tumor cells in vitro and in vivo . The c-kit receptor was identified as a M(r) 145,000 immunoreactive band in human colon cancer cells HT29, HCT8/S11, and HCT116 . Cellular invasion induced by 10 ng/ml stem cell factor (EC(50) = 3 ng/ml) in HT29 cells was blocked by 1 micro M STI571 (IC(50) = 56 nM) and pharmacological inhibitors of several oncogenic signaling pathways, namely, phosphatidylinositol 3-kinase (LY294002), Rho GTPases (Clostridium botulinum exoenzyme C3 transferase), and Rho-kinase (Y27632) . STI571 inhibited HT29 cell proliferation (IC(50) = 6 micro M) and induced apoptosis in vitro . These cellular effects were associated with a decrease in tumor growth . We also demonstrated that stem cell factor is a proangiogenic factor in vivo and in vitro . These encouraging results warrant further preclinical investigations and clinical trials on the use of the c-kit inhibitor STI571 as a chemotherapeutic agent in colon cancer prevention and in treatment of advanced colorectal cancers associated with liver metastases.

Chem Biol Interact, 2002 Aug 15, 140(3), 199 - 213
Lipoyl dehydrogenase catalyzes reduction of nitrated DNA and protein adducts using dihydrolipoic acid or ubiquinol as the cofactor; Chen HJ et al.; Inflamed tissues generate reactive nitrogen oxide species (RNO(x)), such as peroxynitrite (ONOO-)and nitryl chloride (NO2Cl), which lead to formation of nitrated DNA and protein adducts, including 8-nitroguanine (8NG), 8-nitroxanthine (8NX), and 3-nitrotyrosine (3NT) . Once formed, the two nitrated DNA adducts are not stable in DNA and undergo spontaneous depurination . Nitration of protein tyrosine leads to inactivation of protein functions and 3NT has been detected in various disease states . We herein report that reduction of these nitro adducts to their corresponding amino analogues can be catalyzed by lipoyl dehydrogenases (EC 1.8.1.4) from Clostridium kluyveri (ck) and from porcine heart (ph) using NAD(P)H as the cofactor . We also found that dihydrolipoic acid (DHLA) and ubiquinol can be used as effective cofactors for reduction of 8NG, 8NX, and 3NT by these lipoyl dehydrogenases . The reduction efficiency of the mammalian enzyme is higher than the bacterial isozyme . The preference of cofactors by both lipoyl dehydrogenases is DHLA>NAD(P)H>ubiquinol . In all the systems examined, the nitrated purines are reduced to a greater extent than 3NT under the same conditions . We also demonstrate that this lipoyl dehydrogenase/antioxidant system is effective in reducing nitrated purine on NO2Cl-treated double stranded calf thymus DNA, and thus decreases apurinic site formation . The nitroreductase activity for lipoyl dehydrogenase might represent a possible metabolic pathway to reverse the process of biological nitration.

FEMS Microbiol Lett, 2002 Aug 27, 214(1), 119 - 25
Taxonomic identity of type E botulinum toxin-producing Clostridium butyricum strains by sequencing of a short 16S rDNA region; Pourshaban M et al.; Several micro-organisms capable of producing botulinum neurotoxin type E, though phenotypically similar to Clostridium butyricum (a normally non-neurotoxigenic organism), have recently been isolated in Italy and China . Some of these micro-organisms had been implicated in food-borne botulism, a serious neuroparalytic disease . The taxonomic identity of the type E botulinum toxin-producing strains is confirmed here, through sequencing of a genus- and species-specific segment of the 16S rRNA gene . Confirmation leads to the conclusion that neurotoxigenic C . butyricum must be regarded as an emergent food-borne pathogen.

FEMS Microbiol Lett, 2002 Aug 27, 214(1), 77 - 80
Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens; Calcutt MJ et al.; The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching . The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C . perfringens strain 13 . The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins . The C . perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity . To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.

Chemistry, 2002 Aug 16, 8(16), 3769 - 72
Enzyme accessibility and solid supports: which molecular weight enzymes can be used on solid supports? An investigation using confocal Raman microscopy; Kress J et al.; The accessibility of various solid supports (TentaGel, PEGA 1900, and beaded controlled pore glasses (CPGs)) to a range of enzymes was investigated . The different beaded materials were loaded with the peptide 4-cyanobenzamide-Gly-Pro-Leu-Gly-Leu-Phe-Ala-Arg-OH and incubated with the enzymes MMP-12 (22 kDa), thermolysin (35 kDa), MMP-13 (42.5 kDa), clostridium collagenase (68 kDa), and NEP (90 kDa) . The absence/presence of the cyano stretching frequency was measured by means of confocal Raman microscopy . It was found that none of the investigated enzymes could enter the polymer matrices of TentaGel . PEGA 1900 was compatible only with the two smallest enzymes, while beaded CPG was successful even with NEP (90 kDa), proving its superiority over other materials in terms of bio-compatibility.

Clin Infect Dis, 2002 Sep 15, 35(6), 690 - 6 Epub 2002 Aug 26.
Adjunctive intracolonic vancomycin for severe Clostridium difficile colitis: case series and review of the literature; Apisarnthanarak A et al.; Successful treatment of severe Clostridium difficile colitis has been reported with the use of adjunctive intracolonic vancomycin (ICV) therapy . We report a descriptive case series and review the literature on patients with C . difficile colitis who received adjunctive ICV therapy . Nine patients received antibiotics within 6 weeks prior to presentation . Complete resolution of the clinical presentation occurred in 8 patients (88.9%), and eradication of C . difficile cytotoxin production was documented in 3 (75%) of 4 patients who were tested after the completion of adjunctive ICV therapy . One patient (11.1%) died as a result of progressive multisystem organ failure . In the 6 weeks after the completion of treatment for C . difficile colitis, no patient had recurrent disease, required surgical intervention, or experienced complications from adjunctive ICV therapy . In this case series, administration of adjunctive ICV therapy appeared to be a safe, practical, and effective adjunctive therapy for severe C . difficile colitis.

J Biol Inorg Chem, 2002 Sep, 7(7-8), 781 - 90 Epub 2002 Mar 15.
Removal of a cysteine ligand from rubredoxin: assembly of Fe(2)S(2) and Fe(S-Cys)(3)(OH) centres; Cross M et al.; The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)(4) active site . Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli . The C42A protein expresses with a Fe(III)(2)S(2) cluster in place . In contrast, the other proteins are isolated in colourless forms, although a Fe(III)(2)S(2) cluster may be assembled in the C42G protein via incubation with Fe(III)and sulfide . The four mutant proteins were isolated as stable mononuclear Hg(II)forms which were converted to unstable mononuclear Fe(III)preparations that contain both holo and apo protein . The Fe(III)systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies . The dominant Fe(III) form in the C9A preparation is a Fe(S-Cys)(3)(OH) centre, similar to that observed previously in the C6S mutant protein . Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells . In addition to Fe(S-Cys)(3)(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe(III)form in which a ligand appears to be supplied by the protein chain.

J Clin Microbiol, 2002 Sep, 40(9), 3470 - 5
Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates; Spigaglia P et al.; The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC, which are supposed to code for the positive and negative regulators of toxin expression, respectively . Different studies have described variations in C . difficile toxin A and B genes, but little is known about C . difficile variants for the accessory genes . The PaLoc of several C . difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains . Of the toxinogenic C . difficile strains examined, 25% showed variations . No correlation between C . difficile variant strains and key patient groups was found . Interestingly, all of these strains showed a variant tcdC gene . Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids . It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C . difficile strains.

Mol Hum Reprod, 2002 Sep, 8(9), 871 - 9
Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening; Carson DD et al.; High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium . Of approximately 12,000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages . Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold . Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results . In addition, novel genes were identified . Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes . Several genes were studied further by Northern blot analyses . These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased approximately 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition . Immunostaining also revealed strong specific staining for claudin-4/CPE, EP(1) and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage . Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans . Some of these gene products, e.g . OPN, may play direct roles in embryo-uterine interactions during the implantation process.

Blood, 2002 Sep 15, 100(6), 1948 - 56
CCL19 induces rapid dendritic extension of murine dendritic cells; Yanagawa Y et al.; Dendritic cells (DCs) possess numerous dendrites that may be of great advantage to interaction with T cells . However, it has been poorly understood how the dendritic morphology of a DC is controlled . In the present study, using a murine spleen-derived DC line, we analyzed effects of CCR7 ligands, CCL19 and CCL21, on dendritic morphology . Mature DCs, but not immature DCs, showed vigorous migration to either CCL19 or CCL21 . CCL19 also rapidly (within 30 minutes) induced marked extension of dendrites of mature DCs that was maintained at least for 24 hours . On the other hand, CCL21 failed to induce rapid dendritic extension, even though a modest dendritic extension of mature DCs, compared to that by CCL19, was induced 8 or 24 hours after treatment with CCL21 . In addition, pretreatment with a high concentration of CCL21 significantly inhibited the rapid dendritic extension induced by CCL19 . Thus, it is suggested that CCL19 and CCL21 exert agonistic and antagonistic influences on the initiation of dendritic extension of mature DCs . The CCL19-induced morphologic changes were completely blocked by Clostridium difficile toxin B that inhibits Rho guanosine triphosphatase proteins such as Rho, Rac, and Cdc42, but not by Y-27632, a specific inhibitor for Rho-associated kinase . These findings suggest that Rac or Cdc42 (or both), but not Rho, are involved in the CCL19-induced dendritic extension of mature DCs.

Appl Environ Microbiol, 2002 Sep, 68(9), 4292 - 300
The fibronectin type 3-like repeat from the Clostridium thermocellum cellobiohydrolase CbhA promotes hydrolysis of cellulose by modifying its surface; Kataeva IA et al.; Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of Clostridium thermocellum are common among bacterial extracellular glycohydrolases . The function of these domains is not clear . CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn3(1,2)), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain . Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn3(1,2) (more efficient), and Gh9-Fn3(1,2)-CBDIII (greatest efficiency) . Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn3(1,2), and then Gh9-Fn3(1,2)-CBDIII (least stable) . Mixing of Orpinomyces endoglucanase CelE with Fn3(1,2,) or Fn3(1,2)-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper . Scanning electron microscopic studies of filter paper treated with Fn3(1,2), Fn3(1,2)-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn3(1,2) and Fn3(1,2)-CBDIII and to a lesser extent by CBDIII . X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper . CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 micro moles g(-1) and relative affinities (K(r)) of 1.12 and 2.13 liters g(-1), respectively . Fn3(1,2) bound weakly to both celluloses . Fn3(1,2)-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 micro moles g(-1) and K(r)s of 1.14 and 1.98 liters g(-1), respectively . Fn3(1,2) and CBDIII contained 2 and 1 mol of calcium per mol, respectively . The results suggest that Fn3(1,2) aids the hydrolysis of cellulose by modifying its surface . This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn3(1,2) on the cellulose surface.

Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 779 - 84
Diaphorase catalyzed biotransformation of RDX via N-denitration mechanism; Bhushan B et al.; Previously, we hypothesized that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be biotransformed by anaerobic sludge via three different routes: (1) direct ring cleavage via alpha-hydroxylation of a-CH(2) group, (2) reduction of one of the -NO(2) groups to -NO, (3) N-denitration prior to ring cleavage . The present study describes biotransformation of RDX via route 3 by a diaphorase (EC 1.8.1.4) from Clostridium kluyveri using NADH as electron donor . The removal of RDX was accompanied by the formation and accumulation of nitrite ion (NO(2)(-)), formaldehyde (HCHO), ammonium (NH(4)(+)), and nitrous oxide (N(2)O) . None of the RDX-nitroso products were detected . The ring cleavage product methylenedinitramine was detected as the transient intermediate . Product stoichiometry showed that each reacted RDX molecule produced one nitrite ion and the product distribution gave a carbon (C) and nitrogen (N) mass balance of 91 and 92%, respectively, supporting the occurrence of a mono-denitration step prior to the ring cleavage and decomposition . Severe oxygen mediated inhibition (92% inhibition) of RDX biotransformation and superoxide dismutase-sensitive cytochrome c reduction indicated the potential involvement of an anion radical RDX(.-) prior to denitration . A comparative study between native- and apo-enzymes showed the possible involvement of flavin mononucleotide (FMN) in catalyzing the transfer of a redox equivalent (e/H(+)) from NADH to RDX to produce RDX(.-) responsible for secondary decomposition.

Clin Microbiol Infect, 2002 Jul, 8(7), 413 - 8
Detection of toxin production in Clostridium difficile strains by three different methods; Yucesoy M et al.; OBJECTIVE: To compare two immunoassays for detection of toxins produced in vitro by isolates of Clostridium difficile with the standard tissue culture assay, to help in the diagnosis of C . difficile-associated diarrhoea . METHODS: Toxin production was investigated in 42 strains of C . difficile of various serotypes, ribotypes and S-protein types . These included strains from our laboratory collection, strains freshly isolated from stool specimens of patients suspected of suffering from C . difficile-associated disease or of carrying it asymptomatically, and one reference strain (NCTC 11223) . Toxin was assayed by (i) a rapid slide immunoassay (C . difficile toxin A test, Clearview, Oxoid), (ii) an enzyme-linked microplate immunoassay (C . difficile toxin A/B test, Techlab), and (iii) a tissue culture assay . The rapid slide assay and the enzyme immunoassay were performed according to the manufacturers' recommendations . The tissue culture assay was performed using Vero cells . RESULTS: Thirty of the 42 strains (71%) were shown to be positive for toxin A by the slide immunoassay and 34 of the strains (81%) were found to be toxin A/B producers by the enzyme immunoassay . The same 34 strains that were positive in the enzyme immunoassay also produced toxin B (cytotoxin) in the tissue culture assay . The sensitivity, specificity, and positive and negative predictive values for the rapid slide immunoassay method were calculated to be 88.2%, 100.0%, 100.0% and 66.7%, respectively, when compared to tissue culture assay results as the reference method . These values for the enzyme immunoassay method were all 100.0% . In this study eight strains were found to be non-toxin-producing by all methods . It is possible that there were four strains that only produced toxin B (A- B+), and were missed by the rapid A-only assay . CONCLUSIONS: We can recommend the use of the Techlab A + B enzyme immunoassay for the detection of toxin production by C . difficile strains because of its high sensitivity and specificity, its ease of use, and its capability of detecting both A- and B-type toxins.

Acta Crystallogr D Biol Crystallogr, 2002 Sep, 58(Pt 9), 1433 - 41 Epub 2002 Aug 23.
Advantages of high-resolution phasing: MAD to atomic resolution; Schmidt A et al.; The structure of the endoglucanase A from Clostridium thermocellum CelA was re-solved by three-wavelength MAD . Experimental phases were obtained in the resolution range 25-1.0 A . Various structure-solution approaches were tested in order to quantify the contribution of each wavelength . Two-wavelength MAD phasing was sufficient to obtain excellent experimental phases . SAD at the remote wavelength also resulted in interpretable maps . The three-wavelength MAD electron-density map was of excellent quality: for parts of the structure, atom types and bond types could be easily assigned . Double bonds in peptide links and side chains could be located owing to their increased electron density indicating their pi character . Comparison with a previously determined structure of CelA at 1.65 A showed that, apart from a few additional multiple conformers and differently oriented side chains, major differences occur at the protein-solvent interface . A complete additional solvent shell could be observed and the inner shells have been completed . The high accuracy of the structure allowed unambiguous assignment of the protonation state for the active-site catalytic carboxylates.

Kaohsiung J Med Sci, 2002 May, 18(5), 221 - 8
Bacteriology and antimicrobial susceptibility in biliary tract disease: an audit of 10-year's experience; Chang WT et al.; Cholelithiasis, choledocholithiasis and hepatolithiasis are common biliary tract diseases . These diseases may cause severe infection and/or sepsis . In addition to surgical treatments, prompt administration of appropriate antibiotic is important to control the biliary tract infection . The purpose of this study is to illustrate the bacteriology in biliary tract disease and provide information for antibiotic choices . From Jan 1991 to Aug 2000, 1394 patients including gallbladder (GB) stones, common bile duct (CBD) stones, intrahepatic duct (IHD) stones, GB polyps and biliary malignancy were subjects for this retrospective study . The overall positive rate of bile culture is 36% in this study while it was 25%, 66%, 67% and 9% for GB stones, CBD stones, IHD stones and biliary malignancy, respectively . A significantly higher (p = 0.001) positive culture rate was found for GB stones with acute cholecystits (47%) compared with that without inflammation (17%) . Similarly, the culture rate for hepatolithiasis with acute cholangitis was higher than that without cholangitis (75% vs 51%, p = 0.011) . Long-term external biliary drainage in biliary malignancy increased the risk of bacterial culture rate . For gallstone diseases, the most common organisms cultured were Gram negative bacteria (74%), in which Escherichia coli (36%) and Klebsiella (15%) were most commonly found, followed by Gram positive (15%) bacteria such as Enterococcus (6%), Staphylcoccus (3%), Streptococcus (2%) . Bacteroides (5%) and Clostridium (3%) were occasionally found anaerobes (9%) . Polymicrobial infection was encountered in 19%, 31% and 29% for patients with GB stones, CBD stones and IHD stones, respectively; frequency of mixed aerobic and anaerobic infection was 7%, 12% and 9% . In the current study, ampicillin in combination with sulbactam and aminoglycoside is still a suggestive empirical therapy . Antibiotic treatment should be adjusted based on later bacteriological cultures and clinical condition.

Dis Mon, 2002 May, 48(5), 367 - 83
Botulinum toxin type B: an overview of its biochemistry and preclinical pharmacology; Callaway JE et al.; Produced by Clostridium botulinum, botulinum toxins are high molecular weight protein complexes consisting of the neurotoxin and additional nontoxic proteins that function to protect the toxin molecule . The neurotoxin acts to inhibit the release of acetylcholine at the neuromuscular junction, causing muscle paralysis . Purified toxin complexes have found a niche in the treatment of clinical disorders involving muscle hyperactivity . The different serotypes are structurally and functionally similar; however, specific differences in neuronal acceptor binding sites, intracellular enzymatic sites, and species sensitivities suggest that each serotype is its own unique pharmacologic entity . Recently, botulinum toxin type B has been developed as a liquid formulation to avoid the lyophilization (vacuum-drying) and reconstitution processes associated with decreasing the potency and stability of current type A toxin preparations . Biochemical tests were conducted to evaluate the quality of toxin in this formulation . In 3 consecutive manufacturing lots, the botulinum toxin type B complex was found to be highly purified, intact, uniform, and consistent from lot to lot . Also, it showed long-term stability at refrigerator and room temperatures (2 to 25 degrees C) . Electrophysiologic studies in cynomolgus monkeys showed that botulinum toxin type B is effective in paralyzing injected muscle groups, with minimal spread to relatively distant noninjected muscles.

J Bacteriol, 2002 Sep, 184(18), 5088 - 95
Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans; Murashima K et al.; Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome . In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA . EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively . The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed . These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose . The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation . Reactions were also performed by adding different cellulosomes in a sequential manner . When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed . On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed . These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.

J Biochem Mol Biol Biophys, 2002 Apr, 6(2), 147 - 50
Differential conformational environment of tryptophan in epsilon native prototoxin and active toxin from Clostridium perfringens type D; Kumar A et al.; The tryptophan content of Clostridium perfringens type D epsilon protoxin and toxin was found to be one residue per molecule of protein . N-bromosuccinimide in the presence of urea cleaves the tryptophan with total loss of lethality in both toxin and prototoxin . Fluorescence spectroscopy, circular dichroism (CD) and 10% ethylene glycol solvent perturbation studies showed that the tryptophan in epsilon toxin and that in prototoxin have different conformational environments . The tryptophan is more on the surface in the prototoxin than in the toxin molecule . NBS causes total loss of lethality of the toxin with its ellipticity coming to almost zero in the near UV region of the CD.

Infect Control Hosp Epidemiol, 2002 Aug, 23(8), 474 - 7
Undetected vancomycin-resistant Enterococcus stool colonization in a Veterans Affairs Hospital using a Clostridium difficile-focused surveillance strategy; Ray AJ et al.; We examined the point prevalence of undetected vancomycin-resistant Enterococcus (VRE) stool colonization in an institution that screens stool samples submitted for Clostridium difficile testing . Of 112 patients not known to be colonized, 10 (9%) had rectal VRE colonization . A prospective algorithm was effective for identification of colonized patients.

Med Dosw Mikrobiol, 2002, 54(1), 55 - 60
{Enterotoxic strains of Clostridium perfringens and sporulation}; Augustynowicz E et al.; Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning . Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro . A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised . The goal of the work was to determine the level of correlation between CPE synthesis and spores formation . Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels . Sixty-four C . perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed . Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively . No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found . The obtained results indicate that sporulation is not an essential factor for cpe gene expression.

J AAPOS, 2002 Aug, 6(4), 252 - 4
Hyperbaric oxygen therapy in the treatment of orbital gas gangrene; Fielden MP et al.; Clostridial gas gangrene (myonecrosis) is a rapidly progressive, life-threatening infection . The occurrence of clostridial gas gangrene in the orbit is uncommon . We present a case of gas gangrene in the orbit of a child, with Clostridium perfringens isolated from the wound . Our patient was successfully treated with extensive surgical debridement, anti-microbial therapy, and hyperbaric oxygen therapy . Hyperbaric oxygen is established as an adjuvant therapy of clostridial gas gangrene and has been shown to significantly reduce morbidity and mortality . To our knowledge, this is the first reported case of using hyperbaric oxygen therapy in the treatment of orbital gas gangrene.

Arch Latinoam Nutr, 2002 Jun, 52(2), 155 - 9
{Clostridium perfringens in raw and cooked meats and its relation with the environment in Costa Rica}; Rodriguez E et al.; The presence of Clostridium perfringens in eight slaughter houses from Costa Rica was analyzed using the Most Probable Number (MPN) technique, in order to assess the risk of acquiring a food borne intoxication due to consumption of contaminated meat . C . perfringens was detected in 29 (88%) out of 33 soil samples collected from the slaughter house surroundings (average 6.7 x 10(2) MPN/g), as well as in 70 (93%) out of 75 intestinal contents of slaughtered animals (average 3 x 10(4) MPN/g), in 42 (55%) out of 76 samples of slaughtered meat (average 2.2 x 10(4) MPN/g) and in 30 (61%) out of 49 retail meats (average 8 x 10(3) MPN/g) . In addition, the presence of this bacterium was evaluated in ten retail meat markets located in the Metropolitan Area of Costa Rica, where it was isolated from 15 (75%) out of 20 samples of ground meat and from 28 (36%) de 78 stew meat samples (average 1.9 x 10(3) and 7.5 x 10(2) MPN/g, respectively) . Only one out of 35 samples of cooked meat obtained from 32 restaurants that utilize heated water baths (average temperature of 82 degrees C) was positive for C . perfringens (4 MPN/g, temperature 72 degrees C) . Out of 1121 bacterial isolates obtained, 250 were evaluated for enterotoxigenicity . Only 3 (1.2%) of these tested positive for enterotoxin production, probably because most wild strains are not toxin producers, even though they can be induced to produce it as a result of repeated thermal shocks . The present results urge the adoption of adequate preventive measures and high sanitary standards in the meat processing industry in Costa Rica, in order to minimize the risk of food-borne intoxications caused by C . perfringens, due to its widespread distribution and potential human health hazard.

Protein Expr Purif, 2002 Aug, 25(3), 519 - 26
Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum; Iddar A et al.; Clostridium acetobutylicum gapN was cloned and expressed in Escherichia coli BL-21 . The IPTG-induced nonphosphorylating NADP-dependent GAPDH (GAPN) has been purified about 34-fold from E . coli cells and its physical and kinetic properties were investigated . The purification method consisted of a rapid and straightforward procedure involving anion-exchange and hydroxyapatite chromatographies . The purified protein is an homotetrameric of 204kDa exhibiting absolute specificity for NADP . Chromatofocusing analysis showed the presence of only one acidic GAPN isoform with an acid isoelectric point of 4.2 . The optimum pH of purified enzyme was 8.2 . Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65 degrees C with activation energy of 18KJmol(-1) . The apparent K(m) values for NADP and D-glyceraldehyde-3-phosphate (D-G3P) or DL-G3P were estimated to be 0.200+/-0.05 and 0.545+/-0.1 mM, respectively . No inhibition was observed with L-D3P . The V(max) of the purified protein was estimated to be 78.8 U mg(-1) . The Cl . acetobutylicum GAPN was markedly inhibited by sulfhydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfhydryl groups in the catalytic activity.

J Food Prot, 2002 Aug, 65(8), 1267 - 70
Influence of pH and temperature on the growth of and toxin production by neurotoxigenic strains of Clostridium butyricum type E; Anniballi F et al.; Strains of Clostridium butyricum that produce botulinal toxin type E have been implicated in outbreaks of foodborne botulism in China, India, and Italy, yet the conditions that are favorable for the growth and toxinogenesis of these strains remain to be established . We attempted to determine the temperatures and pH levels that are most conducive to the growth of and toxin production by the six strains of neurotoxigenic C . butyricum that have been implicated in outbreaks of infective and foodborne botulism in Italy . The strains were cultured for 180 days on Trypticase-peptone-glucose-yeast extract broth at various pHs (4.6, 4.8, 5.0, 5.2, 5.4, 5.6, and 5.8) at 30 degrees C and at various temperatures (10, 12, and 15 degrees C) at pH 7.0 . Growth was determined by checking for turbidity; toxin production was determined by the mouse bioassay . We also inoculated two foods: mascarpone cheese incubated at 25 and 15 degrees C and pesto sauce incubated at 25 degrees C . The lowest pH at which growth and toxin production occurred was 4.8 at 43 and 44 days of incubation, respectively . The lowest temperature at which growth and toxin production occurred was 12 degrees C, with growth and toxin production first being observed after 15 days . For both foods, toxin production was observed after 5 days at 25 degrees C . Since the strains did not show particularly psychrotrophic behavior, 4 degrees C can be considered a sufficiently low temperature for the inhibition of growth . However, the observation of toxin production in foods at room temperature and at abused refrigeration temperatures demands that these strains be considered a new risk for the food industry.

J AOAC Int, 2002 Jul-Aug, 85(4), 952 - 9
French laboratory proficiency testing program for food microbiology; Augustin JC et al.; The proficiency testing program in food microbiology (Reseau d'Analyses et d'Echanges en Microbiologie des Aliments; RAEMA), created in 1988, currently includes 440 participating laboratories . The program establishes proficiency in detection of Salmonella and Listeria monocytogenes, as well as quantitation of aerobic microorganisms, Enterobacteriaceae, coliforms, Escherichia coli, Clostridium perfringens, coagulase-positive Staphylococcus, and Listeria monocytogenes . Twice a year, 5 test samples are sent to participants to assess their precision and trueness for enumeration and detection of microorganisms . Results show an increasing involvement of food microbiology laboratories in quality assurance programs and use of standard and validated analytical methods . However, the percentage of laboratories obtaining questionable and unsatisfactory microbiological results remains relatively constant.

J Biol Chem, 2002 Oct 18, 277(42), 39463 - 8 Epub 2002 Aug 12.
Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes; Miyata S et al.; Clostridium perfringens epsilon-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization . Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs . To test this idea, we examined the distribution of radiolabeled epsilon-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes . When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs . The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs . Furthermore, cholesterol depletion by methyl-beta-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells . When epsilon-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs . These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.

Toxicon, 2002 Jun, 40(6), 797 - 802
Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F; Poli MA et al.; Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum . The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin . Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F) . Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum . Variation between triplicates was typically 5-10% . Less than 1% cross-reactivity occurred between other serotypes A, B, E or F) . When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F . However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences . We believe these differences arise from trypsin activation of the toxin . These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity . Copright 2002 Elsevier Science Ltd.

J Appl Microbiol, 2002, 93(3), 512 - 9
Characterization of a bacteriocin produced by a newly isolated Bacillus sp . Strain 8 A; Bizani D et al.; AIMS: The aim of this research was to investigate the production of bacteriocins by Bacillus spp . isolated from native soils of south of Brazil . METHODS AND RESULTS: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified . The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase . A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus . Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited . Bacteriocin was stable at 80 degrees C, but the activity was lost when the temperature reached 87 degrees C . It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6.0-9.0 . CONCLUSIONS: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food . SIGNIFICANCE AND IMPACT OF STUDY: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.

Clin Infect Dis, 2002 Sep 1, 35(Suppl 1), S101 - 5
An outbreak of necrotizing enterocolitis associated with a novel clostridium species in a neonatal intensive care unit; Alfa MJ et al.; An outbreak of necrotizing enterocolitis (NEC) occurred in 6 neonates within a 2-month period . Blood cultures from 3 of these neonates grew the same strain of what appears to be a novel clostridial species for which the name "Clostridium neonatale" has been proposed . A point-prevalence survey that used rectal swabs was performed in our intensive-care and intermediate-care nurseries, and it indicated that 20.8% of neonates carried this same "C . neonatale" strain despite having no evidence of NEC . In conclusion, we describe an outbreak of NEC associated with the novel species, and we suggest that, in larger neonates, carriage of this type of Clostridium species may be a necessary step in the multistage pathogenesis of NEC.

Clin Infect Dis, 2002 Sep 1, 35(Suppl 1), S93 - S100
The role of clostridial toxins in the pathogenesis of gas gangrene; Stevens DL et al.; Clostridium perfringens gas gangrene is, without a doubt, the most fulminant necrotizing infection that affects humans . In victims of traumatic injury, the infection can become well established in as little as 6-8 h, and the destruction of adjacent healthy muscle can progress several inches per hour despite appropriate antibiotic coverage . Shock and organ failure are present in 50% of patients and, among these, 40% die . Despite modern medical advances and intensive-care regimens, radical amputation remains the single best life-saving treatment . Over the past century, much has been learned about the pathogenesis of this disease, and novel therapies are on the horizon for patients with this devastating infection.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 455 - 61 Epub 2002 Jun 29.
Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module; Kurokawa J et al.; The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B {Kurokawa J et al . (2001) Biosci Biotechnol Biochem 65:548-554} in pKS305 . The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510 . The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains . CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined . CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan . The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC . Immunological analysis indicated that CelT is a catalytic component of the C . thermocellum F1 cellulosome . This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

Water Res, 2002 Jul, 36(12), 3023 - 8
Regrowth and survival of indicator microorganisms on the surfaces of household containers used for the storage of drinking water in rural communities of South Africa; Momba MN et al.; The present study covered two rural communities of South Africa: Ncera and Ntselamanzi villages . Raw water from Ncera river is used by the community of Ncera village for drinking, while the community of Ntselamanzi receives their drinking water from Alice purification system . Treated water is supplied to the community by a public standpipe system . In rural communities of South Africa, many households use polyethylene (PE) and galvanized steel (GS) containers for the storage of their drinking water . To investigate the regrowth and survival of indicator microorganisms on the surface of household containers during the storage of drinking water, PE and GS slides were suspended in the appropriate household containers for a period of 48 h . This period of 48 h was chosen as the study period because results from the questionnaire indicated that the largest percentage (62%) of households store their water for that length of time . The experiment was performed to test drinking water as it is collected and stored by rural communities . No disinfection of household containers or slides was done during the study period . Attached coliphages (F-RNA (FP) and somatic phage (SP), coliform bacteria (total coliform (TC), presumptive Escherichia coli (EC), Salmonella (Sal) and Clostridium perfringens (CP) were measured during the study period . With the exception of CP, attached indicator microorganisms consisted of TC, presumptive E . coli and Salmonella, somatic and F-RNA coliphages, although the yield (average count) for the last four groups (EC: < 1-3 cfu cm(-2), Sal: < 1-15 cfu cm(-2), FP: < 1-7 pfu cm(-2), SP: < 1-7pfu cm(-2)) was lower than that of TC (3-183 cfu cm(-2)) . However, the lowest yield of indicator microorganisms was noted for presumptive E . coli . Whereas the occurrence and survival of TC was noted on the surface of household containers during the entire period of the experimental study, other indicator microorganisms occurred from time to time . The regrowth of indicator microorganisms occurred 48 h after the exposure of slides to both types of test waters . This length of time mostly resulted in the regrowth of TC (with an increase in bacterial counts) while the persistence of other indicator organism groups on the surface of the slides was apparent . A comparison between PE and GS containers showed that more TC (average count) regrew on PE than on GS containers (for river water, PE: from 36 to 55 cfu cm(-2), GS: from 25 to 26 cfu cm(-2); for standpipe water, PE: from 147 to 183 cfu cm(-2), GS from 3 to 4 cfu cm(-2)) . This study revealed that both types of household containers supported the growth and survival of indicator microorganisms due to the bad quality of the intake water before storage . The storage of drinking water for 48 h mainly resulted in the regrowth of TC . Nevertheless, the persistence of other indicator microorganisms was observed on the surface of household containers.

J Med Microbiol, 2002 Aug, 51(8), 635 - 40
Haemophilus segnis polymicrobial and monomicrobial bacteraemia identified by 16S ribosomal RNA gene sequencing; Lau SK et al.; This paper reports a case of Haemophilus segnis polymicrobial bacteraemia and a case of H . segnis monomicrobial bacteraemia identified by 16S ribosomal RNA gene sequencing . In the first case, a gram-negative aerobic coccobacillus was isolated with Streptococcus intermedius and S . sanguis from the blood culture of a 32-year-old intravenous drug addict with left thoracic empyema . In the second case, a gram-negative aerobic coccobacillus was isolated from the blood culture of an 82-year-old woman with Clostridium difficile colitis and septicaemic shock . Both gram-negative coccobacilli grew on chocolate agar as colonies of 1 mm in diameter after incubation for 24 h at 37 degress C in air with CO2 5%, but only to pinpoint sizes on blood agar under the same incubation conditions . Both strains were factor V-dependent, but not factor X-dependent . For the first isolate, the Vitek system (NHI) showed that it was 56% likely to be Actinobacillus actinomycetemcomitans and 40% Neisseria subflava; whereas the API system (NH) showed that it was 58% likely to be H . aphrophilus/paraphrophilus and 42% H . parainfluenzae . For the second isolate, the Vitek system (NHI) showed that it was 95% likely to be H . influenzae VIII; whereas the API system (NH) showed that it was 58% likely to be H . aphrophilus/paraphrophilus and 42% H . parainfluenzae . 16S rRNA gene sequencing showed that there were four base differences between isolate 1 and H . segnis and two base differences between isolate 2 and H . segnis, indicating that both isolates most closely resembled a strain of H . segnis . Only two cases of H . segnis bacteraemia were found in the English scientific literature, one in a case of infective endocarditis and the other in a case of pancreatic abscess . Including the present two cases, the overall mortality of H . segnis bacteraemia was 50%.

J Neurochem, 1976 Nov, 27(5), 1035 - 42
On the activation of plasma membrane ecto-enzymes by treatment with neuraminidase; Trams EG et al.; It had been proposed that sialyl-residues on the surface of the cell control the activity of certain plasma membrane ecto-enzymes . We have tested the effects of several established (or presumptive) ecto-enzymes in tissue cultures of CNS-derived cells . Application of neuraminidases to cultured mouse neuroblastoma (N-18), neonatal Syrian hamster astrocytes (NN), human astrocytoma (Cox clone) and two lines of primary mouse astroblasts failed to change the activity of ecto-ATPase and 5'-nucleotidase . Only two of the seven neuraminidase preparations produced marked or moderate increases in inorganic pyrophosphatase, p-nitrophenylphosphatase and cholinesterase . We have concluded that the stimulation of these enzymes was not due to removal of sialyl-residues . We suggest that contaminants (haemolysins?) in neuraminidase preparations of Clostridium perfringens increased membrane permeability and facilitated substrate-product translocation.

Harefuah, 2002 May, 141 Spec No, 73 - 7, 120
{Botulism disease}; Marcus N et al.; Botulism is caused by a neurotoxin produced from the anaerobic, spore forming bacteria--clostridium botulinum . The disease is usually caused by toxins type A, B and E . Since the disease was first recognized in the beginning of the nineteenth century as food poisoning, different forms of intoxication were described . Infantile botulism, wound botulism, infectious botulism and inadvertent botulism are all clinical syndromes caused by the same toxin . The attempt to use the botulinum toxin as biological warfare agent is well known . Recently the potential terrorist use of botulinum toxin has become a real concern . Botulism is characterized by its classic triad: 1) symmetric descending flaccid paralysis with prominent bulbar palsies 2) afebrile patient 3) clear sensorium . The paralysis usually begins in the cranial nerves where blurred vision, dysarthia and dysphagia are the initial complaints . Diagnosis is based on clinical findings, history of suspicious exposure and supportive ancillary testing to rule out other causes of neurologic dysfunction that mimic botulism such as the Guillain-Barre syndrome, Myasthenia Gravis or cerebrovascular stroke . Laboratory confirmation of suspected cases is usually delayed and treatment should begin before confirmation is completed . The treatment includes supportive care, and the administration of antitoxin which reduces mortality if given early . Since community and emergency room physicians may be the first to treat patients with any type of botulinum intoxication, they must know how to diagnose and treat this rare but potentially lethal disease.

Theor Popul Biol, 2002 Jun, 61(4), 423 - 34
Critical issues in bacterial phylogeny; Gupta RS et al.; To understand bacterial phylogeny, it is essential that the following two critical issues be resolved: (i) development of well-defined (molecular) criteria for identifying the main groups within Bacteria, and (ii) to understand how the different main groups are related to each other and how they branched off from a common ancestor . These issues are not resolved at present . We have recently described a new approach, based on shared conserved inserts and deletions (indels or signature sequences) found in various proteins, that provides a reliable means for understanding these issues . A large number of conserved indels that are shared by different groups of bacteria have been identified . Using these indels, and based simply on their presence or absence, all of the main groups within Bacteria can be defined in clear molecular terms and new species could be assigned to them with minimal ambiguity . The analysis of these indels also permits one to logically deduce that the various main bacterial groups have branched off from a common ancestor in the following order: Low G+C Gram-positive ==> High G+C Gram-positive ==> Clostridium-Fusobacteria-Thermotoga ==> Deinococcus-Thermus-Green nonsulfur bacteria ==> Cyanobacteria ==> Spirochetes ==> Chlamydia-Cytophaga-Bacteroides-Green sulfur bacteria ==> Aquifex ==> Proteobacteria 1 (epsilon and delta) ==> Proteobacteria-2 . (alpha) ==> Proteobacteria-3 (beta) and ==> Proteobacteria-4 (gamma) . The validity of this approach was tested using sequence data from bacterial genomes . By making use of 18 conserved indels, species from all 60 completed bacterial genomes were assigned to different groups . The observed distribution of these indels in different species was then compared with that predicted by the model . Of the 936 observations concerning the placement of these indels in various species, all except one were in accordance with the model . The placement of bacteria into different groups using this approach also showed excellent correlation with the 16S rRNA phylogenies with nearly all of the species assigned to the same groups by both methods . These results provide strong evidence that the genes containing these indels have not been affected by factors such as lateral gene transfers . However, such events are readily detected by this means and some examples are provided . The approach described here thus provides a reliable and internally consistent means for understanding various critical and long outstanding issues in bacterial phylogeny .

Toxicon, 2002 Aug, 40(8), 1135 - 140
Clostridium botulinum C2 toxin: binding studies with fluorescence-activated cytometry; Stiles BG et al.; Clostridium botulinum C2 enterotoxin consists of two unlinked proteins designated as C2II, which recognizes a cell-surface glycoprotein and translocates an ADP-ribosyltransferase, C2I, into the cytosol of a targeted cell . Fluorescence-activated cytometry was used to study the cellular interactions of Alexa488-labeled C2I (C2I-A488) and proteolytically activated C2II (C2IIa-A488) . The binding of C2IIa-A488 (4 degrees C/10 min) to Chinese hamster ovary (CHO) and African green monkey kidney (Vero) cells yielded a signal/noise ratio of 7:1 and 4:1, respectively . C2I-A488 binding required C2IIa and resulted in a 4:1 (CHO) and 10:1 (Vero) signal/noise ratio that was readily competed by unlabeled C2I . Neither C2I nor C2IIa bound to a CHO line (RK14), lacking the receptor for C2IIa . C2I-A488 did not dock with the heterologous cell-binding component (iota b) of Clostridium perfringens iota toxin, a binary toxin closely related to C2 . Pretreatment of wild-type CHO or Vero cells with pronase or papain (37 degrees C/30 min) prevented a cell-associated C2IIa-specific signal . However, CHO and Vero cells pretreated with papain at 25 degrees C had a 1.5- to 2.3-fold increase in C2IIa-specific fluorescence versus untreated cells incubated with C2IIa-A488 . Overall, these studies further demonstrated the utility of fluorescence-activated cytometry for studying the binding characteristics of bacterial binary toxins like C2.

Int J Food Microbiol, 2002 Aug 25, 77(3), 187 - 97
Time-to-detection, percent-growth-positive and maximum growth rate models for Clostridium botulinum 56A at multiple temperatures; Zhao L et al.; We previously developed models for the influence of inoculum size on the growth kinetics (time-to-detection and maximum growth rate) and percent-growth-positive samples of Clostridium botulinum 56A with factors of inoculum size (1, 100, and 10,000 spores/sample) . pH (5.5 . 6.0 and 6.5) and sodium chloride concentration (0.5%, 2% and 4%) at 30 degrees C . In this present study, data were collected at two more temperatures (15 and 22 degrees C), making the final design a complete 3 X 3 X 3 X 3 factorial with a total of 81 conditions . Growth was followed hourly as change in A620 . The Gompertz equation was fit to the growth data, and the parameters derived were used to calculate the maximum growth rate and time-to-detection . Linear regression with polynomial terms was used to analyze the effect of environmental factors on time-to-detection and maximum growth rate . Logistic regression with polynomial terms was used to analyze the data for percent-growth-positive . Despite the fact that the variance is larger in this extended data set (which includes two temperatures that are further away from the optimum), the inoculum size effect is clearly demonstrated . When inoculum size increased, the percent-growth-positive samples increased and the time-to-detection decreased . When the inoculum was 1000 spores/sample or higher, little additional effect on time-to-detection was observed . Inoculum size might influence results through simple probability or quorum sensing . Our results show that the observed effect of inoculum size from the previous report at a single temperature is not restricted to a specific growth condition, but rather a general phenomenon . The maximum growth rate was independent of inoculum levels, confirming our previous results.

J Biochem (Tokyo), 2002 Aug, 132(2), 237 - 43
Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp . TS12; Sumida T et al.; We report here the molecular cloning and characterization of a glucocerebrosidase {EC 3.2.1.45} from Paenibacillus sp . TS12 . The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues . The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus . The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin . The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively . Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases . This is the first report of the molecular cloning and characterization of a glucocerebrosidase from a procaryote.

J Biochem (Tokyo), 2002 Aug, 132(2), 197 - 203
Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli; Kim YK et al.; A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail . The enzyme reaction proceeded via an ordered bi bi mechanism, in which P(i) bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose . The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P(i) . In the synthetic reaction, the enzyme showed three times higher activity with beta-D-glucose than with alpha-D-glucose, and also showed weak activity with 1,5-anhydro-D-glucitol, indicating that the beta-anomeric hydroxyl group of D-glucose is highly required . However, even when it is removed enzyme activity remains . The substrate specificity and kinetic studies revealed that the configurations of the C3 and C4 hydroxyl groups were strictly required for the enzyme activity, whereas those of C2 and C6 could be substituted or deleted . The mechanism of substrate inhibition by D-glucose was studied in detail and it was concluded that D-glucose competed with G 1-P for its binding site in the synthetic reaction.

Microbiol Immunol, 2002, 46(6), 371 - 82
Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries; Lan PT et al.; The chicken cecum contains a great many bacteria, most of which are strict anaerobes . A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken . A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed . Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle . The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria . Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes . Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria . Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp . (24%), and Bacteroides spp . (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum . The 16S rDNA sequences of Lactobacillus acidophilus, L . crispatus, L . salivarius, and L . reuteri were the most frequently found in the Lactobacillus group in chicken cecum.

J Vet Diagn Invest, 2002 Jul, 14(4), 281 - 7
A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus; Yaeger M et al.; This survey was undertaken to determine the relative frequency of agents that are currently associated with neonatal diarrhea in swine, including Clostridium difficile and porcine reproductive and respiratory syndrome virus (PRRSV) . The subjects for this study were the first 100 live 1-7-day-old piglets submitted to the Iowa State University Veterinary Diagnostic Laboratory with a clinical signalment of diarrhea, beginning on January 1, 2000 . The evaluation of each pig included bacterial culture of a section of ileum, 2 sections of jejunum, and a single section of colon; a fluorescent antibody test (FAT) or immunohistochemistry (IHC) for transmissible gastroenteritis virus (TGEV); ELISA's for rotavirus and C . difficile toxins; IHC for PRRSV; and microscopic examination of ileum, midjejunum, spiral colon, liver, spleen, and lung . Survey results demonstrate a decline in the relative number of diagnoses of TGEV, Escherichia coli, and Clostridium perfringens type C compared with retrospective data . The combined case frequency rate for these 3 pathogens dropped from 70% in 1988 to 21% in 2000 . This survey also demonstrated the emergence of C . difficile as an important pathogen of neonatal swine . Clostridium difficle toxin was detected in the colon contents of 29% of the piglets, and at least 1 toxin-positive animal was identified in 55% of the cases . All 29 C . difficile toxin-positive piglets had mesocolonic edema, and colitis was observed in 21 of 29 toxin-positive animals . PRRSV-positive macrophages were detected in the lamina propria of intestinal villi by IHC in 10 piglets with diarrhea . In 6 of these cases, PRRSV was the only pathogen detected . Gross and microscopic lung lesions were not a reliable indicator of PRRSV infection in these neonatal pigs with diarrhea . The addition of tests for C . difficile and PRRSV to a routine neonatal diarrhea diagnostic protocol resulted in a significant increase in thediagnostic success rate on both individual animal and case bases.

Postgrad Med J, 2002 Jun, 78(920), 366 - 7
Yoghurt biotherapy: contraindicated in immunosuppressed patients?
MacGregor G, Smith AJ, Thakker B, Kinsella J.
A fatal case of Lactobacillus rhamnosus septicaemia after prolonged oral vancomycin for recalcitrant Clostridium difficile infection is reported . The patient was immunosuppressed with cyclophosphamide and steroids for Sjogren's syndrome . The administration of Lactobacillus spp as "biotherapy" may be hazardous in such circumstances.

Diagn Microbiol Infect Dis, 2002 Aug, 43(4), 257 - 63
The diagnosis of Clostridium difficile-associated diarrhea: comparison of Triage C . difficile panel, EIA for Tox A/B and cytotoxin assays; Alfa MJ et al.; This study prospectively compared; Triage(R) C . difficile test (TCT), TechLab C . difficile toxin A-B enzyme immuno-assay (EIA), and cell-culture cytotoxin test (CT) . Of the 400 stools tested, 99 were positive by any test with 92, 41 and 58 detected by TCT, EIA and CT, respectively . Culture of discordant samples indicated that 52 contained C . difficile (42 toxigenic, 10 non-toxigenic), 10 contained Clostridium species and 2 had no detectable clostridium isolates . There were 21/42 toxigenic C . difficile isolates from 17 patients whose stools were negative when originally tested by CT . Review of available patient charts indicated that 12/14 did not previously or currently have C . difficile associated diarrhea, whereas 2 patients developed disease within a few days . Compared to CT as the gold standard, the sensitivity and specificity were; 93%, 89% and 66%, 99% for TCT and EIA respectively . The 8 stool samples with Toxin A(-) Toxin B(+) isolates were detected in 8, 4, and 6 samples by TCT, EIA and CT, respectively . In summary, TCT as a screening test allowed reliable reporting for 85% of stools on the day of receipt . For the 15% of stools requiring further testing we recommend the use of CT.

Przegl Epidemiol, 2002, 56(1), 49 - 56
{Epidemiology of Clostridium difficile infection}; Szczesny A et al.; Pseudomembranous colitis (PMC), antibiotic-associated diarrhoea (AAD), and colitis (AAC) caused by Clostridium difficile are recognized as complications of antibiotic treatment (cephalosporins, penicillins, clindamycin and others) . Two groups are particularly at risk: older and immunocompromised patients . In recent years C . difficile has been recognized as a common nosocomial pathogen . To understand the epidemiology of the C . difficile infection, many outbreaks have been investigated by various methods . In the paper we reviewed different methods of C . difficile typing and discussed the epidemiology of C . difficile-associated infections in light of recent publications.

Curr Gastroenterol Rep, 2002 Aug, 4(4), 279 - 86
Clostridium difficile-associated diarrhea: current strategies for diagnosis and therapy; Moyenuddin M et al.; Clostridium difficile, a spore-forming toxigenic bacterium, is one of the most common causes of infectious diarrhea and colitis in the United States . Most patients with C . difficile infection have recently received antimicrobial therapy--usually clindamycin, cephalosporins, or the extended-spectrum penicillins . Clinical presentation varies from asymptomatic colonization to mild diarrhea to severe colitis . The mainstay of diagnosis is detection of C . difficile toxin A, toxin B, or both with a cytotoxin test or enzyme immunoassay of the stool of patients who have received antibiotic therapy and have features of C . difficile-associated diarrhea . Enzyme immunoassays that detect both toxins are preferred because of their higher diagnostic accuracy . If the first assay is negative and C . difficile-associated diarrhea is strongly suspected, a second assay may be performed . Ten days of oral metronidazole is the preferred therapy for most initial infections . Vancomycin is considered second-line therapy because of its cost and potential to select for vancomycin resistance . About 20% to 25% of patients experience reinfection or relapse after initial therapy and require retreatment . The disease can best be prevented by limiting the use of broad-spectrum antibiotics and adhering to control techniques.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1155 - 60
Clostridium phytofermentans sp . nov., a cellulolytic mesophile from forest soil; Warnick TA et al.; An obligately anaerobic, mesophilic, cellulolytic bacterium, strain ISDgT, was isolated from forest soil . Cells of this isolate stained Gram-negative, despite possessing a Gram-positive cell-wall ultrastructure, and were motile, straight rods that formed spherical terminal spores that swelled the sporangium . Cellulose, pectin, polygalacturonic acid, starch, xylan, arabinose, cellobiose, fructose, galactose, gentiobiose, glucose, lactose, maltose, mannose, ribose and xylose supported growth . The major end products of fermentation were ethanol, acetate, CO2 and H2; formate and lactate were minor products . The optimum temperature for growth was 35-37 degrees C . Phylogenetic analyses based on 16S rRNA sequence comparisons showed that strain ISDgT was related to a group of anaerobes that included Clostridium herbivorans, Clostridium polysaccharolyticum and Clostridium populeti . The G+C content of this strain was 35.9 mol% . On the basis of numerous genotypic and phenotypic differences between strain ISDgT and its close relatives, strain ISDgT is proposed as a novel species in the genus Clostridium, for which the name Clostridium phytofermentans sp . nov . is proposed . The type strain is ISDgT (= ATCC 700394T).

Biochemistry, 2002 Aug 6, 41(31), 9795 - 802
A novel mechanism for Clostridium botulinum neurotoxin inhibition; Eswaramoorthy S et al.; Clostridium botulinum neurotoxins are zinc endopeptidase proteins responsible for cleaving specific peptide bonds of proteins of neuroexocytosis apparatus . The ability of drugs to interfere with toxin's catalytic activity is being evaluated with zinc chelators and metalloprotease inhibitors . It is important to develop effective pharmacological treatment for the intact holotoxin before the catalytic domain separates and enters the cytosol . We present here evidence for a novel mechanism of an inhibitor binding to the holotoxin and for the chelation of zinc from our structural studies on Clostridium botulinum neurotoxin type B in complex with a potential metalloprotease inhibitor, bis(5-amidino-2-benzimidazolyl)methane, and provide snapshots of the reaction as it progresses . The binding and inhibition mechanism of this inhibitor to the neurotoxin seems to be unique for intact botulinum neurotoxins . The environment of the active site rearranges in the presence of the inhibitor, and the zinc ion is gradually removed from the active site and transported to a different site in the protein, probably causing loss of catalytic activity.

Gastroenterology, 2002 Aug, 123(2), 505 - 15
Corticotropin-releasing hormone antagonists possess anti-inflammatory effects in the mouse ileum; Wlk M et al.; BACKGROUND & AIMS: Corticotropin-releasing hormone (CRH) released at local sites of inflammation promotes inflammation in the periphery . We investigated its effects in the intestinal responses caused by toxin A from Clostridium difficile, the causative agent of antibiotic-associated colitis . METHODS: Ileal loops were injected with 10 microg of toxin A, and enterotoxic responses were measured at various time points . RESULTS: Pretreatment of mice with 2.5 microg/kg of the CRH receptor antagonist alpha-helical CRH((9-41)) that blocks both CRH receptor subtypes reduced toxin A-mediated ileal secretion, epithelial cell damage, mucosal edema, neutrophil infiltration, and mucosal content of interleukin 1 beta and tumor necrosis factor alpha . Pretreatment with the specific CRH(1) receptor antagonist antalarmin (20 mg/kg, IP) also inhibited toxin A-induced fluid secretion and toxin A-associated histologic changes . CRH messenger RNA and protein were increased in mouse ileum 30 minutes after intraluminal toxin A administration . In situ hybridization and immunohistochemistry demonstrated that toxin A at 1 hour caused a substantial increase in the expression of both CRH receptor subtypes in the ileal mucosa . CONCLUSIONS: Peripheral CRH may play a proinflammatory role in toxin A-induced intestinal secretion and inflammation and that CRH(1) receptor, at least in part, is important in the mediation of these responses.

Gastrointest Endosc, 2002 Aug, 56(2), 209 - 12
Contamination of single-use biopsy forceps: a prospective in vitro analysis; Kinney TP et al.; BACKGROUND: It has been suggested that single-use biopsy forceps prevent interpatient transmission of infection during endoscopy . Passage of sterile forceps through the accessory channel of the endoscope may lead to contamination, however, if the endoscope has been inadequately processed . The potential for contamination of single-use biopsy forceps at various stages of endoscope reprocessing was prospectively evaluated . METHODS: A total of 50 disposable biopsy forceps were passed through the accessory channels of 10 colonoscopes at the following stages of reprocessing: (1) before use in patients to establish a baseline of high-level disinfection, (2) directly after colonoscopy to confirm contamination with use, (3) after manual cleaning and flushing of the accessory channel to support the claim that manual cleaning significantly decreases bioburden, (4) after manual cleaning and a 2-minute soak in 2% glutaraldehyde to assess for contamination after an inadequate cleaning time, and (5) after manual cleaning and a 20-minute soak in 2% glutaraldehyde . The forceps were then sealed in sterile plastic bags after adding 20 mL of thioglycollate broth medium . The suspension was passed through a 0.2-micron vacuum filter and the filters were cultured . All cultures were incubated more than 48 hours . RESULTS: Biopsy forceps underwent a total of 50 aerobic and 50 anaerobic cultures . Colony-forming units too numerous to count of GI flora, including Escherichia coli, Klebsiella, Pseudomonas, and Clostridium species, grew on 19 of 20 culture plates from biopsy forceps passed through colonoscopes immediately after use . One plate in this group grew 3 colony-forming units of E coli . Persistence of GI flora was noted on 5 of 20 plates after manual cleaning of the colonoscopes . No GI flora were found on forceps after the colonoscopes after soaking in gluteraldehyde for 2 and 20 minutes . Environmental contaminants including diptheroids, Staphylococcus, and Streptococcus species grew on 16 culture plates . CONCLUSIONS: (1) Single-use biopsy forceps are highly susceptible to contamination during passage through the accessory channels of improperly cleaned endoscopes . (2) Disinfection of the colonoscopes in this study prevented contamination of the forceps at baseline and after reprocessing . (3) Proper endoscope reprocessing may be the most important factor in preventing biopsy forceps-related interpatient infection.

Mol Cell Probes, 2002 Jun, 16(3), 179 - 83
One-step cloning and expression of Clostridium difficile toxin B gene (tcdB); Tang-Feldman YJ et al.; The toxin genes of Clostridium difficile have been previously cloned by reconstructing the entire gene in a series of steps in sequence using several cloned fragments . Amplification of a 7.9 kb fragment corresponding to the toxin B gene (tcdB) was obtained with EXPAND Long Template PCR system . The amplified fragment was inserted into the E . coli expression vector pBAD and cloned into competent E . coli TOP 10 cells . tcdB gene sequences representing the complete toxin gene were detected in 3/120 (2.5%) clones analyzed . Culture filtrates of 2/3 clones were found to have cytotoxic activity in human lung fibroblasts . The recombinant protein expressed in E . coli was identified as toxin B by Western immunoblot analysis using C . sordellii antitoxin . This rapid cloning method may be useful in determining the role that individual genes in the pathogenicity locus (PaLoc) play in the virulence of C . difficile . Our results also suggest that the activity of toxin B is independent of other genes in the PaLoc.

South Med J, 2002 Jul, 95(7), 681 - 3
Clostridium difficile-associated diarrhea and chronic renal insufficiency; Yousuf K et al.; BACKGROUND: Clostridium difficile-associated diarrhea (CDAD) is a common cause of mortality and morbidity in hospitalized patients . Some case reports have implicated renal failure as a risk factor for CDAD . The aim of this study was to assess whether chronic renal insufficiency is a risk factor for CDAD and whether it increases mortality and morbidity . METHOD: We reviewed charts of 385 patients with diarrhea for CDAD, chronic renal insufficiency, mortality, and recurrence of CDAD . RESULTS: Seventy-seven patients had infection due to C difficile . There was no difference in the chronic renal insufficiency, mortality, and other comorbid conditions between patients who had C difficile infection and those who did not . The patients with CDAD and chronic renal insufficiency had significantly higher mortality and recurrence of CDAD than patients without chronic renal insufficiency . CONCLUSIONS: Chronic renal insufficiency is not a risk factor for CDAD, but its presence with CDAD increases mortality and recurrence of CDAD.

J Cereb Blood Flow Metab, 2002 Jul, 22(7), 869 - 77
Impairment of autoregulatory vasodilation by NAD(P)H oxidase-dependent superoxide generation during acute stage of subarachnoid hemorrhage in rat pial artery; Shin HK et al.; This study assessed the mechanism(s) by which the autoregulatory vasodilation of rat pial artery in response to acute hypotension during the acute phase of subarachnoid hemorrhage (SAH) was markedly blunted . Increased superoxide production from the cerebral vessels in response to NAD(P)H at 24 hours after SAH + NG-nitro-l-arginine methyl ester (l-NAME) (10 mg/kg) was inhibited by intracisternal administration of a tyrosine kinase inhibitor genistein (10 micromol/L) and Rac inhibitor Clostridium difficile toxin B (1 ng/mL) and a flavoenzyme inhibitor diphenyleneiodonium (10 micromol/L) . The expression of gp91phox was enhanced by SAH + l-NAME from 12 to 24 hours, which was inhibited by genistein and toxin B, but not the p22phox . Increased membrane translocation of Rac after SAH + l-NAME was attenuated by both genistein and toxin B, whereas increased tyrosine kinase activity was blocked by genistein, but not by toxin B . The blunted autoregulatory vasodilation to acute hypotension was effectively recovered by genistein and C . difficile toxin B as well as by diphenyleneiodonium . In conclusion, SAH during acute stage causes an increase in NAD(P)H oxidase-dependent superoxide formation in cerebral vessels, which is due to activation of tyrosine phosphorylation-dependent increased expression of gp91phox mRNA and translocation of Rac protein, thereby resulting in a significant reduction of autoregulatory vasodilation.

J Bacteriol, 2002 Aug, 184(16), 4359 - 68
Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation; Zimmer M et al.; Two temperate viruses, phi3626 and phi8533, have been isolated from lysogenic Clostridium perfringens strains . Phage phi3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination . For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined . The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales . Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3'-protruding cohesive ends of nine residues . Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters . The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region . A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence . These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA . The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of phi3626 with onset of sporulation in C . perfringens . We found that the phi3626 attachment site attP lies in a noncoding region immediately downstream of int . Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3' end of a guaA homologue . This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.

Circ Res, 2002 Jul 26, 91(2), 173 - 9
Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice; Vecchione C et al.; 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) improve endothelial function . We determined whether withdrawal of statin therapy affects endothelium-dependent relaxation in mice and studied the underlying mechanism . Mice were treated with daily injections of cerivastatin (2 mg/kg per day SC), atorvastatin (1 and 10 mg/kg per day SC), or placebo . Vascular reactivity was studied in aortic rings from these mice after 10 days of treatment and after cessation of therapy for several days . Both statins improved endothelium-dependent relaxation to acetylcholine . Compared with control, withdrawal of statin treatment transiently (from day 4 to 7) attenuated endothelium-dependent relaxation . In vessels from animals subjected to atorvastatin withdrawal, the antioxidant tiron restored relaxations . Vascular superoxide anion generation was unaffected by statin therapy but was increased during withdrawal . In mice lacking the gp91phox subunit of the NADPH oxidase, no attenuation of acetylcholine-induced relaxation and no increase in superoxide generation were observed after withdrawal of atorvastatin . In human umbilical vein endothelial cells, statins, which decrease the membrane association of NADPH oxidase-activating Rac-1, increased the activity of this GTPase in whole-cell lysates . Withdrawal of statins induced a translocation of Rac-1 from the cytosol to the membrane and transiently increased NADPH-induced lucigenin chemiluminescence in membrane preparations . Rac-1 inactivation by Clostridium difficile toxin B inhibited the cerivastatin-induced oxygen radical production in human umbilical vein endothelial cells . These observations indicate that the withdrawal of statins induces endothelial dysfunction . The underlying mechanism involves activation of a gp91phox-containing NADPH oxidase by Rac-1 and the subsequent scavenging of endothelium-derived NO by superoxide anions generated from this enzyme.

Emerg Infect Dis, 2002 Aug, 8(8), 802 - 7
Antecedent treatment with different antibiotic agents as a risk factor for vancomycin-resistant Enterococcus; Carmeli Y et al.; We conducted a matched case-control study to compare the effect of antecedent treatment with various antibiotics on subsequent isolation of vancomycin-resistant Enterococcus (VRE); 880 in-patients; 233 VRE cases, and 647 matched controls were included . After being matched for hospital location, calendar time, and duration of hospitalization, the following variables predicted VRE positivity: main admitting diagnosis; a coexisting condition (e.g., diabetes mellitus, organ transplant, or hepatobiliary disease); and infection or colonization with methicillin-resistant Staphylococcus aureus or Clostridium difficile within the past year (independent of vancomycin treatment) . After controlling for these variables, we examined the effect of various antibiotics . Intravenous treatment with third-generation cephalosporins, metronidazole, and fluoroquinolones was positively associated with VRE . In our institution, when we adjusted the data for temporo-spatial factors, patient characteristics, and hospital events, treatment with third-generation cephalosporins, metronidazole, and fluoroquinolones was identified as a risk factor for VRE . Vancomycin was not a risk factor for isolation of VRE.

Mol Microbiol, 2002 Aug, 45(3), 617 - 26
Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD; Murashima K et al.; Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes . In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique . The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous . To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening . We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities . One of the mutants possessed a sevenfold higher thermostability than EngB . The possible mechanisms of thermostabilization are discussed.

Brief Bioinform, 2002 Jun, 3(2), 181 - 94
Exact mapping of prokaryotic gene starts; Baytaluk MV et al.; It is known that while the programs used to find genes in prokaryotic genomes reliably map protein-coding regions, they often fail in the exact determination of gene starts . This problem is further aggravated by sequencing errors, most notably insertions and deletions leading to frame-shifts . Therefore, the exact mapping of gene starts and identification of frame-shifts are important problems of the computer-assisted functional analysis of newly sequenced genomes . Here we review methods of gene recognition and describe a new algorithm for correction of gene starts and identification of frame-shifts in prokaryotic genomes . The algorithm is based on the comparison of nucleotide and protein sequences of homologous genes from related organisms, using the assumption that the rate of evolutionary changes in protein-coding regions is lower than that in non-coding regions . A dynamic programming algorithm is used to align protein sequences obtained by formal translation of genomic nucleotide sequences . The possibility of frame-shifts is taken into account . The algorithm was tested on several groups of related organisms: gamma-proteobacteria, the Bacillus/Clostridium group, and three Pyrococcus genomes . The testing demonstrated that, dependent or a genome, 1-10 per cent of genes have incorrect starts or contain frame-shifts . The algorithm is implemented in the program package Orthologator-GeneCorrector.

Microbiol Immunol, 2002, 46(5), 353 - 8
Design of species-specific primers to identify 13 species of Clostridium harbored in human intestinal tracts; Kikuchi E et al.; The genus Clostridium is dominant in human intestinal tracts and plays an important role in human health . We designed species-specific primers to identify 13 species of Clostridium (C . perfringens, C . paraputrificum, C . bifermentans, C . difficile, C . clostridiiforme, C . nexile, C . sphenoides, C . indolis, C . ramosum, C . cocleatum, C . butyricum, C . sordellii, and C . innocuum) easily and rapidly . The PCR annealing temperature was set at a uniform 60 C for application to all strains at the same time . To confirm the specificities of these primers, 85 intestinal bacteria in total, including type strains, reference strains, and isolates were used . Ten primers (including those for C . perfringens to C . cocleatum) indicated high specificities . Although there were some cross-reactions with the other three primers, the target species were distinguishable from other bacteria by the different sizes of PCR products.

Microbiol Immunol, 2002, 46(5), 333 - 42
Differential expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cancer cell lines in response to Clostridium difficile toxin A; Kim JM et al.; Intestinal epithelial cells are the initial sites of host response to Clostridium difficile infection and can play a role in signaling the influx of inflammatory cells . To further explore this role, the regulated expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cells were investigated . An expression of the CXC chemokines, including IL-8 and growth-related oncogene (GRO)-alpha, and the CC chemokine monocyte chemoattractant protein (MCP)-1 from HT-29 cells increased in the 1-6 hr following C . difficile toxin A stimulation, assessed by quantitative RT-PCR . In contrast, the expression of neutrophil activating protein-78 (ENA-78) was delayed for 18 hr . The up-regulated mRNA expression of chemokines was paralleled by the increase of protein levels . However, the expression of macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated on activation normal T cells expressed and secreted), and interferon-gamma-inducible protein-10 (IP-10) was not changed in HT-29 or Caco-2 cells stimulated with toxin A . Upon stimulation of the polarized Caco-2 epithelial cells in a transwell chamber with toxin A, CXC and CC chemokines were released predominantly into the basolateral compartment . Moreover, the addition of IFN-gamma and TNF-alpha to toxin A stimulated Caco-2 cells increased the basolateral release of CC chemokine MCP-1 . In contrast, IFN-gamma and TNF-alpha had no effect on the expression of the CXC chemokines IL-8 and GRO-alpha . These results suggest that a CXC and CC chemokine expression from epithelial cells infected with C . difficile may be an important factor in the mucosal inflammatory response.

J Biol Chem, 2002 Oct 11, 277(41), 38133 - 40 Epub 2002 Jul 22.
Adhesion-related kinase repression of gonadotropin-releasing hormone gene expression requires Rac activation of the extracellular signal-regulated kinase pathway; Allen MP et al.; Recent studies suggest that adhesion-related kinase (Ark) plays a role in gonadotropin-releasing hormone (GnRH) neuronal physiology . Ark promotes migration of GnRH neurons via Rac GTPase and concomitantly suppresses GnRH gene expression via homeodomain and myocyte enhancer factor-2 (MEF2) transcription factors . Here, we investigated the signaling cascade required for Ark inhibition of the GnRH promoter in GT1-7 GnRH neuronal cells . Ark repression was blocked by the MEK/ERK pathway inhibitor, PD98059, and dominant negative MEK1 but was unaffected by dominant negative Ras . Inhibitors of the Rho family GTPases, Clostridium difficile toxin B (Rho/Rac/Cdc42 inhibitor) and Clostridium sordellii lethal toxin (Rac/Cdc42 inhibitor), blocked Ark inhibition of GnRH transcription . Moreover, dominant negative Rac blunted both Ark activation of ERK and repression of the GnRH promoter, demonstrating an essential role for Rac in coupling Ark to ERK activation . Like Ark, a constitutively active mutant of Rac suppressed GnRH transcription in an ERK-dependent manner . Finally, Ark-mediated repression was significantly attenuated by a dominant negative MEF2C, whereas repression induced by constitutively active Rac was unaffected, indicating that MEF2 proteins are not targets of the Ark --> Rac --> MEK --> ERK cascade . The data suggest that Ark suppresses GnRH gene expression via the coordinated activation of a Rac --> ERK signaling pathway and a distinct MEF2- dependent mechanism.

J Rheumatol, 2002 Jul, 29(7), 1426 - 9
Detection of bacterial DNA in Latin American patients with reactive arthritis by polymerase chain reaction and sequencing analysis; Cuchacovich R et al.; OBJECTIVE: Bacteria and/or their antigens are thought to play a role in the pathogenesis of reactive arthritis (ReA) . Polymerase chain reaction (PCR) using the 16S ribosomal RNA-PCR method was used to identify bacterial DNA in synovial fluid (SF) and tissue (ST) in a well defined group of patients with chronic ReA . In addition, species found were identified by means of sequence analysis . METHODS: We examined 15 ST and 5 SF samples of 15 patients with ReA, 5 ST samples of 5 patients with osteoarthritis (OA), and 8 SF from 8 patients with closed traumatic knee injuries using a nested PCR with universal 16S rRNA primers . In addition, a nested PCR was developed to detect DNA sequences of Salmonella sp . and Mycoplasma sp . Automated sequencing and comparative data analysis (GenBank) were also performed to identify the species . RESULTS: Bacterial DNA was identified in 8 cases, 5 ST and 3 SF; Chlamydia trachomatis (n = 2), Pseudomonas sp . (n = 3), and Bacillus cereus (n = 2) were the most common microorganisms identified . A variety of microorganisms including Clostridium sp., Lactobacillus sp., Pseudomonas migulae, P . fluorescens, and P . putida, and Neisseria meningitidis serogroup B were also identified . In half of the cases (4/8) 2 to 3 bacterial antigens were identified simultaneously . CONCLUSION: Bacterial DNA is present in the joints in patients with chronic ReA . A wide spectrum of bacteria including some not previously associated with ReA were identified . Further studies are needed to establish their exact role in the pathogenesis of ReA and related arthritides.

Protein Expr Purif, 2002 Jul, 25(2), 219 - 28
Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A; Chaddock JA et al.; Clostridium botulinum neurotoxin type A is a potently toxic protein of 150 kDa with specific endopeptidase activity for the SNARE protein SNAP-25 . Proteolytic cleavage of BoNT/A with trypsin leads to removal of the C-terminal domain responsible for neuronal cell binding . Removal of this domain result in a catalytically active, non-cell-binding derivative termed LH(N)/A . We have developed a purification scheme to prepare LH(N)/A essentially free of contaminating BoNT/A . LH(N)/A prepared by this scheme retains full enzymatic activity, is stable in solution, and is of low toxicity as demonstrated in a mouse toxicity assay . In addition, LH(N)/A has minimal effect on release of neurotransmitter from a primary cell culture model . Both the mouse bioassay and in vitro release assay suggest BoNT/A is present at less than 1 in 10(6) molecules of LH(N)/A . This represents a significant improvement on previously reported figures for LH(N)/A, and also the light chain domain, previously purified from BoNT/A . To complement the preparation of LH(N)/A from holotoxin, DNA encoding LH(N)/A has been introduced into Escherichia coli to facilitate expression of recombinant product . Expression and purification parameters have been developed to enable isolation of soluble, stable endopeptidase with a toxicity profile enhanced on that of LH(N)/A purified from BoNT/A . The recombinant-derived material has been used to prepare antisera that neutralise a BoNT/A challenge . The production of essentially BoNT/A-free LH(N)/A by two different methods and the possibilities for exploitation are discussed.

Eur J Biochem, 2002 Jul, 269(14), 3425 - 32
Fluorescent analogs of UDP-glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile; Bhattacharyya S et al.; Uridine-5'-diphospho-1-alpha-d-glucose (UDP-Glc) is a common substrate used by glucosyltransferases, including certain bacterial toxins such as Toxins A and B from Clostridium difficile . Fluorescent analogs of UDP-Glc have been prepared for use in our studies of the clostridial toxins . These compounds are related to the methylanthraniloyl-ATP compounds commonly used to probe the chemistry of ATP-dependent enzymes . The reaction of excess methylisatoic anhydride with UDP-Glc in aqueous solution yields primarily the 2' and 3' isomers of methylanthraniloyl-UDP-Glc (MUG) . As the 2' and 3' isomers readily interconvert, this isomeric mixture was copurified by HPLC away from the other isomeric products, and was characterized by a combination of NMR, fluorescence and mass spectrometric methods . TcdA binds MUG competitively with respect to UDP-Glc with an affinity of 15 +/- 2 microm in the absence of Mg2+ . There is currently no evidence that the fluorescent substrate analog is turned over by the toxin in either glucosyltransferase or glucosylhydrolase reactions . Using a competition assay, the affinity of UDP-Glc was determined to be 45+/-10 microm in the absence of Mg2+ . The binding of UDP-Glc and Mg2+ are highly coupled with Mg2+ affinities in the range of 90-600 microm, depending on the experimental conditions . These results imply that one of the significant roles of the metal ion might be to stabilize the enzyme-substrate complex prior to initiation of the transferase chemistry.

Am J Gastroenterol, 2002 Jul, 97(7), 1769 - 75
Breaking the cycle: treatment strategies for 163 cases of recurrent Clostridium difficile disease; McFarland LV et al.; OBJECTIVE: There is currently uncertainty as to the best treatment for patients with recurrent episodes of Clostridium difficile disease (RCDD) . Our objective was to evaluate the success of treatment strategies in a cohort of 163 RCDD patients . METHODS: Data were used from patients who had participated in the placebo arm in two national referral clinical trials evaluating a new combination treatment . Patients with active RCCD were enrolled, prescribed either vancomycin or metronidazole, and randomized to either the investigational biological or a placebo . All patients were observed for at least 2 months for a subsequent episode of RCCD . RESULTS: Of the 163 cases, 44.8% recurred . A tapering course of vancomycin resulted in significantly fewer recurrences (31%, p = 0.01), as did pulsed dosing of vancomycin (14.3%, p = 0.02) . A trend (p = 0.09) for a lower recurrence frequency was observed for high-dose (> or =2 g/day) vancomycin and low-dose (< or =1 g/day) metronidazole . Vancomycin was significantly more effective in clearing C . difficile culture and/or toxin by the end of therapy than metronidazole (89% vs 59%, respectively; p < 0.001) . CONCLUSIONS: These data show that tapered or pulsed dosing regimens of vancomycin may result in a significantly better cure of RCDD . The persistence of C . difficile spores suggests that additional strategies to restore the normal colonic microflora may also be beneficial.

Crit Care, 2002 Jun, 6(3), 205 - 11 Epub 2002 May 09.
Clinical review: bacteremia caused by anaerobic bacteria in children; Brook I; This review describes the microbiology, diagnosis and management of bacteremia caused by anaerobic bacteria in children . Bacteroides fragilis, Peptostreptococcus sp., Clostridium sp., and Fusobacterium sp . were the most common clinically significant anaerobic isolates . The strains of anaerobic organisms found depended, to a large extent, on the portal of entry and the underlying disease . Predisposing conditions include: malignant neoplasms, immunodeficiencies, chronic renal insufficiency, decubitus ulcers, perforation of viscus and appendicitis, and neonatal age . Organisms identical to those causing anaerobic bacteremia can often be recovered from other infected sites that may have served as a source of persistent bacteremia . When anaerobes resistant to penicillin are suspected or isolated, antimicrobial drugs such as clindamycin, chloramphenicol, metronidazole, cefoxitin, a carbapenem, or the combination of a beta-lactamase inhibitor and a penicillin should be administered . The early recognition of anaerobic bacteremia and administration of appropriate antimicrobial and surgical therapy play a significant role in preventing mortality and morbidity in pediatric patients.

Cancer Detect Prev, 2001, 25(6), 548 - 57
Clostridium as a tumor-specific delivery system of therapeutic proteins; Theys J et al.; The feasibility of gene therapy strategies in cancer treatment still has important pitfalls . Transfer of therapeutic proteins to the hypoxic/necrotic 'extracellular' microenvironment of solid tumors, based on the engineering of nonpathogenic clostridia is proposed as an alternative methodology . Using the rat rhabdomyosarcoma R1 in vivo tumor model, we demonstrated that Clostridium species colonized the tumors, whereas proliferation of these bacteria was absent in normal tissues . C . acetobutylicum was genetically engineered to express and secrete either mTNF-alpha or the E . coli cytosine deaminase . Quantitative in vitro data showed stability of the vectors, and significant levels of biologically active therapeutic proteins in lysates and supernatants of recombinant clostridia . Administration of either of these recombinant Clostridium strains to tumor-bearing rats resulted in the presence of active proteins in the tumor tissue . Based on these data and supported by its selective colonization pattern and safety, the Clostridium gene transfer system offers a potential application in anti-cancer therapies.

Schweiz Arch Tierheilkd, 2002 Jun, 144(6), 263 - 73
{The necrotizing enteritis by Clostridium perfringens type C in piglets: practical observations, control and epidemiology}; Luginbuhl A; Necrotizing enteritis in piglets is caused by the spore-forming anaerobe Clostridium perfringens type C . The pathology is believed to be due to the production of beta-toxin by the agent and the infection tends to persist in affected herds despite appropriate hygiene measures . During the period 1989 to 2001, 35 outbreaks were observed in 15 herds in a limited geographic region in northwestern Switzerland in the canton of Fribourg . Initial outbreaks of acute disease were followed by chronic manifestations of necrotizing enteritis in eleven herds . Since clinical symptoms in case of chronic necrotizing enteritis and of mixed infections were rather non-specific, diagnosis in all herds was confirmed by gross pathological analysis, intestinal histology and bacteriological culture . After initial evaluation in 135 litters (1500 piglets), metaphylaxis consisted of penicillin for outbreaks of acute disease or of amoxycillin-clavulanic acid pending results of laboratory confirmation . Vaccination with a C . perfringens toxoid vaccine (Gletvax 6, also containing E . coli pilus antigens) together with penicillin chemoprophylaxis was used in 2400 litters (27,000 piglets) . In 12 to 17% of litters disease recurred during this combined prophylaxis . Necrotizing enteritis persisted in all affected herds throughout the follow-up period.

Tijdschr Diergeneeskd, 2002 Jul 1, 127(13), 422 - 4
{Botulism poisoning in cattle, a case report, diagnosis and prevention}; van Wuijckhuise L et al.; On a dairy farm 22 animals die in 14 days . After 10 days the clinical diagnosis is confirmed: clostridium botulinum type D intoxication . The clinical, diagnosis, therapy and prevention are discussed.

Transpl Infect Dis, 2002 Mar, 4(1), 41 - 5
Legionellosis in a lung transplant recipient obscured by cytomegalovirus infection and Clostridium difficile colitis; Nichols L et al.; A 52-year-old-white male underwent double lung transplantation for severe emphysema due to alpha-1-antitrypsin deficiency and heavy tobacco use . Following a postoperative course complicated by renal insufficiency, pulmonary emboli, and Clostridium difficile colitis, he was discharged in stable condition . Two months later, he was admitted to a local hospital with a fever, abdominal pain, diarrhea, nausea, and dyspnea . Computerized tomography (CT) of the chest revealed bilateral pleural effusions . Sigmoidoscopy was grossly normal but biopsy demonstrated cytomegalovirus (CMV) colitis, and the patient was placed on intravenous ganciclovir . Over the next week, he became progressively hypoxemic and was transferred to the University of Pittsburgh Medical Center (post-transplant day 81) for further evaluation . His medications on transfer included: ganciclovir, prednisone, tacrolimus, dapsone, fluconazole, ondansetron, lansoprazole, digoxin, and coumadin.

J Perinat Med, 2002, 30(3), 197 - 208
Cutaneous and subcutaneous infections in newborns due to anaerobic bacteria; Brook I; This review describes the microbiology and management of the major cutaneous and subcutaneous infections in newborns where anaerobic bacteria predominate: omphalitis, necrotizing fasciitis, breast abscess, and scalp infection following intrauterine fetal monitoring . The predominant bacteria known to cause these infections are group B streptococcus, group D enterococcus, group A streptococcus, Staphylococcus aureus, Enterobacteriaceae, and anaerobic bacteria . All of these agents can colonize or infect the mother and subsequently colonize or infect the fetus or newborn either intrauterinely or during the passage through the birth canal . Infections due to anaerobes are often polymicrobial, and include also aerobic and facultative bacteria . The anaerobes recovered from these infections are Bacteroided fragilis group, Fusobacterium spp., Peptostreptococcus spp . and Clostridium spp . Early recognition and effective medical and surgical therapy are essential to recovery . Managements of these infections include surgical debridement and drainage when appropriate as well as topical and systemic use of antimicrobial agents effective against both aerobic and anaerobic bacteria.

Am J Physiol Gastrointest Liver Physiol, 2002 Aug, 283(2), G282 - 91
PGF(2alpha)-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle; Cao W et al.; Lower esophageal sphincter (LES) tone depends on PGF(2alpha) and thromboxane A(2) acting on receptors linked to G(i3) and G(q) to activate phospholipases and produce second messengers resulting in muscle contraction . We therefore examined PGF(2alpha) signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES . In Eso, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(13) and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum . A {(35)S}GTPgammaS-binding assay confirmed that G(13), RhoA, and ARF1 were activated by PGF(2alpha) . Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor . In LES, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(q) and G(i3), and a {(35)S}GTPgammaS-binding assay confirmed that G(q) and G(i3) were activated by PGF(2alpha) . PGF(2alpha)-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol . At low PGF(2alpha) concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B . Thus, in Eso, PGF(2alpha) activates a PLD- and protein kinase C (PKC)-dependent pathway through G(13), RhoA, and ARF1 . In LES, PGF(2alpha) receptors are coupled to G(q) and G(i3), activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C . At low concentrations, PGF(2alpha) activates PKC . At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.

J Am Chem Soc, 2002 Jul 24, 124(29), 8667 - 72
Genetic construction of truncated and chimeric metalloproteins derived from the alpha subunit of acetyl-CoA synthase from Clostridium thermoaceticum; Loke HK et al.; In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha(2)beta(2) tetrameric 310 kDa bifunctional enzyme . ACS catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-CoA from CO (or CO(2) in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP) . ACS contains seven metal-sulfur clusters of four different types called A, B, C, and D . The B, C, and D clusters are located in the 72 kDa beta subunit, while the A-cluster, a Ni-X-Fe(4)S(4) cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa alpha subunit . The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined . Acetyl-CoA synthase catalytic activity remained when the entire beta subunit was removed, as long as CO, rather than CO(2) and a low-potential reductant, was used as a substrate . Truncating an approximately 30 kDa region from the N-terminus of the alpha subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties . Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of {Fe(4)S(4)}(2+) clusters . Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the alpha subunit . The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters . This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

Bioresour Technol, 2002 Sep, 84(3), 213 - 20
Effects of anaerobic/aerobic incubation and storage temperature on preservation and deodorization of kitchen garbage; Wang Q et al.; To develop a garbage recycling system for the purpose of the production of lactic acid (LA) to use as raw material for producing biodegradable plastics, the preservation and deodorization of garbage during storage are very important . Anaerobic incubation (i.e., storage) was prove to be more suitable than aerobic incubation during the garbage storage in terms of concentration of LA and soluble sugar, pH value, viable bacteria counts and offensive odour substances . This difference is due to a fact that the growth of putrefactive bacteria such as coliforms and Clostridium spp . appeared to be inhibited by anaerobic fermentation during the storage, because the fermentation caused a drop of garbage pH and generated inhibitory substances, i.e., bacteriocins . Under anaerobic condition, LA concentration in the stored garbage was found to be higher in the order: 37 > 25 > 50 > 5 degrees C, and the concentration of sugar accumulated during the 50 degrees C-storage was the highest . Among the conditions employed, the optimum condition for the storage of kitchen garbage was anaerobic at 5 degrees C.

Infect Immun, 2002 Aug, 70(8), 4353 - 61
Clostridium septicum alpha-toxin is active against the parasitic protozoan Toxoplasma gondii and targets members of the SAG family of glycosylphosphatidylinositol-anchored surface proteins; Wichroski MJ et al.; As is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of Toxoplasma gondii tachyzoites . The mechanisms by which T . gondii GPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown . As a first step in developing tools to study these processes, we show here that Clostridium septicum alpha-toxin, a pore-forming toxin that targets GPI-anchored protein receptors on the surface of mammalian cells, is active against T . gondii tachyzoites (50% effective concentration, 0.2 nM) . Ultrastructural studies reveal that a tight physical connection between the plasma membrane and the underlying membranes of the inner membrane complex is locally disrupted by toxin treatment, resulting in a massive outward extension of the plasma membrane and ultimately lysis of the parasite . Toxin treatment also causes swelling of the parasite endoplasmic reticulum, providing the first direct evidence that alpha-toxin is a vacuolating toxin . Alpha-toxin binds to several parasite GPI-anchored proteins, including surface antigen 3 (SAG3) and SAG1 . Interestingly, differences in the toxin-binding profiles between the virulent RH and avirulent P strain were observed . Alpha-toxin may prove to be a powerful experimental tool for molecular genetic analysis of GPI anchor biosynthesis and GPI-anchored protein trafficking in T . gondii and other susceptible protozoa.

Infect Immun, 2002 Aug, 70(8), 4261 - 72
Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates; Miyamoto K et al.; Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C . perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene . In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates . Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid . The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus . Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested . Similar dcm sequences were also detected in several cpe-negative C . perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene . Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested . Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene . Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C . perfringens plasmid . The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C . perfringens type A strains.

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