Clostridia

Ginecol Obstet Mex, 1992 Jun, 60, 162 - 70
{Gynecologic and obstetric infections caused by aerobic bacteria}; Figueroa-Damian R et al.; The anaerobic bacteria (AB) are between the most numerous microorganisms (mo) that constitute the flora of the female genital tract, so they can participate in the etiology of obstetric and gynecologic infections (OGI) . The objective of this study was to investigate the frequency of AB isolations and the clinical characteristics of the anaerobic infections (AI) in patients of the National Institute of Perinatology, from January 1st, 1988 to May 31, 1991 . AB were isolated from 117 patients who developed 163 infections; 167 anaerobic and 83 aerobic bacteria were recovered from these infections . The 99.2% were obstetric patients . The 85.5% of the isolations of AB were done from patients with endometritis, and 8.5% from postsurgical wound abscesses . Most of the AI were polymicrobial with a mean of 2.1 mo by infection . Peptostreptococcus, Clostridium and Bacteroids were the AB most frequent recovered . The majority of the patients had resolution of the infection within the first 5 days of antimicrobial treatment . There was no mortality in this group . We concluded that the AB have an important role in the etiology of OGI, then it is necessary that the treatment of these infections include antibiotics that cover AB.

Poult Sci, 1992 Jun, 71(6), 959 - 69
Barley inclusion and avoparcin supplementation in broiler diets . 1 . Effect on small intestinal bacterial flora and performance; Hofshagen M et al.; Diets based on barley or corn without avoparcin supplementation were associated with high counts of Clostridium perfringens in the contents of the small intestine of the birds at the age of 2 to 4 wk . The weight gain of birds younger than 2 wk and the body weight of 4-wk-old birds were significantly lower, and the feed conversion ratio at slaughter was significantly higher, in birds on barley diets than in birds on corn diets . The frequency of birds with sticky droppings on Day 21 was significantly higher for barley diets . The number of C . perfringens, and the feed conversion ratio at slaughter were significantly lower but the number of coliform bacteria, weight gain during the 3rd wk, and body weight of 4-wk-old chickens were significantly higher when the diets were supplemented with 7.5 mg avoparcin/kg feed . The effect of avoparcin on the feed conversion ratio was statistically significant only on a barley diet.

J Clin Invest, 1992 Jun, 89(6), 1866 - 74
Production of the Pseudomonas aeruginosa neuraminidase is increased under hyperosmolar conditions and is regulated by genes involved in alginate expression; Cacalano G et al.; The pathogenesis of Pseudomonas aeruginosa infection in cystic fibrosis (CF) is a complex process attributed to specific characteristics of both the host and the infecting organism . In this study, the properties of the PAO1 neuraminidase were examined to determine its potential role in facilitating Pseudomonas colonization of the respiratory epithelium . The PAO1 neuraminidase was 1000-fold more active than the Clostridium perfringens enzyme in releasing sialic acid from respiratory epithelial cells . This effect correlated with increased adherence of PAO1 to epithelial cells after exposure to PAO1 neuraminidase and was consistent with in vitro studies demonstrating Pseudomonas adherence to asialoganglioside receptors . The regulation of the neuraminidase gene nanA was examined in Pseudomonas and as cloned and expressed in Escherichia coli . In hyperosmolar conditions neuraminidase expression was increased by 50% (P less than 0.0004), an effect which was OmpR dependent in E . coli . In Pseudomonas the osmotic regulation of neuraminidase production was dependent upon algR1 and algR2, genes involved in the transcriptional activation of algD, which is responsible for the mucoid phenotype of Pseudomonas and pathognomonic for chronic infection in CF . Under the hyperosmolar conditions postulated to exist in the CF lung, nanA is likely to be expressed to facilitate the initial adherence of Pseudomonas to the respiratory tract.

Eur J Biochem, 1992 Jun 1, 206(2), 547 - 52
(R)-lactyl-CoA dehydratase from Clostridium propionicum . Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA; Hofmeister AE et al.; 1 . A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed . The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine . No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes . Both preparations exhibited similar activities towards (R)-lactyl-CoA as well as towards (R)-2-hydroxybutyryl-CoA, the latter being the superior substrate . 2 . Three species of (2R)-2-hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S-position . By incubation of these species with acetyl-CoA, propionate CoA-transferase and lactyl-CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was release as 3HOH . The data indicate that stereospecific abstraction of the 3Si hydrogen of (2R)-2-hydroxybutyryl-CoA during the dehydration . 3 . The identity of the product of the dehydration as crotonyl-CoA was established by the combined action of the enzymes crotonase and (S)-3-hydroxyacyl-CoA dehydrogenase . The results indicate that the elimination of water from (R)-2-hydroxybutyryl-CoA occurs in a syn mode . 4 . All enzyme activities necessary for the conversion of L-threonine via (R)-2-hydroxybutyryl-CoA to butyrate were detected in cell-free extracts of C . propionicum . 5 . A new mechanism for the dehydration of lactyl-CoA is proposed.

Eur J Biochem, 1992 Jun 1, 206(2), 537 - 46
Rac1, a low-molecular-mass GTP-binding-protein with high intrinsic GTPase activity and distinct biochemical properties; Menard L et al.; Rac1, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst . To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography . Rac1 protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concentrations . Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady-state GTPase activity of 18 nmol.min-1.mg protein-1 (turnover number approximately 0.39 min-1 at 37 degrees C), which is 40-fold higher than H-ras . The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min-1 for rac1 vs 0.007 min-1 for H-ras (at 20 degrees C) . Rac1 also released bound GDP faster than H-ras (koff.GDP = 1.02 min-1 for rac1 vs 0.33 min-1 for H-ras at 20 degrees C) . In contrast, rac1 released bound guanosine 5'-{gamma-thio}triphosphate (GTP{S}) at a slower rate than H-ras (koff.GTP{S} approximately 0.04 min-1 for rac1 vs 0.31 min-1 for H-ras at 20 degrees C) . Rac1 was a very good substrate for in vitro geranylgeranylation (C20) but not for farnesylation (C15), whereas the converse is true for H-ras . Surprisingly, rac1 was a very poor substrate for in vitro ADP-ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA . As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine . This protein (referred to as {Thr115----Asn}rac1) contains the consensus amino acids of all four GTP-binding domains of H-ras . The koff.GDP of {Thr115----Asn}rac1 was reduced to that of H-ras, but {Thr115----Asn}rac1 exhibited essentially identical kcat (0.13 min-1 at 20 degrees C) and koff-GTP{S} (0.03 min-1 at 20 degrees C) values as the wild-type protein . Thus, the region(s) in rac1 which control the dissociation of GTP{S} (and presumably GTP) do not entirely coincide with those controlling GDP dissociation . Biochemical analysis of {Thr115----Asn}rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains.

J Trop Pediatr, 1992 Jun, 38(3), 129 - 31
Pathogens in neonatalomphalitis; Airede AI; During a 3-year study period, 33 neonates with omphalitis (with proven cultures) were encountered; aerobic and anaerobic cultures were obtained . An incidence of 2/1000 live births was recorded, with a high prevalence rate of 15.6/1000 admissions noted . Aerobes were isolated from 23 (70 per cent) specimens, whilst anaerobes were recovered alone in five (15 per cent) cases . There were 40 (1.2 per specimen) and 31 (0.9 per specimen) aerobic and anaerobic isolates, respectively . There was a significant detection of anaerobic pathogens such as the B . fragilis group (14), Gram-positive cocci (4) and Clostridium perfringens (3) . Beta lactamase production was seen in 25 isolates, recovered from 25 newborns.

Clin Otolaryngol, 1992 Jun, 17(3), 223 - 4
Management of facial synkinesis with Clostridium botulinum toxin injection; Mountain RE et al.; Associated movements after facial paralysis (synkinesis), due to unphysiological co-innervation of the facial muscles, often complicates the rehabilitation of patients following facial palsy . Clostridium botulinum toxin is a neurotoxin that interferes with the release of acetylcholine from motor nerve end plates, causing skeletal muscular paralysis . This paper concentrates on its clinical use in treating synkinesis affecting orbicularis oculi function and documents the results of treatment in 4 patients . Control of synkinesis, achieved in all 4 patients, was effective within a few days and lasted for 4-6 months . 2 patients developed transient diplopia and ptosis shortly after injection . However, no lasting complications or systemic side-effects were noted . All patients reported a significant improvement in their symptoms and reinjection at 7 months was carried out successfully.

J Pediatr Surg, 1992 Jun, 27(6), 744 - 6
Perforated pseudomembranous colitis in the breast-fed infant; Harmon T et al.; Pseudomembranous colitis (PMC) is uncommon in the infant and complications requiring surgical intervention are rare . All prior cases have involved the direct administration of antibiotics to the child . A 2-month-old girl required bowel resection for perforation of a thickened and inflamed left colon . Findings were consistent with PMC and the stool was Clostridium difficile toxin positive . The patient was treated with vancomycin and did well . The patient's mother later admitted to self-administration of ciprofloxacin while breast-feeding her infant . Oral doses of this drug are concentrated in breast milk at levels higher than serum . Antibiotics derived from breast milk can cause complicated PMC in the infant . A directed history can establish an early diagnosis and help distinguish PMC from other more common infantile enterocolitides.

J Diarrhoeal Dis Res, 1992 Jun, 10(2), 87 - 92
Colonisation with digoxin-reducing strains of Eubacterium lentum and Clostridium difficile infection in nursing home patients; Bennett RG et al.; Stool specimens obtained from 77 residents of a nursing home were analysed to determine the relationship between colonisation with digoxin-reducing strains of Eubacterium lentum and infection with Clostridium difficile . Patients were categorised according to previous antibiotic treatment, prescription of enteral feedings, and pattern of bowel habits . Colonisation with digoxin-reducing E . lentum was less common in subjects infected with C . difficile, in those treated with antibiotics previously, and in those prescribed enteral feedings . Normal bowel habits were more common in those without C . difficile . The lowest incidence of diarrhoea was seen in patients without C . difficile who were colonised with digoxin-reducing species . This study establishes an inverse relationship between the presence of C . difficile and E . lentum that reduce digoxin which is related to previous treatment with antibiotics and prescription of enteral feedings . Bacterial markers may prove to be a useful tool for predicting clinical disturbances in bowel function.

Int J Food Microbiol, 1992 Jun, 16(2), 117 - 21
Detection of Clostridium botulinum in natural sweetening; Nakano H et al.; Various sugar products were examined for contamination with C . botulinum spores . Type A, B and C spores were detected in three of 56 samples of sugar for apiculture, which may attest the significance of bee-feed as a source of contamination of honey . The heavy contamination of honey with C . botulinum spores sometimes encountered, however, can not be explained unless some other factors, e.g., that allowing germination and multiplication of the spores somewhere during honey production, are found . Type A spores were detected in some samples of raw sugar and molasses and also in two of 41 samples of brown sugar lump, but not in refined sugar or other various samples taken at a sugar factory or in sugar cane left on the field in Okinawa . The fact that some natural sweetenings are contaminated with C . botulinum spores, even in low concentrations, may be food-hygienically important.

Antimicrob Agents Chemother, 1992 Jun, 36(6), 1332 - 5
Effects of antibiotics and other drugs on toxin production in Clostridium difficile in vitro and in vivo; Barc MC et al.; In an attempt to understand more completely why patients treated with phenothiazines (chlorpromazine and cyamemazine), methotrexate, and certain antibiotics such as clindamycin have an increased risk of developing pseudomembranous colitis, the production of toxins A and B by Clostridium difficile in the presence of these drugs was measured in vitro as well as in vivo by using axenic mice . None of the drugs tested increased the production of toxins either in vitro or in vivo.

J Clin Microbiol, 1992 Jun, 30(6), 1544 - 50
Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays; Muller F et al.; Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized . Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule . No MAb was able to neutralize cytotoxic activity significantly . The two IgG1 MAbs were purified and applied to various immunodiagnostic assays . MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay . An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.

Infect Immun, 1992 Jun, 60(6), 2488 - 92
Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A; Frey SM et al.; The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule . We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units . In the present study, we subcloned a series of fragments from this portion of the gene . The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding . Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.

Biochem Biophys Res Commun, 1992 May 29, 185(1), 341 - 9
Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2{4Fe-4S} ferredoxin; Davasse V et al.; A gene encoding the exact sequence of Clostridium pasteurianum 2{4Fe-4S} ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli . The synthetic gene is efficiently expressed in E . coli and its product has been purified and characterized . The N-terminal sequence is identical to that of the protein isolated from C . pasteurianum and the recombinant ferredoxin contains the exact amount of {4Fe-4S} clusters (2 per monomer) expected for homogeneous holoferredoxin . It displays reduction potential and kinetic parameters as electron donor to C . pasteurianum hydrogenase I identical to those determined for the native ferredoxin . All of these properties demonstrate that the 2{4Fe-4S} ferredoxin expressed in E . coli is identical to the parent clostridial protein.

J Biol Chem, 1992 May 25, 267(15), 10274 - 80
Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum; Just I et al.; We purified a novel ADP-ribosyltransferase produced by a Clostridium limosum strain isolated from a lung abscess and compared the exoenzyme with Clostridium botulinum ADP-ribosyltransferase C3 . The C . limosum exoenzyme has a molecular weight of about 25,000 and a pI of 10.3 . The specific activity of the ADP-ribosyltransferase is 3.1 nmol/mg/min with a Km for NAD of 0.3 microM . Partial amino acid sequence analysis of the tryptic peptides revealed about 70% homology with C3 . The novel exoenzyme modifies selectively the small GTP-binding proteins of the rho family in human platelet membranes presumably at the same amino acid (asparagine 41) as known for C3 . Recombinant rhoA and rhoB serve as substrates for C3 and the C . limosum exoenzyme . Whereas recombinant rac1 protein is only marginally ADP-ribosylated by C3 or by the C . limosum exoenzyme in the absence of detergent, in the presence of 0.01% sodium dodecyl sulfate rac1 is modified by C3 but not by the C . limosum exoenzyme . Recombinant CDC42Hs protein is a poor substrate for C . limosum exoenzyme and is even less modified by C3 . The C . limosum exoenzyme is auto-ADP-ribosylated in the presence of 0.01% sodium dodecyl sulfate by forming an ADP-ribose protein bond highly stable toward hydroxylamine . The data indicate that ADP-ribosylation of small GTP-binding proteins of the rho family is not unique to C . botulinum C3 ADP-ribosyltransferase but is also catalyzed by a C3-related exoenzyme from C . limosum.

Eur J Biochem, 1992 May 15, 206(1), 79 - 85
Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum; Schrader T et al.; Protein PC of the glycine reductase from Eubacterium acidaminophilum was purified to homogeneity by chromatography on phenyl-Sepharose and Sepharose S . The apparent molecular mass of the native protein, which showed an associating/dissociating behaviour, was about 420 kDa . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of protein PC revealed two protein bands corresponding to 48 and 57 kDa, indicating an alpha 4 beta 4 composition . The smaller subunit was identified as an acetyl-group-transferring protein, the 57-kDa protein was hydrophobic . N-terminal amino acid sequences were determined for both subunits . Antibodies raised against the 48-kDa subunit showed cross-reactions with extracts of E . acidaminophilum grown on different substrates and with extracts from other glycine-utilizing anaerobic bacteria such as Clostridium purinolyticum, C . sticklandii, and C . sporogenes . The respective protein from the former two organisms corresponded in molecular mass . When protein PA was chemically carboxymethylated by iodo{2-14C}acetate and incubated with protein PC, acetyl phosphate was a reaction product, thus establishing it as the product of the glycine reductase reaction by using homogeneous preparations of these two proteins from E . acidaminophilum.

Eur J Biochem, 1992 May 15, 206(1), 151 - 9
The glutamate dehydrogenase gene of Clostridium symbiosum . Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli; Teller JK et al.; The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques . The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein . The recombinant plasmid complemented the nutritional lesion of an E . coli glutamate auxotroph . There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%) . The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme . The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme . The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host . The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes . The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.

Eur J Pharmacol, 1992 May 12, 226(1), 87 - 91
ADP-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and compound 48/80; Koch G et al.; The amphiphilic agents melittin, mast cell degranulating peptide and compound 48/80 inhibit the ADP-ribosylation of the small GTP-binding proteins rho by Clostridium botulinum exoenzyme C3 . Half-maximal and maximal inhibition (greater than 90%) of ADP-ribosylation occurred at about 8 and 25 micrograms/ml for compound 48/80, at 10 and 45 microM for mast cell degranulating peptide and at 15 and 50 microM for melittin, respectively . In addition, these compounds increase the steady state GTP hydrolysis and the association and dissociation rate of GTP-binding of rho proteins through an increase of GDP/GTP exchange . The data suggest that the amphiphilic agents tested interact with small GTP-binding proteins of the rho protein family.

J Biol Chem, 1992 May 5, 267(13), 8719 - 22
Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction; Hirata K et al.; In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner . Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction . GTP gamma S alone did not induce the contraction in the absence of Ca2+ . This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily . The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN . smg p21B, another small GTP-binding protein, was inactive . EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle . The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction . smg p21B was inactive . These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction.

J Gen Microbiol, 1992 May, 138 ( Pt 5), 861 - 9
Autolysis of Clostridium acetobutylicum ATCC 824; Croux C et al.; The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined . Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer . The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth . Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations . The autolysin of C . acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase . The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence . The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol . A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.

Mol Gen Genet, 1992 May, 233(1-2), 260 - 8
Comparative sequence analysis of the Clostridium difficile toxins A and B; von Eichel-Streiber C et al.; The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCd13 cover the tox locus of Clostridium difficile VPI 10463 . This region of 19 kb of chromosomal DNA contains four open reading frames including the complete toxB and toxA genes . The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera . A special feature of ToxA and ToxB is their repetitive C-termini . We define herein 19 individual CROPs (combined repetitive oligopeptides of 20-50 aa length) in the ToxB C-terminus, which are separable into five homologous groups . Comparison of the aa sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved aa in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa . The probable function of these domains is discussed . Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf).

J Med Microbiol, 1992 May, 36(5), 307 - 11
Proteolytic activity of Clostridium difficile; Seddon SV et al.; Ten isolates of Clostridium difficile expressing different degrees of toxigenicity and virulence in an animal model were assayed for the production of proteolytic enzymes by various methods . All strains demonstrated some activity in one or more of the assay systems . There was no direct correlation between toxigenic status and enzyme production . However, those strains known to be highly virulent in a hamster model were the most proteolytic . The most commonly detected enzyme was cell associated, and its substrate specificity suggested it was a trypsin-like enzyme . Initial purification of the enzyme from strain VPI 10463 gave a 10% yield with a 14-fold increase in purity . Inhibition studies on this preparation indicated that the enzyme was a thiol protease . The enzyme has pH and temperature optima of 7.5 and 37 degrees C, respectively . These characteristics suggest that the enzyme is more related to clostripain, the thiol clostridio-peptidase of C . histolyticum, than to trypsin . Whilst the role of this enzyme remains unclear, it is possible that it may be a contributory factor in the virulence of the organism as described for other clostridial infections.

Mol Biol Evol, 1992 May, 9(3), 495 - 506
Nucleotide sequence of nifD from Frankia alni strain ArI3: phylogenetic inferences; Normand P et al.; The complete nucleotide sequence of the nifD gene encoding the alpha subunit of component I of nitrogenase from Frankia alni strain ArI3 was determined . The coding region is 1,458 bp in length and encodes a polypeptide of 486 residues with a predicted molecular weight of 53,500 . Phylogenetic inferences with 12 complete published nifD sequences were drawn using a variety of approaches . Frankia nifD clusters with proteobacteria rather than with Clostridium pasteurianum, the other Gram-positive bacterium studied . Extant eubacterial nif genes seem to have at least three distinct evolutionary origins as a result of ancient gene duplications . Within the Gram-positive bacterial phylum, functional nif genes descend from different duplicates.

J Clin Microbiol, 1992 May, 30(5), 1085 - 8
Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A; De Girolami PC et al.; The Premier Clostridium difficile toxin A enzyme immunoassay (PTA EIA) (Meridian Diagnostics, Inc., Cincinnati, Ohio) for rapid diagnosis of antibiotic-associated colitis (AAC) was evaluated in a multicenter study . Stool samples from 421 patients suspected of having AAC were tested for toxin A by the PTA EIA and for toxin B by three tissue culture assays (TCA) employing WI-38 cells (New England Deaconess Hospital) in conventional tubes or foreskin fibroblasts (Children's Hospital) or Vero cells (Beth Israel Hospital) in microwells . The tubes and plates were examined at 24 and 48 h for cytotoxicity . Clinical criteria, repeat testing at another site, and culture of frozen stool samples for C . difficile were used to evaluate discrepant results . Of 504 samples, 66 were positive and 409 were negative by both tests . Eight samples had indeterminate PTA EIA results and were excluded from this analysis . Of 21 discrepancies, 9 were PTA EIA positive and TCA negative and 12 were PTA EIA negative TCA positive . Following resolution of the discrepancies, 11 of 12 PTA EIA-negative-TCA-positive and 5 of 9 PTA EIA-positive-TCA-negative samples were considered true positive for AAC . The sensitivity and specificity were, respectively, 86.6 and 99.0% for the PTA EIA and 93.9 and 99.8% for TCA . The predictive values of positive and negative tests were, respectively, 94.7 and 97.4% for the PTA EIA and 98.7 and 98.8% for TCA . We conclude that the PTA EIA is a rapid, simple EIA technique whose accuracy in detecting enterotoxin A approaches that of reference TCA methods for detection of cytotoxin B.

Arch Pathol Lab Med, 1992 May, 116(5), 517 - 20
Evaluation of the latex agglutination test for detection of Clostridium difficile; Kelly WF et al.; We compared two Clostridium difficile latex agglutination tests, Meritec from Meridian Diagnostic (Cincinnati, Ohio) and CDT from Becton-Dickinson (Cockeysville, Md), on 289 specimens submitted for tissue culture cytotoxicity using MRC-5 cells . When compared with CDT, the Meritec latex agglutination test had a sensitivity of 90% (26/29), a specificity of 97% (251/260), and a correlation of 96% . Meritec was compared with tissue culture cytotoxicity on 357 specimens . Meritec had a sensitivity of 77% (30/39), a specificity of 93% (298/318), and a correlation of 92% . Clinical review of 10 Meritec +/- tissue culture cytotoxicity minus patients revealed one likely, two probable, and seven doubtful cases of C difficile disease . In contrast, review of 10 Meritec +/- tissue culture cytotoxicity plus patients showed seven likely and three probable cases of C difficile disease . The Meritec is comparable with the CDT latex agglutination test, but is not nearly as sensitive as either tissue culture assay or culture for detection of C difficile disease . A positive latex agglutination test should be confirmed by a tissue culture cytotoxicity assay.

J Bacteriol, 1992 May, 174(10), 3290 - 9
Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene; Narberhaus F et al.; The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors . By using the E . coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C . acetobutylicum, respectively, were isolated . The cloned fragments partially overlapped, and together they spanned 4,083 bp of the clostridial genome that were completely sequenced . On one strand, four open reading frames of which the last was obviously truncated were identified . The last three genes showed high homology to the grpE, dnaK, and dnaJ heat shock genes of E . coli, respectively . They were preceded by an open reading frame (orfA) without any homology to sequences available in the EMBL or GenBank data bases . Typical translational start sites could be found in front of all four genes . Northern (RNA) blot analysis revealed transcripts of this region with a maximum length of 5.0 kb . Thus, these genes are probably organized in an operon . A transcription terminator could be found between the dnaK and dnaJ genes . By primer extension analysis, a major heat-inducible transcription start site was identified 49 bases upstream of orfA . This site was preceded by a region (5'-TTGACA{17 bp}TATTTT) that exhibited high homology to the consensus promoter sequences of gram-positive bacteria as well as sigma 70-dependent E . coli . Between this promoter and the initiation codon of orfA, a hairpin-loop structure with a possible regulatory role in the expression of these genes was found . Additional heat-inducible transcription start sites were located 69 bases upstream of orfA and 87 bases upstream of grpE; the corresponding promoter regions showed less similarity to other known promoter sequences . Maximum mRNA levels of this heat shock operon were found about 15 min after a heat shock from 30 to 42 degrees C . Our results indicate that orfA codes for an unknown heat shock protein.

J Bacteriol, 1992 May, 174(9), 2874 - 80
Binding of DNA to alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species prevents formation of cytosine dimers, cytosine-thymine dimers, and bipyrimidine photoadducts after UV irradiation; Fairhead H et al.; Small, acid-soluble proteins (SASP) of the alpha/beta-type from spores of Bacillus and Clostridium species bind to DNA; this binding prevents formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of the spore photoproduct, an adduct between adjacent thymine residues . alpha/beta-Type SASP also bound to poly(dG).poly(dC) and poly(dA-dG).poly(dC-dT) . While UV irradiation of poly(dG).poly(dC) produced cyclobutane-type cytosine dimers as well as fluorescent bipyrimidine adducts, the yields of both types of photoproduct were greatly reduced upon irradiation of alpha/beta-type SASP-poly(dG).poly(dC) complexes . UV irradiation of poly(dA-dG).poly(dC-dT) produced a significant amount of a cyclobutane dimer between cytosine and thymine, as well as a 6-4 bipyrimidine adduct . Again, binding of alpha/beta-type SASP to poly(dA-dG).poly(dC-dT) greatly reduced formation of these two photoproducts, although formation of the cytosine-thymine analog of the spore photoproduct was not observed . These data provide further evidence for the dramatic change in DNA structure and photoreactivity which takes place on binding of alpha/beta-type SASP and suggest that binding of these proteins to DNA in vivo prevents formation of most deleterious photoproducts upon UV irradiation.

Toxicon, 1992 May-Jun, 30(5-6), 653 - 68
Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B; Schranner I et al.; The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography . The alpha-toxin has a mol . wt of 190,000 and does not contain any disulfide cross-linkages . It consists of a single polypeptide chain . The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC . The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice . Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5) . One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol . wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test . The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain . The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine . By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary.

Plasmid, 1992 May, 27(3), 207 - 19
Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid; Sloan J et al.; A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled . The vector, pJIR418, contains the replication regions from the C . perfringens replicon pIP404 and the E . coli vector pUC18 . The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E . coli . Both chloramphenicol and erythromycin resistance can be selected in C . perfringens and E . coli since pJIR418 carries the C . perfringens catP and ermBP genes . Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms . The versatility of pJIR418 and its applicability for the cloning of toxin genes from C . perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.

Indian J Med Res, 1992 May, 95, 148 - 51
Bacteriology of ophthalmic infections with special reference to anaerobes; Aggarwal R et al.; A total of 138 eye specimens were processed for aerobic and anaerobic cultures . Clinical data were obtained from 50 patients with unilateral ophthalmic infection . Cultures from the uninfected eye of 38 of these 50 patients were also processed for comparison . In addition, 50 cultures were obtained from one or both eyes of 30 healthy controls who had no eye infection . Anaerobes and aerobes were isolated from infected eyes of 6 (12%) and 37 (74%) patients respectively . No growth was observed in infected eye of 8 (16%) patients . A mixture of aerobes and anaerobes were recovered only in 2 cases . Of the total 47 aerobic isolates from infected eye specimens, Staphylococcus aureus (11), coagulase negative staphylococci (12) and Streptococcus pneumoniae (9) were predominant isolates . Six anaerobes isolates included Gram positive nonsporing anaerobic bacilli (4 including Propionibacterium acne) as predominant isolates . Clostridium perfringens was isolated from a case of post operative endoophthalmitis . From the uninfected eye of same patients though the number and types of aerobic bacteria were similar, none grew any anaerobes . Aerobic and anaerobic bacteria were isolated in 70 and 6 per cent of eye swabs respectively from the healthy controls.

Pharmacol Res, 1992 May-Jun, 25(4), 373 - 81
In vitro activity of sulphimidazole alone and in association with trimethoprim against enteric pathogens; Castelli M et al.; Sulphimidazole is a new sulphonamide belonging to the class of intestinal sulphonamides and characterized by the fact that it is active even in vitro . It has the heterocyclic ring of 5-nitroimidazoles on amidic nitrogen . Its antibacterial activity is similar to that of the classical sulphonamides but differs in that it also combats certain anaerobic bacteria such as Clostridium botulinum . This effect is completely absent in the case of sulphadiazine and sulphamethoxazole . Also, since p-amino-benzene-sulphonamide is present in the molecule, the drug acts in synergism with trimethoprim against certain aerobic or facultative strains of enteric pathogens.

Infect Immun, 1992 May, 60(5), 2110 - 4
Mapping of functional regions of Clostridium perfringens type A enterotoxin; Hanna PC et al.; Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE) . Removal of the N-terminal 25 amino acids of CPE increased the primary cytotoxic effect of CPE but did not affect binding . CPE sequences required for at least four epitopes were also identified.

Biosci Biotechnol Biochem, 1992 May, 56(5), 788 - 93
Cloning and nucleotide sequence of a frxC-ORF469 gene cluster of Synechocystis PCC6803: conservation with liverwort chloroplast frxC-ORF465 and nif operon; Ogura Y et al.; A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase . Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known . Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469-ORF248) was isolated from the cyanobacterium Synechocystis PCC6803 . In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively . Synechocystis frxC showed 31% amino acid sequence identity with nifH1 from Clostridium pasteurianum . Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C . pasteurianum nifK product, which is the beta subunit of a molybdenum-iron protein of a nitrogenase complex . Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.

J Bacteriol, 1992 May, 174(10), 3282 - 9
Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum; Narberhaus F et al.; The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E . coli groESL . Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome . Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C . acetobutylicum, respectively . The 3' end of a third gene (orfZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases . The length of the groESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C . acetobutylicum are organized in a bicistronic operon . From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E . coli was deduced . Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42 degrees C . An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism.

Eur J Clin Microbiol Infect Dis, 1992 May, 11(5), 458 - 62
In vitro activity of the new glycopeptide decaplanin; Neu HC et al.; The activity of decaplanin, a new glycopeptide, was compared to that of vancomycin, teicoplanin and daptomycin . Decaplanin was two- to four-fold less active than vancomycin, telcoplanin and daptomycin against Staphylococcus aureus and Staphylococcus epidermidis, with an MIC90 of 2 micrograms/ml for methicillin-susceptible and 4 micrograms/ml for methicillin-resistant isolates . Decaplanin had activity similar to that of vancomycin against Streptococcus pyogenes, Streptococcus agalactiae, group C and G streptococci, with an MIC90 of 0.12 micrograms/ml . It was less active than the other agents against the viridans group streptococci (MIC90 4 micrograms/ml) . The activity of decaplanin against enterococci (MIC90 4 micrograms/ml) was similar to that of vancomycin . Clostridium spp . were inhibited by 0.5 micrograms/ml, peptostreptococci and peptococci by 0.25 microgram/ml . Decaplanin was active from pH 5.5 to 7.5 . Inoculum size had a minimal effect on MICs, and increased concentrations of Ca2+ and Mg2+ and 50% serum did not alter MICs or MBCs.

J Pediatr, 1992 May, 120(5), 747 - 9
Electrodiagnosis reliability in the diagnosis of infant botulism; Graf WD et al.; Infant botulism is confirmed by isolation of Clostridium botulinum from stool culture or by toxin assay . Although electrodiagnosis has been described as a diagnostic tool in infant botulism, our 11-year review of toxin-confirmed cases suggests that electrodiagnosis is not a reliable tool . In the case report presented, results of electrodiagnosis were negative but enema effluent contained adequate concentrations of organism and toxin to confirm the diagnosis.

J Mol Biol, 1992 Apr 20, 224(4), 1181 - 4
Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum . Evidence for a ligand-induced conformational change; Stillman TJ et al.; A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion . The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate . The crystals diffract well and X-ray photographs have established that they are in the space group R32 . Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit . Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell . Such a conformational rearrangement may represent an important step in the catalytic cycle.

Eur J Biochem, 1992 Apr 15, 205(2), 799 - 808
Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens; Gerwig GJ et al.; The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit . The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC . The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry . The following carbohydrate structures could be established: {formula: see text} The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B . cellulosolvens and of Clostridium thermocellum {Gerwig, G . J., Kamerling, J . P., Vliegenthart, J . F . G., Morag (Morgenstern), E., Lamed, R . & Bayer, E . A . (1991) Eur . J . Biochem . 196, 115-122}, having 3, 5 and 6 as common elements . The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B . cellulosolvens.

Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3483 - 7
Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A; Shoseyov O et al.; The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced . When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed . The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da . The open reading frame is preceded by a Gram-positive-type ribosome binding site . A signal peptide sequence of 28 amino acids is present at its N terminus . The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues . There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids . These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex.

Eur J Biochem, 1992 Apr 15, 205(2), 767 - 73
Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri; Michel C et al.; The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively . Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa) . Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin . This preparation could be reactivated to 95-100% by addition of adenosylcobalamin . The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity . 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin . Upon incubation of the mutase with {5'-3H}adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity . During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin . EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme . Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose . The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.

Eur J Biochem, 1992 Apr 15, 205(2), 759 - 65
Glutamate mutase from Clostridium cochlearium . Purification, cobamide content and stereospecific inhibitors; Leutbecher U et al.; Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified . Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit . The enzyme proved to be very similar to that of C . tetanomorphum as described by Barker et al . {Barker, H . A., Rooze, V., Suzuki, F . & Iodice, A . A . (1964) J . Biol . Chem . 239, 3260-3266} but component E of C . cochlearium was more stable and led to the first pure preparation . The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-{alpha-(aden-9-yl)}-cob(II)amide . A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy . A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis . Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM) . (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory . The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis . However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.

Burns, 1992 Apr, 18(2), 167 - 9
Massive haemorrhage due to rectosigmoid ulcers in a patient with extensive burns; Law EJ et al.; A 36-year-old white-skinned male was admitted with 45.5 per cent burns, mostly of full skin thickness . Severe rectal bleeding from rectal ulcerations developed on postburn day 12 . Various conservative attempts at management failed, and after multiple transfusions, abdominoperineal resection was carried out with eventual complete recovery . Complications during his acute phase included Pseud . aeruginosa sepsis and Clostridium difficile diarrhoea . Extensive skin grafts were required . The cause of the rectal ulcerations is unclear.

Mayo Clin Proc, 1992 Apr, 67(4), 369 - 72
An unusual case of myxedema megacolon with features of ischemic and pseudomembranous colitis; Patel R et al.; Myxedema megacolon is rare; usually, it manifests with abdominal distention, flatulence, and constipation . Herein we describe a 72-year-old man who had intermittent diarrhea, bloating, and abdominal pain for more than a year . Cultures of stool specimens for Clostridium difficile enterotoxin were variably positive and negative . Colonoscopic biopsy specimens were thought to be consistent with chronic ischemia . Thyroid function tests showed severe hypothyroidism; the patient's symptoms resolved with thyroid hormone replacement . We hypothesize that gross dilatation of the colon, attributed to myxedema, was followed by intestinal ischemia and complicated by recurrent episodes of pseudomembranous colitis . A review of the relevant literature is provided . This unusual manifestation of myxedema should be considered in the differential diagnosis when a patient has diarrhea, bloating, and abdominal pain.

Biochim Biophys Acta, 1992 Apr 6, 1130(3), 259 - 66
The use of chimeric gene constructs to express a bacterial endoglucanase in mammalian cells; Hall J et al.; The synthesis and secretion of a truncated Clostridium thermocellum endoglucanase (EGE') encoded by the celE' gene was investigated in Chinese hamster ovary (CHO) cells . Fusion genes consisting of the human growth hormone (hGH) gene and celE', transcribed from the SV40 early enhancer/promoter, were constructed and stably transfected into CHO cells . A gene consisting of celE' inserted into the first exon of the hGH gene resulted in the synthesis of truncated proteins (less than or equal to 22 kDa) lacking endoglucanase activity . Cloning celE' into the second exon of the hGH gene, resulted in the synthesis and secretion of a 50 kDa protein with endoglucanase activity . A 50 kDa protein was also synthesised by cells transfected with celE' cloned into the fifth exon of the hGH gene . However, despite a 5-fold increase in enzyme activity compared to the exon 2 transfected cell line less than 40% of the protein was secreted . Constructs devoid of introns, in which celE' was fused to the SV40 early promoter and to the rabbit beta-globin polyadenylation sequence resulted in a 2-18-fold increase in endoglucanase activity compared to the constructs containing introns . In addition more than 75% of the synthesised protein was secreted . Analyses of EGE' encoded mRNA from the transfected cell lines suggests that the presence of introns results in the aberrant splicing of message by the use of cryptic splice sites in the celE' gene . These results demonstrate that introns are not required for the efficient expression of a bacterial endoglucanase in mammalian cells, rather introns appear to reduce expression of the encoded protein.

Actas Urol Esp, 1992 Apr, 16(4), 354 - 6
{Bacteremia caused by Clostridium perfringens as a complication of ureterosigmoidostomy}; Batista-Miranda JE et al.; A 63 year old male underwent cystectomy and ureterosigmoidostomy after diffuse carcinoma "in situ" of the bladder was discovered, and thereafter, various episodes of pyelonephritis and metabolic imbalance, in one of them, a left pneumo-ureter and a positive blood culture for Clostridium Perfringens and enterococci was detected . Empiric therapy with Aztreonam was started, and changed after to high-dose intravenous amoxicillin . Two months later the ureterosigmoidostomy was converted to an ileal conduit . The patient has remained asymptomatic on subsequent controls.

Avian Dis, 1992 Apr-Jun, 36(2), 469 - 73
High mortality of domestic turkeys associated with Ascaridia dissimilis; Norton RA et al.; Third- and fourth-stage Ascaridia dissimilis larvae were isolated from commercial white turkey intestinal scrapings from two farms that were experiencing high mortality . Lesions consisted of a necrotic-like enteritis that was most severe in the jejunum . Subsequent bacteriological isolation yielded heavy growth of Escherichia coli and Clostridium perfringens . The rate of mortality declined rapidly when the turkeys were administered 18 ppm fenbendazole for 7 days.

Toxicon, 1992 Apr, 30(4), 419 - 26
Sensitivity in culture of epithelial cells from rhesus monkey kidney and human colon carcinoma to toxins A and B from Clostridium difficile; Torres J et al.; The effect of toxins A and B from Clostridium difficile on human colon carcinoma cells (HT-29, epithelial), rhesus monkey kidney cells (MA-104, epithelial) and green monkey kidney cells (VERO, fibroblast) was studied . Both toxins caused rounding of HT-29 cells and rounding with projections remaining attached to the substrate in MA-104 and VERO cells; however, the sensitivity to each toxin varies considerably . Toxin A was detected in ng by VERO, pg by HT-29 and fractions of pg by MA-104 cells; for toxin B, pg were detected by VERO, ng by MA-104 and micrograms by HT-29 cells . HT-29 cells were grown with galactose to allow their differentiation to enterocytes, and their sensitivity to the toxins during the process was studied . At early stages, the sensitivity to both toxins was similar, and as the differentiation proceeded, the response to both toxins decreased continuously, and after 16 days no evident morphological effect was observed, even with micrograms amounts of either toxin . In contrast to all cell lines reported to date, HT-29 and MA-104 epithelial cells are exquisitely sensitive to toxin A and less responsive to toxin B . The rounding of HT-29 by these toxins depends on the degree of differentiation of the cell.

FEMS Microbiol Lett, 1992 Apr 1, 71(1), 5 - 9
Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers; McMillin DE et al.; Clostridium difficile is the causative agent for pseudomembranous colitis in humans . Toxic strains of C . difficile produce two toxins, toxin A and toxin B . A reliable and definitive method of typing the toxic strains of C . difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities . A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study . The C . difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains . These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined . The use of this typing scheme in clinical applications is discussed.

Vet Microbiol, 1992 Apr, 31(1), 89 - 99
Development of double sandwich ELISA for Clostridium perfringens beta and epsilon toxins; el Idrissi AH et al.; Specific, double-sandwich ELISAs for beta and epsilon toxins were developed by coating wells of microplates with specific sheep antitoxin IgG and using specific rabbit antitoxin IgG as detecting antibodies . The assay for beta toxin detected a minimum level of 8 ng/ml of purified toxin . The assay for epsilon toxin was capable of detecting 2 ng/ml of purified toxin . When applied to detect the toxin in intestinal contents using 50% fetal bovine serum as diluent the lowest amounts detected were about at least 30 ng/ml for beta toxin and 4 ng/ml for epsilon toxin . Clear differences in ELISA readings of both assays have been found between culture filtrates from toxin and non-toxin producing strains . These results suggested that both assays described in this study could detect their respective toxin in buffers, culture supernatants or in intestinal contents.

Nippon Yakurigaku Zasshi, 1992 Apr, 99(4), 191 - 203
{ras oncogene-related small molecular weight GTP-binding protein, rho gene product and botulinum C3 ADP-ribosyltransferase}; Morii N et al.; The ras oncogene products (ras p21s) are 21-KDa proteins with activities of GTP binding and hydrolysis . A number of proteins homologous to ras p21 have been discovered and collectively named small molecular weight GTP-binding proteins . These proteins undergo post-translational modification with isoprenoid residues attached to cysteine in their carboxyl terminal . With this modification, they attach to cellular membranes . The biochemical activities of these proteins, i.e., GTP hydrolysis and binding, are regulated by various regulatory factors such as GDP-GTP exchange proteins and GTPase-activating proteins, but little is known about the cellular functions and physiological pathways through which they regulate these functions . Botulinum C3 ADP-ribosyltransferase, a 23-KDa exoenzyme secreted from certain strains of types C and D Clostridium botulinum, specifically ADP-ribosylates the rho family of these GTP-binding proteins . This ADP-ribosylation occurs at a specific asparagine residue in their putative effector domain, and presumably interferes with their interaction with a putative effector molecule downstream in signal transduction . C3 exoenzyme, when incubated with or microinjected into cultured cells, ADP-ribosylates a rho gene product in the cells, and causes profound cell rounding with loss of adhesion plaques and collapse of stress fiber . Microinjection of an activated mutant of rho A protein, on the contrary, induced extensive adhesion and actin assembly in cultured cells . These results suggest that the rho family of proteins are involved in morphogenesis and motility of cells via assembly and disassembly of cytoskeletal systems, and botulinum ADP-ribosyltransferase is a useful tool for clarifying the molecular mechanism of these processes.

Mol Microbiol, 1992 Apr, 6(7), 873 - 84
Cloning, sequencing and distribution of the Salmonella typhimurium LT2 sialidase gene, nanH, provides evidence for interspecies gene transfer; Hoyer LL et al.; The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli . Sialidase expression was regulated positively by cAMP . In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity . A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S . typhimurium LT2 . The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi . Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S . typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event . At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases . The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase . From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S . typhimurium obtained its nanH copy most recently from Salmonella arizonae . S . typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH . These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.

Appl Environ Microbiol, 1992 Apr, 58(4), 1411 - 4
Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C; Garcia-Alvarado JS et al.; Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C . Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C . There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).

Appl Environ Microbiol, 1992 Apr, 58(4), 1195 - 1200
Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914; Macfarlane GT et al.; Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture . In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1 . At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa . High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa . Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process . Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4 . The optimal pH of protease activity was 7.0 . Although a wide variety of proteins were hydrolyzed by C . bifermentans proteases, none of the enzymes were collagenolytic . Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed . With the exception of Ca2+, divalent metal ions inhibited proteolysis . Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.

Appl Environ Microbiol, 1992 Apr, 58(4), 1075 - 81
Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan; Croux C et al.; An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824 . The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24% . The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8 . It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp . The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1 . Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline . The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain . The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall.

Am J Physiol, 1992 Apr, 262(4 Pt 2), F572 - 7
Involvement of C3 exotoxin-sensitive G proteins (rho/rac) in PTH signal transduction in OK cells; Reshkin SJ et al.; Parathyroid hormone (PTH) in opossum kidney (OK) cells leads to inhibition of Na-Pi cotransport, to the generation of inositol trisphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) and to a phosphorylation of proteins present in an enriched apical membrane fraction (27, 28; for review see Ref . 23) . In the present report we have identified two of these phosphoproteins with molecular weights of approximately 22,000 and approximately 24,000, respectively, as guanosine 5'-triphosphate (GTP)-binding proteins, ADP-ribosylated by the Clostridium botulinum exotoxin C3 and recognized by an anti-rho polyclonal antibody but not by pan-ras monoclonal antibody; as suggested by Western-blot analysis the content of the proteins recognized by the anti-rho antibody did not alter in the membrane fraction as a function of treatment with PTH . Transient permeabilization of OK cells using streptolysin O and including the C3 exotoxin attenuated PTH-dependent inhibition of Na-Pi cotransport at hormone concentrations higher than 10(-10) M; residual PTH-dependent inhibition is equal to that observed after pharmacological activation of protein kinase A and protein kinase C, respectively . C3 exotoxin did not alter PTH-dependent generation of cAMP but modified production of IP3; it was increased at 10(-11) M and reduced at 10(-8) M PTH, respectively . It is suggested that protein kinase A may be involved in the phosphorylation of C3 exotoxin-sensitive G proteins (rho/rac) . These proteins could be involved in PTH signal transduction.

Am J Phys Med Rehabil, 1992 Apr, 71(2), 102 - 7
Diarrhea in hospitalized patients; Yablon SA et al.; Clostridium difficle has been associated with diarrhea in hospitalized patients receiving antibiotic therapy and may be nosocomially acquired . Rehabilitation hospital inpatients may require frequent antibiotic intervention and are thus at risk, although few reports of epidemics at such centers have been published . This study describes the impact of C . difficle-related disease among rehabilitation hospital inpatients . A retrospective review was conducted of all inpatients evaluated for diarrhea in two freestanding rehabilitation hospitals over a 13-month period . Clostridium difficle was determined to be the etiologic agent of diarrhea in 36% of the 33 patients, and no other etiologies were identified . Four patients were transferred to acute care because of the severity of symptoms . A total of 120 altered or canceled therapy sessions were observed to occur during the rehabilitative hospital course among studied patients, of which 90% (108) occurred during periods when patients were documented to have been symptomatic for diarrhea . Diarrhea and C . difficile-related disease thus appear to exert an important and adverse impact on the hospital course of these patients, both in terms of medical complications and therapy attendance . Physicians should therefore possess a heightened index of suspicion for C . difficile infection when evaluating patients with diarrhea in this setting . Diagnostic evaluation of rehabilitation hospital inpatients with diarrhea should include C . difficile toxin assay . If the results of the toxin assay are positive, appropriate therapy, including initiation of oral vancomycin or metronidazole and avoidance of antimotility drugs, should be instituted promptly to minimize risk of potential sequelae.

Ann Emerg Med, 1992 Apr, 21(4), 434 - 6
Clostridial myonecrosis resulting from subcutaneous epinephrine suspension injection; Hallagan LF et al.; A 32-year-old asthmatic man developed a life-threatening Clostridium perfringens infection after subcutaneous epinephrine suspension injection . Extensive surgical debridement, including forequarter amputation, hyperbaric oxygen therapy, and appropriate antibiotics were used as life-saving measures . The patient made a fair functional recovery . We could find no previously reported cases of gas gangrene following subcutaneous injection in otherwise healthy individuals . The pathogenesis and treatment of this infection are discussed.

Am J Gastroenterol, 1992 Apr, 87(4), 526 - 9
Emphysematous gastritis secondary to gastric infarction; Binmoeller KF et al.; We report a 72-yr-old female hospitalized for an upper gastrointestinal hemorrhage who developed emphysematous gastritis and gas in the portal vein . Endoscopy of the stomach revealed severe circumferential erythema, erosions, exudates, and friability of irregularly thickened proximal gastric folds . The patient became septic on the third day of hospitalization and deteriorated rapidly . Computerized tomographic scan of the abdomen revealed extensive collections of gas within the gastric wall and in the intrahepatic portal veins . Autopsy revealed severe atherosclerotic disease and a stenosis with thrombus at the origin of the celiac artery . Clostridium welchii was isolated in blood cultures prior to the patient's death . Postmortem review of endoscopic biopsies of the stomach revealed changes of incipient gastric infarction.

Surg Gynecol Obstet, 1992 Apr, 174(4), 291 - 6
Clostridial septicemia in an urban hospital; Myers G et al.; The records of 56 patients at an urban hospital who had positive blood cultures for clostridia were reviewed . Each patient was classified as immunologically normal or immunosuppressed . Data were collected on clinical history, type of clostridial bacteremia, physical and laboratory determinants of infection, therapeutic intervention, clinical course and outcome . Of the 56 patients, 22 were determined to be immunosuppressed . Among all 56 patients, 28 had a malignancy, usually gastrointestinal or hematologic in origin . Fever, leukocytosis and abdominal pain were common in both groups . Clostridial bacteremia almost always heralded clostridial septicemia . A gastrointestinal source of infection, particularly carcinoma of the colon or rectum or enterocolitis, was evident or presumed in 43 of the 56 patients . Clostridium perfringens was the most frequently isolated microorganism, but C . septicum was associated with more complications and a higher mortality rate . Septic complications and mortality were higher among the patients with immunosuppression.

J Bacteriol, 1992 Apr, 174(7), 2236 - 40
Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70; Messner P et al.; The surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was shown to contain a new type of glycan chain . Different from all known eubacterial glycoproteins, the saccharide moiety consists only of six sugar residues without any repeat sequences . Proteolytic digestion of purified S-layer glycoprotein resulted in isolation of several glycopeptide fractions . These are composed of the same hexasaccharide portion but are linked to oligopeptides of different length . One of them contains only a single amino acid . As concluded from chemical analyses and proton and carbon nuclear magnetic resonance spectroscopy of this preparation, the hexasaccharide moiety is linked via a novel O-glycosidic linkage . This is a beta-D-glucose residue linked to the phenolic hydroxyl group of tyrosine in intact S-layer glycoprotein.

J Bacteriol, 1992 Apr, 174(7), 2199 - 207
Physical and genetic mapping of the genomes of five Mycoplasma hominis strains by pulsed-field gel electrophoresis; Ladefoged SA et al.; We present the complete maps of five Mycoplasma hominis genomes, including a detailed restriction map and the locations of a number of genetic loci . The restriction fragments were resolved by field inversion gel electrophoresis or by the contour-clamped homogeneous-electric-field system of pulsed-field gel electrophoresis . All the ApaI, SmaI, BamHI, XhoI, and SalI restriction sites (total of 21 to 33 sites in each strain) were placed on the physical map, yielding an average resolution of 26 kb . The maps were constructed using three different approaches: (i) size determination of DNA fragments partially or completely cleaved with one or two restriction enzymes, (ii) hybridization analysis with purified restriction fragments and specific probes, and (iii) use of linking clones . A genetic map was constructed by hybridization with gene-specific probes for rpoA, rpoC, rrn, tuf, gyrB, hup, ftsY, the unc operon, the genes for two M . hominis-specific antigenic membrane proteins, and one gene encoding a protein with some homology to Escherichia coli alanyl-tRNA synthetase . The positions of mapped loci were partially conserved in the five strains except in one strain in which a 300-kb fragment was inverted . The numbers and order of mapped restriction sites were only partly conserved, and this conservation was restricted to certain regions . The gene order was compared with the gene order established for other bacteria and was found to be identical to that of the phylogenetically related Clostridium perfringens . The genome size of the M . hominis strains varied from 704 to 825 kb.

J Appl Bacteriol, 1992 Apr, 72(4), 309 - 14
Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak; Mahony DE et al.; Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens . Ninety-two colonies of Cl . perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis . They were also tested for the in vitro production of bacteriocin and enterotoxin . Sixteen of the 21 stool specimens were tested directly for enterotoxin . This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers . The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype.

Eur J Cell Biol, 1992 Apr, 57(2), 292 - 7
Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT; Fiorentini C et al.; Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now . It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation . Here, we describe some effects of toxin A on the leukemic T cell line JURKAT . Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin . However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent . After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter . These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment . These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation . The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.

Br J Ophthalmol, 1992 Apr, 76(4), 252 - 3
Metastatic endophthalmitis caused by Clostridium perfringens; Nangia V et al.; A case of metastatic endophthalmitis due to Clostridium perfringens originating from the biliary tract is reported . The grave visual prognosis and the importance of early detection and treatment of the primary source of infection are emphasised.

Int J Syst Bacteriol, 1992 Apr, 42(2), 312 - 4
Phylogenetic analysis of the pathogenic anaerobe Clostridium perfringens using the 16S rRNA nucleotide sequence; Canard B et al.; Clostridium perfringens, the first pathogenic clostridium examined, was placed in the nonmycoplasma subgroup of the low-dG+dC-content gram-positive cluster on the basis of the results of a phylogenetic analysis in which we used 16S rRNA comparisons . The closest relative that has been identified to date is Clostridium pasteurianum.

Rev Latinoam Microbiol, 1992 Apr-Jun, 34(2), 87 - 9
{Diagnosis of blackleg by direct immunofluorescence in bovine bone marrow}; de Guzman AM et al.; On smears of marrow-bone death bovines, presumptively diagnostic of black-leg, we have carried out the direct immunofluorescence test (IF) . We used two labelled immunosera with fluorescein isothiocyanate . The immunosera wer prepared with the reference strain 5078 of Clostridium chauvoei as following: a) a cellular extract obtained with veronal buffer 0.045 M pH = 8.6, and b) a flagellar suspension obtained by agitation with glass beads, centrifuged at 3,500 x g, and centrifuged again at 16,000 x g during 20 min at 4 degrees C . Both antigenic preparations were injected into rabbits, five doses (0.2, 0.4, 0.8, 1.6 and 3.2 ml) each by i.v . route . Control positive strain of C . chauvoei were used, and control negative strains of C . septicum and C . perfringens were used too . Of all 56 examined samples, 26 (47.5%) gave positive IF test . These results had a 100% of correlation by culture and biochemical identification.

J Am Vet Med Assoc, 1992 Apr 1, 200(7), 991 - 4
Enteric Clostridium perfringens infection associated with parvoviral enteritis in dogs: 74 cases (1987-1990); Turk J et al.; Parvovirus infection was confirmed by fluorescent antibody staining of or viral isolation from specimens of small intestine in 181 (17%) of 1,110 dogs necropsied between July 1, 1987 and Dec 31, 1990 . Clostridium perfringens was isolated from 74 (69%) of 108 dogs with parvovirus infection from which specimens of jejunum also had been obtained for culture of anaerobic bacteria . Gram-positive bacilli in association with focal to diffuse necrosis of the superficial portions of the villi were observed in histologic sections of specimens of small intestine from 56 (98%) of 57 dogs from which parvovirus and C perfringens had been identified . These findings indicate that C perfringens frequently proliferates in dogs with parvovirus infection.

J Bacteriol, 1992 Apr, 174(7), 2312 - 22
Interaction between DNA and alpha/beta-type small, acid-soluble spore proteins: a new class of DNA-binding protein; Setlow B et al.; DNA in spores of Bacillus and Clostridium species is associated with small, acid-soluble proteins (SASP) of the alpha/beta type; the presence of these proteins is a major factor in causing spore resistance to UV light, alpha/beta-type SASP did not bind to single-stranded DNA, single- or double-stranded RNA, or DNA-RNA hybrids in vitro . However, these proteins bound a variety of double-stranded DNAs and conferred protection against DNase cleavage . The binding of alpha/beta-type SASP to DNA saturated at a protein/DNA ratio (wt/wt) of 4:1 to 5:1, which is approximately 1 SASP per 4 bp . alpha/beta-type SASP-DNA interaction did not require divalent cations, was independent of pH between 6 and 8, and, for some SASP-DNA pairs, was relatively insensitive to salt up to 0.3 M . The relative affinity of alpha/beta-type SASP for different DNAs was poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than plasmid pUC19 greater than poly(dA-dT).poly(dA-dT), with poly(dA).poly(dT) giving no detectable binding . This order in alpha/beta-type SASP-DNA affinities parallels the facility with which the DNAs adopt an A-like conformation, the conformation in alpha/beta-type SASP-DNA complexes . An oligo(dG).oligo(dC) of 12 bp was bound by alpha/beta-type SASP . While a 26-bp oligo(dG).oligo(dC) bound more tightly than the 12-mer, there was no significant increase in affinity for alpha/beta-type SASP with further increase in size of oligo(dG).oligo(dC) . In contrast, binding of alpha/beta-type SASP to oligo(dA-dT).oligo(dA-dT) was minimal up to at least a 70-mer, and binding to poly(dA-dT).poly(dA-dT) was very cooperative . In addition to blocking DNase digestion, binding of alpha/beta-type SASP to DNA blocked (i) cleavage of the DNA backbone by hydroxyl radicals and orthophenanthroline-Cu2+, (ii) DNA cleavage by restriction enzymes, in particular those with specificity for GC-rich sequences; and (iii) in vitro transcription of some but not all genes . However, methylation of dG residues by dimethyl sulfate was not affected by alpha/beta-type SASP binding.

Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 931 - 6
ADP-ribosylation of small GTP-binding proteins by Bacillus cereus; Just I et al.; A human pathogenic strain of Bacillus cereus produces an exoenzyme which selectively ADP-ribosylates 20-25 kDa GTP-binding proteins in platelet membranes . Pre-ADP-ribosylation of rho proteins of human platelet membranes with Clostridium botulinum exoenzyme C3 or Clostridium limosum exoenzyme inhibits subsequent ADP-ribosylation by the exoenzyme from B . cereus indicating similar substrate specificity of the transferases . The ADP-ribosyltransferase from B . cereus reveals no immunological cross-reactivity with C . botulinum C3 and C . limosum exoenzyme.

Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 1273 - 9
The complete nucleotide sequence of the gene coding for the nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin; Tsuzuki K et al.; The structural gene for a nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin was found to exist on a 7.8 kb DNA fragment obtained from a type C phage DNA . The gene existed between the neurotoxin and hemagglutinin genes, and consisted of an 3588 bp open reading frame (1196 amino acid residues) . It was speculated that this gene and the neurotoxin gene were transcribed by the same mRNA (polycistronic transcription) in C . botulinum organisms.

Biochim Biophys Acta, 1992 Mar 24, 1130(2), 203 - 8
Cloning of HSP70 (dnaK) gene from Clostridium perfringens using a general polymerase chain reaction based approach; Galley KA et al.; A general method to clone the HSP70 gene from any species employing polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of this protein family is described . Using this method, a clone containing the entire coding sequence for the HSP70 gene from Clostridium perfringens, has been isolated and sequenced . The HSP70s from C . perfringens as well as other Gram-positive groups of bacteria contain a large deletion of 25 amino acids, near the N-terminal end, which is not seen in HSP70 from other sources.

Presse Med, 1992 Mar 21, 21(11), 509 - 14
{Prevention of acute otitis media . Amoxicillin versus glycoproteins from Klebsiella pneumoniae . Study in children under 5 years of age}; Cohen R et al.; Several studies in the English language literature have shown that continuous antibiotic prophylaxis is more effective than a placebo in preventing recurrent otitis media . In this prospective, randomized trial the effectiveness of continuous amoxicillin therapy was compared with that of glycoproteins from Klebsiella pneumoniae (GKP) . The two treatments were administered during 3 months to children aged 1 to 5 years, who had at least 3 episodes of otitis media within the 3 months preceding their admission to the trial . The trial lasted from February 1989 to July 1990 . It involved 60 children (mean age: 22.6 months) who had been seen as out-patients and had recovered from their latest episode of otitis . Thirty-three children received amoxicillin 25 mg/kg/day divided into two doses, during 3 months . Twenty-seven children were given GKP 2 tablets per day during 8 days in the 1st month and 1 tablets per day during 8 days in the following 2 months . During the study period, 14 cases of otitis media were observed in children under amoxicillin, as against 22 cases in children under GKP . When the type of day-nursery was taken into account as adjustment factor, the results showed a significantly lower percentage of failures in the amoxicillin group (P less than 0.03) . Both treatments were well tolerated (no child was excluded from the trial for intolerance) . No Clostridium difficile toxin developed in the amoxicillin group.

J Burn Care Rehabil, 1992 Mar-Apr, 13(2 Pt 2), 276 - 80
Changing flora in burn and trauma units: historical perspective--experience in the United States; Smith DJ Jr et al.; Changes in patient management, increased survival rates, and widespread use of antimicrobials have altered the flora that are found to colonize the wounds of patients with burns and trauma-related injuries . Enterococci have emerged as a prominent cause of wound infection and are capable of producing significant morbidity . Staphylococcus aureus, although it remains a common colonizer, has developed resistance to several antimicrobial agents . Recent reports suggest that the incidence of Pseudomonas infections is decreasing, whereas multiple antimicrobial resistance has emerged in a number of other gram-negative organisms that were not heretofore considered major pathogens . Candida organisms appear to be the prominent pathogens among opportunistic yeasts and fungi, and Bacteroides and Clostridium species appear to be the most common causes of infection by anaerobes . Patterns of viral infection in patients with burns and trauma-related injuries have not been investigated in detail . Overall, it appears that rates of infection and associated mortality have decreased in some patient populations; progress in this regard can be attributed to improvements in antimicrobial therapy, would management, and nutrition.

J Biol Chem, 1992 Mar 5, 267(7), 4472 - 8
Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD; Chauvaux S et al.; 12 carboxyl residues of the Clostridium thermocellum endoglucanase CelD were mutated to alanine . The specific activity of five of the mutated proteins was 1% or less that of wild type . The Ca2+ binding isotherms of these five were similar to those of wild type CelD, consistent with the fact that none of the mutated residues is observed to be directly involved in Ca2+ binding in the three-dimensional structure of the protein (Juy, M., Anit, A . G., Alzuri, P . M., Poljak, R . J., Claeyssens, M., Beguin, P., and Aubert, J.-P., manuscript in preparation) and suggesting that the mutations did not result in gross alterations of the tertiary structure . Analysis of the physico-chemical and enzymatic properties of the five purified mutated proteins and consideration of their position in the three-dimensional structure suggest that carboxyl groups identified may play roles as a general acid catalyst and a source of negative charge in stabilizing a carbonium ion intermediate . Among mutated residues, Glu-555 appears as the most likely candidate for participating in the catalytic mechanism of endoglucanase CelD.

Kansenshogaku Zasshi, 1992 Mar, 66(3), 302 - 5
{Studies on Clostridium absonum isolates from clinical specimens}; Masaki T et al.; Two strains of Clostridium absonum isolated from clinical specimens and a type strain were tested for heat resistance of spores, biochemical properties and neutralization of their lecithinase by anti-C . perfringens alpha toxin antiserum . In addition, the usefulness of RapID ANA (AMUCO) and ANIDENT (API) kits for the identification of C . absonum was examined . The results were summarized as follows: 1 . C . absonum isolates from clinical specimens were resistant against heating at 70 degrees C for 10 min and a type strain against heating at 85 degrees C for 10 min, less resistant than those of C . perfringens BP6K . 2 . C . absonum differentiated from C . perfringens in fermentation of trehalose, melibiose and raffinose, and hydrosis of esculin and starch . 3 . Lecithinase of C . absonum was not completely neutralized by anti-C . perfringens alpha toxin antiserum . 4 . Both RapID ANA and ANIDENT kits were useful for differentiation of C . absonum from C . perfringens but not for the identification of C . absonum.

Biochem Int, 1992 Mar, 26(4), 577 - 85
1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum; Ganadu ML et al.; The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR . Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented . Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.

Eur J Clin Microbiol Infect Dis, 1992 Mar, 11(3), 263 - 5
Susceptibility of anaerobic bacteria to tosufloxacin; Nord CE et al.; The in vitro activity of tosufloxacin against anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, Bacteroides spp . and fusobacteria was determined by the agar dilution method . This activity was compared with that of ciprofloxacin, piperacillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol . Tosufloxacin, imipenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested . Tosufloxacin has an antibacterial activity that warrants investigation in clinical trials.

Eur J Clin Microbiol Infect Dis, 1992 Mar, 11(3), 246 - 9
Evaluation of a new commercial Clostridium difficile toxin A enzyme immunoassay using diarrhoeal stools; Delmee M et al.; A new, commercially available enzyme immunoassay for the detection of toxin A in stool specimens, the Premier Clostridium difficile Toxin A test (Meridian Diagnostics), was evaluated using 228 diarrhoeal stool specimens . Using a cytotoxin assay on HeLa cells as the reference method, this new test resulted in a sensitivity of 88% and a specificity of 95% . Using the presence or absence of a toxigenic strain in the stools as the reference method, the sensitivity was similar to that of the cytotoxin assay (71.7+ versus 70.5%) and the overall correlation was even better (89.4% versus 82%) . The Premier Clostridium difficile Toxin A assay is rapid and easy to perform and is an excellent alternative to the usual toxin B assay.

Biol Chem Hoppe Seyler, 1992 Mar, 373(3), 123 - 32
The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase; Strobl G et al.; Purification of aldehyde oxidoreductase from C . thermoaceticum, the first detected enzyme able to reduce reversibly non-activated carboxylic acids to the corresponding aldehydes (White, H., Strobl, G., Feicht, R . & Simon, H . (1989) Eur . J . Biochem . 184, 89-96), results in the generation of multiple forms of the enzyme . The specific activities for the viologen-mediated dehydrogenation of butyraldehyde for the two main forms of the purification procedure are 530 and 450 U/mg . Two forms of the enzyme composed of alpha,beta- and alpha,beta,gamma-subunits, can be differentiated . The latter binds to red-Sepharose and can be eluted very specifically with NADPH . In contrast to the alpha,beta-types the trimeric forms also catalyse the reversible reduction of oxidised viologen with NADPH (VAPOR activity) . The dimer alpha,beta can oligomerize and the alpha,beta,gamma-trimer can easily form various oligomers or split off the gamma-subunit . The apparent molecular masses of the subunits alpha,beta and gamma are 64, 14 and 43 kDa . The alpha,beta-form reveals an apparent molecular mass of 86 kDa containing about 29 iron, 25 acid-labile sulphur, 0.8 tungsten and forms about 1 mol pterine-6-carboxylic acid by permanganate oxidation . The corresponding values of the trimer showing a mass of 300 kDa, are about 82 Fe, 54 S, 3.4 W and 2.5 pterine-6-carboxylic acid . In addition, 1.7 mol of FAD could be found which seems to be a component of the gamma-subunit . The aldehyde oxidoreductase from C . thermoaceticum and that from C . formicoaceticum (White, H., Feicht, R., Huber, C., Lottspeich, F . & Simon, H . (1991) Biol . Chem . Hoppe-Seyler 372, 999-1005) show qualitative similarities as far as the Fe, S, W and pterin content and the broad substrate specificity are concerned . However, there are also surprisingly marked differences with respect to composition and amino-acid sequence.

J Appl Bacteriol, 1992 Mar, 72(3), 244 - 51
Pseudomonas fluorescens subsp . cellulosa: an alternative model for bacterial cellulase; Hazlewood GP et al.; Pseudomonas fluorescens subsp . cellulosa, a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy . Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose . These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface . Cells of Ps . fluorescens subsp . cellulosa do not adhere to cellulose . In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose . Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps . fluorescens subsp . cellulosa and expressed in Escherichia coli, are synthesized by the pseudomonad under a variety of conditions . These enzymes appear to be post-translationally modified, probably through glycosylation . Overall, it appears that the cellulase/hemicellulase system of Ps . fluorescens subsp . cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum.

J Med Microbiol, 1992 Mar, 36(3), 190 - 7
A non-haemagglutinating form of Clostridium difficile toxin A; Kamiya S et al.; Analysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q . From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A') of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography . Analysis of toxins A and A' from cultures of C . difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A' was not merely toxin A coupled to a component of the growth medium . Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A' had the same Mr . Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A' in denaturing but non-reducing conditions, and in reducing conditions eight bands (38-175 Kda) were seen in toxin A but not A' . Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A' respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A'.

J Bacteriol, 1992 Mar, 174(6), 1848 - 53
Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition; Johnston NC et al.; The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids . Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains . Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings . When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium . The acyl chains reflected the palmitic acid content of the medium more closely . Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains . The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased . However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid . In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid . These results indicate that C . acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides . These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains . Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids . The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids.

Biochem J, 1992 Mar 1, 282 ( Pt 2), 511 - 6
Elucidation of the topological parameters of N-acetylneuraminic acid and some analogues involved in their interaction with the N-acetylneuraminate lyase from Clostridium perfringens; Zbiral E et al.; A series of neuraminic acid derivatives modified in the side chain or at C-3, C-4 or C-5 were tested as substrates of inhibitors of N-acetylneuraminate lyase (EC 4.1.3.3) from Clostridium perfringens . The results, together with Km and Ki values reported previously, indicate that the region most important for the binding of sialic acids is an equatorial zone reaching from C-8 via the ring oxygen atom to C-4 of the sugar molecule, whereas the substituents at C-9 and C-5 may be varied to a higher extent without significantly disturbing enzyme action . It is shown that stereo-electronic factors are responsible for the immediate heterolytic fragmentation of the cyclic sialic acid into pyruvic acid and 2-acetamidomannose or a related C-6 sugar.

Infect Immun, 1992 Mar, 60(3), 784 - 90
Purification and characterization of the lethal toxin (alpha-toxin) of Clostridium septicum; Ballard J et al.; Clostridium septicum lethal (alpha-toxin) was purified and found to be a basic protein (pI 8.4) of approximately 48 kDa that is both lethal and hemolytic . The alpha-toxin had a hemolytic activity of approximately 2 x 10(7) hemolytic units per mg and a 50% lethal dose of approximately 10 micrograms/kg of body weight for mice . The alpha-toxin formed concentration-dependent, sodium dodecyl sulfate-resistant aggregates of approximately 230 kDa . Mice immunized with alpha-toxin showed a significant increase in survival time over mock-immunized mice when challenged with C . septicum . Rabbit polyclonal antibody was generated against the purified toxin and was used to confirm that toxin with the same molecular weight was present in seven different C . septicum isolates . No proteins in the supernatants from cultures of Clostridium perfringens, Clostridium histolyticum, Clostridium chauvoei, or Clostridium difficile were found to react with the C . septicum alpha-toxin-specific antibody.

Infect Immun, 1992 Mar, 60(3), 1237 - 40
High-affinity binding of Clostridium perfringens epsilon-toxin to rat brain; Nagahama M et al.; 125I-epsilon-toxin showed high affinity to rat brain homogenates and synaptosomal membrane fractions, having single binding phases with dissociation constants (Kds) of 2.5 and 3.3 nM, respectively . Treatment of synaptosomal membrane fractions with pronase and neuraminidase lowered the binding of the labeled toxin, whereas treatment with trypsin and phospholipase C did not . Heating of the fractions resulted in a decrease in the binding of the toxin . These data suggest that interaction of epsilon-toxin with cell membranes in the brain is facilitated by a sialoglycoprotein . On the other hand, treatment of the membrane fractions with lipase resulted in complete loss of binding, suggesting that the interaction may require an appropriate lipid environment . These data suggest the presence of specific binding sites in brain tissue for epsilon-toxin.

Eur J Biochem, 1992 Mar 1, 204(2), 831 - 9
1H-NMR studies on partially and fully reduced 2(4Fe-4S) ferredoxin from Clostridium pasteurianum; Bertini I et al.; The ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated through 1H-NMR spectroscopy in the reduced and partially oxidized states . The 1H-NMR spectrum of fully reduced ferredoxin, obtained by addition of stoichiometric amounts of dithionite, has been characterized . One- and two-dimensional NMR saturation transfer experiments on partially reduced samples have allowed the isotropically shifted signals of the reduced form to be correlated to those of the oxidized form, for which the complete assignment of the beta-CH2 cysteinyl residues is available . In addition, observation of the 1H-NMR signals of the intermediate species with characteristic chemical shift values for each cluster allowed us to assign all the Cys beta-CH2 signals to cluster I or cluster II and to calculate the difference in redox potential between them . Starting from these results, reanalysis of the 1H-NMR features of the two clusters in the oxidized form showed that they are strikingly similar, supporting the idea of a high degree of internal symmetry between them, in agreement with crystallographic results on an homologous ferredoxin . On the other hand, the 1H-NMR properties of the two clusters in the reduced form deviate considerably from each other, suggesting that reduction of the clusters brings about different structural changes and loss of internal symmetry . A theoretical approach is reported to account for the isotropic shifts and the temperature dependence of the NMR signals of the reduced protein.

Eur J Biochem, 1992 Mar 1, 204(2), 657 - 67
The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene; Whelan SM et al.; The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR) . Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene . Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date . The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%) . The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity . In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively . Comparisons with partial amino acid sequences of the light chain of BoNT/E from C . botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case . Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity . The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.

Infection, 1992 Mar-Apr, 20(2), 58 - 60
Low levels of coagulation inhibitors in patients with Clostridium difficile infection; Aronsson B et al.; To investigate levels of coagulation inhibitors in sera from patients with Clostridium difficile-associated diarrhoea and colitis, commercially available antigen assays were used for immunochemical determination of antithrombin III, protein C and free protein S . Sera from patients with Clostridium difficile-associated diarrhoea and colitis showed significantly lowered levels of all measured inhibitors as compared to controls (Student's t test) . Protein C (mean +/- SD): 0.70 +/- 0.30 vs . 1.28 +/- 0.23, t = 6.61, p less than 0.001; antithrombin: 0.70 +/- 0.21 vs . 0.90 +/- 0.17, t = 3.12, p less than 0.01; free protein S: 0.27 +/- 0.06 vs . 0.37 +/- 0.08, t = 3.7, p less than 0.001 . Infection with C . difficile may lead to loss of coagulation inhibitors and constitutes a risk for thromboembolic complications.

Toxicon, 1992 Mar, 30(3), 323 - 30
Effect of p-chloromercuribenzoate on Clostridium perfringens beta toxin; Sakurai J et al.; p-Chloromercuribenzoate (PCMB) was shown to bind to Clostridium perfringens beta toxin . Treatment of the toxin with N-ethylmaleimide (NEM), 5,5'-dithio-bis(2-nitro-benzoic acid) (DTNB), o-iodosobenzoate (OIBA) and metal ions such as Cu2+ and Ag+ decreased the lethal activity, but PCMB did not affect the lethal activity . On the other hand, the binding of PCMB to the toxin was inhibited by DTNB and NEM in a dose-dependent manner . Furthermore, the lethal activity of beta toxin pretreated with PCMB was not blocked by treatment with NEM, DTNB, OIBA, Cu2+ and Ag+ . However, the PCMB-treated toxin treated with reduced glutathione, dithiothreitol, 2-mercaptoethanol, liver homogenate or serum from mice was inactivated by NEM.

Antibiot Khimioter, 1992 Mar, 37(3), 20 - 2
{Eremomycin in the treatment of antibiotic-associated colitis in golden hamsters}; Rukhlina AA et al.; The efficacy of eremomycin, a new glycopeptide antibiotic, was studied on a model of antibiotic-associated colitis in golden hamsters . The colitis was induced by intraperitoneal or intragastric administration of lincomycin . In a dose of 100 mg/kg administered orally once a day for 5 days eremomycin protected the animals from the lincomycin-induced colitis: some animals survived, the others died in later periods . When the animals were infected with a pathogenetic strain of Clostridium difficile followed by exposure to lincomycin the use of eremomycin produced the similar effect.

Indian J Exp Biol, 1992 Mar, 30(3), 193 - 200
Nitroimidazoles, Part XXIII--activity of satranidazole series against anaerobic infections; Nagarajan K et al.; A large number of nitroimidazoles have been examined for in vitro activity against three anaerobes - Bacteroides fragilis (Bf), a strain of Bf resistant to metronidazole (16a) and Clostridium perfringens and many found to be active . Among these may be mentioned 1-methyl-5-nitroimidazoles carrying N - bound hetetocycles at position 2, such as satranidazole 1a, 1b, 1c, 1k, 1n and 1v which are at least twice as active as metronidazole (16a), ornidazole (16b) and tinidazole (16c) . Even more active are 5-nitroimidazolyl benzimidazole 5d, -thiazolidinone 6b and thiadiazolidine dioxide 8a . Many other types of compounds derived from 1-methyl-2-amino-5-nitroimidazole are feebly active . Among 5-nitroimidazoles with a carbon substituent at position 2, 16a, 16b and 16c are equiactive while dimetridazole 14f is more active than 16a against Bf . Some 2-vinyl derivatives are very potent, with 18f and 18i being outstanding . Activity better than that of metronidazole is seen for nitroimidazooxazepines, e.g . 29d . 5-Nitroimidazoles are more active against anaerobes than 4-nitro isomers . Antianaerobic and antiamoebic activities generally run parallel in these classes of compounds . The study has led to the elaboration of the antianaerobic profile of satranidazole 1a.

Can J Microbiol, 1992 Mar, 38(3), 215 - 21
Cloning of tetracycline-resistance genes from various strains of Clostridium perfringens and expression in Escherichia coli; Saksena NK et al.; One hundred strains of Clostridium perfringens and 52 strains of other clostridia of human and animal origins were screened for tetracycline resistance . Fifty-six strains were resistant to tetracycline in the C . perfringens group . Ten strains were selected for their high level of resistance . In all of them, the tetracycline-resistance genes were found to be residing in large plasmids of about 50 kb, all showing homologies . Several tetracycline-resistance genes from plasmids of various strains of C . perfringens were cloned in plasmid pUC19 and the resistance was expressed in Escherichia coli . Hybridization analysis showed these genes to be homologous among themselves and also to tetP gene from the PCW3-type plasmid.

J Pediatr Surg, 1992 Mar, 27(3), 288 - 90; discussion 291
Recombinant human granulocyte colony-stimulating factor promotes wound healing in a patient with congenital neutropenia; Besner GE et al.; We report a patient with congenital neutropenia or Kostmann's Syndrome who suffered many complications after presenting with Clostridium septicum enterocolitis, including absence of wound healing . Because of several reports of the use of granulocyte colony-stimulating factor (G-CSF) in patients with various complications of neutropenia, we treated this patient with recombinant human (rh) G-CSF . We found that once rhG-CSF restored neutrophil counts to normal, progressive wound healing followed . Thus, rhG-CSF therapy may be useful in treating neutropenic patients with wound complications.

J Gen Microbiol, 1992 Mar, 138 ( Pt 3), 537 - 42
Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies; Tamura Y et al.; Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface . Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria . In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament . These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C . chauvoei possessing flagella . Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes . Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C . chauvoei.

Muscle Nerve, 1992 Mar, 15(3), 273 - 6
In vitro microelectrode study of neuromuscular transmission in a case of botulism; Maselli RA et al.; We performed in vitro microelectrode studies in the anconeus muscle biopsy of a 6-week-old infant intoxicated with Clostridium botulinum toxin B . The most striking abnormalities were the severe reduction of the endplate potential (EPP) quantal content and the marked variability of EPP latencies . The increased variability was often limited to a "single quantum" component of the EPP . Neither the amplitudes nor the frequencies of spontaneous miniature endplate potentials (MEEPs) were decreased . However, there was a wide range of amplitudes and frequencies of MEPPs . This unique combination of electrophysiologic findings indicates a severe presynaptic failure of neuromuscular transmission, which appears to result from an impairment of the process of synaptic vesicle release taking place after the stimulus induced influx of calcium into the motor nerve terminals.

Biochem Biophys Res Commun, 1992 Feb 28, 183(1), 107 - 13
Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755); Poulet S et al.; Recently, it has been shown that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755), isolated from cases of infant botulism, produce a botulinal neurotoxin type E (BoNT/E) . Here we have determined the nucleotide sequences of the BoNT/E genes of these two C . butyricum strains and from C . botulinum E strain Beluga . We show that the sequences of the BoNT/E genes from the two C . butyricum strains are identical and differ in only 64 positions resulting in 39 amino acid changes (97% identity at the amino acid level) from that derived from C . botulinum . Our data suggest a transfer of the BoNT/E gene from C . botulinum to the originally nontoxigenic C . butyricum strains.

Biochim Biophys Acta, 1992 Feb 28, 1130(1), 90 - 4
Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach; Rusanganwa E et al.; Using degenerate oligonucleotide primers for conserved regions of HSP60, a 0.6 kilobase fragment of Clostridium perfringens DNA was amplified by the polymerase chain reaction . The amplified fragment was used as a probe to isolate a genomic clone containing the C . perfringens HSP60 operon . The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) family of bacterial and eukaryotic proteins as well as other upstream and downstream sequences . The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone HSP60 gene/cDNA from any species.

FEBS Lett, 1992 Feb 24, 298(2-3), 185 - 7
Specific binding of nucleotides and NAD+ to Clostridium difficile toxin A; Lobban MD et al.; Binding of nucleotides, a tetrapolyphosphate, and NAD+ to purified toxin A of Clostridium difficile was determined by monitoring changes in intrinsic fluorescence following excitation at 280 nm, and recording emissions at 340 nm . Binding was specific for concentrations over the range 5 to 100 microM for ATP, GTP, and their respective non-hydrolysable analogues AMP-PNP and Gpp(NH)p, tetrapolyphosphate and NAD+.

Eur J Biochem, 1992 Feb 15, 204(1), 13 - 9
Structure of the Clostridium thermocellum gene licB and the encoded beta-1,3-1,4-glucanase . A catalytic region homologous to Bacillus lichenases joined to the reiterated domain of clostridial cellulases; Schimming S et al.; The nucleotide sequence of the Clostridium thermocellum gene licB, coding for a thermoactive beta-1,3-1,4-glucanase, has been determined . The gene is located downstream, but in opposite orientation to the beta-glucosidase gene bglA . A coding region of 1002 bp is flanked by canonical promoter and transcription terminator sequences . The primary translation product of the licB gene has a predicted molecular mass of 37,896 Da . The protein sequence can be divided into several discrete segments: an N-terminal signal peptide, a catalytic region, a segment rich in Pro and Thr residues and a C-terminal reiterated domain . The catalytic region shows close similarity to lichenases of bacilli (52-58% identity) and Fibrobacter succinogenes (35% identity), but is unrelated to barley beta-1,3-1,4-glucanases . It consists of two domains, which in the case of the F . succinogenes lichenase are arranged in reversed order to that of C . thermocellum and Bacillus lichenases . The C-terminal reiterated domain of C . thermocellum lichenase is homologous to the duplicated non-catalytic domain of endo-beta-1,4-glucanases and xylanase Z from the same organism . This domain is considered a characteristic feature of clostridial cellulases organized as multienzyme complex (cellulosome) . The beta-1,3-1,4-glucanase encoded by the licB gene might therefore be an additional enzyme component of the C . thermocellum cellulosome.

Biochem J, 1992 Feb 15, 282 ( Pt 1), 243 - 7
Sphingosine enhances platelet aggregation through an increase in phospholipase C activity by a protein kinase C-independent mechanism; Hashizume T et al.; Sphingosine (a potent inhibitor of protein kinase C) at 5-10 microM, which are concentrations lower than those that inhibit this enzyme activity, enhanced the aggregation of rabbit platelets induced by low concentrations of U46619, platelet-activating factor, thrombin and arachidonic acid, whereas H-7 and staurosporine, other protein kinase C inhibitors, failed to do so . Of the sphingosine analogues which also inhibit protein kinase C, psychosine and lyso-GM3 did not show such an enhancing effect . Sphingosine promoted both Ins(1,4,5)P3 formation and an increase in the cytoplasmic free Ca2+ concentration in response to all the agonists used . Furthermore, the hydrolytic action of exogenously added phospholipase C (from Clostridium perfringens) on platelet membrane phospholipids was dose-dependently enhanced by pretreatment of the platelets with sphingosine . These results imply that sphingosine, at relatively low concentrations, brings about hyperaggregability of the platelets by the agonists employed, probably owing to enhancement of the phospholipase C activity . Such an effect appears to be induced by a mechanism independent of protein kinase C inhibition . We suggest that sphingosine might act as a positive modulator for the stimulus-response coupling in the platelets.

J Chromatogr, 1992 Feb 7, 591(1-2), 121 - 8
Analysis of pH-dependent protein interactions with gel filtration medium; Golovchenko NP et al.; A prepacked Superose 12 HR 10/30 column was used to study the effects of elution ionic strength and pH on the chromatographic behaviour of a strong hydrophobic Clostridium thermocellum endoglucanase (1) and two weak hydrophobic proteins, Clostridium thermocellum endoglucanase C and egg white lysozyme . Ion-exclusion or ion-exchange interactions between weakly hydrophobic proteins and the gel matrix were observed at low ionic strength, depending on whether the pH of the elution buffer was higher or lower than the pI values of the proteins . These interactions were due to the presence of negatively charged groups on the surface of Superose and could be eliminated at any pH by adding electrolyte at a concentration determined by its chemical identity . The optimum results were observed with sodium sulphate at a concentration of 100 mM . The chromatographic behaviour of strong hydrophobic endoglucanase (1) on a Superose column as a function of pH was much more complex because of two interplaying effects, electrostatic and hydrophobic . Ideal size-exclusion chromatography could be achieved only in a narrow range of the conditions: first, the mobile phase must contain a weak salting-out electrolyte such as NaCl, and second, the mobile phase pH must be high enough that hydrophobic interactions between the solute and support are balanced by their electrostatic repulsion . At pH greater than pI, the retardation of endoglucanase (1) gradually increased with decreasing pH as a result of lowering of repulsive electrostatic interactions whether or not the buffer ionic strength was high . At pH less than pI a drastic increase in the capacity factor k' was observed owing to the additivity of hydrophobic and ion-exchange effects.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1992 Feb 5, 267(4), 2600 - 4
Epidermal cell differentiation inhibitor ADP-ribosylates small GTP-binding proteins and induces hyperplasia of epidermis; Sugai M et al.; Epidermal cell differentiation inhibitor (EDIN) is a recently discovered protein which inhibits terminal differentiation of cultured keratinocytes (Sugai, M., Enomoto, T., Hashimoto, K., Matsumoto, K., Matsuo, Y., Ohgai, H., Hong, Y.-M., Inoue, S., Yoshikawa, K., and Suginaka, H . (1990) Biochem . Biophys . Res . Commun . 173, 92-98) . The amino acid sequenced deduced from the EDIN gene has revealed that EDIN shares high amino acid sequence homology with the exoenzyme C3 of Clostridium botulinum (Inoue, S., Sugai, M., Murooka, Y., Paik, S.-Y., Hong, Y.-M., Ohgai, H., and Suginaka, H . (1991) Biochem . Biophys . Res . Commun . 174, 459-464), which has been shown to ADP-ribosylate the rho/rac proteins (members of the small GTP-binding protein family) . We show here that EDIN ADP-ribosylates rhoB p21 in time- and dose-dependent manners in a cell-free system . Kinetic studies of the ADP-ribosylation and peptide mapping of the reaction products of rhoB p21 by EDIN and C3 suggest that the mode of action of the ADP-ribosylation by EDIN is quite similar to that by C3 and that the ADP-ribosylation site of rhoB p21 by EDIN is presumably the same as that by C3 . Proteins in epidermal membranes and keratinocyte homogenate with Mr values of about 22,000 are ADP-ribosylated by EDIN or C3 . Treatment of cultured human keratinocytes by EDIN or C3 results in an inhibition of terminal differentiation and a stimulation of growth of the cells . Moreover, EDIN and C3 injected into adult mouse skin induce hyperplasia of epidermis . These results suggest that EDIN and C3 affect growth and differentiation of keratinocytes by ADP-ribosylation of protein(s) with a Mr of about 22,000, which may be the rho/rac proteins or related proteins.

Biochemistry, 1992 Feb 4, 31(4), 949 - 53
An organic radical in the lysine 2,3-aminomutase reaction; Ballinger MD et al.; Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K . Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor . The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine . Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine . The radical signal is complex, having multiple hyperfine transitions . The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine . The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state . Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical . The results indicate that lysine radicals participate in the rearrangement mechanism.

FEBS Lett, 1992 Feb 3, 297(1-2), 95 - 9
ADP-ribosylation by Clostridium botulinum C3 exoenzyme increases steady-state GTPase activities of recombinant rhoA and rhoB proteins; Mohr C et al.; ADP-ribosylation of recombinant rhoA and rhoB proteins by Clostridium botulinum C3 exoenzyme increased steady-state GTP hydrolysis by 50 to 80% . ADP-ribosylation and increase in GTP hydrolysis occurred at similar concentrations of C3, depended on the presence of NAD and were prevented by anti-C3 antibody or heat inactivation of C3 . In contrast, GTP hydrolysis by Ile-41 rhoA or Ha-ras, which are no substrates for the transferase, were not affected by C3 . ADP-ribosylation facilitated the {3H}GDP release and subsequently, the binding of {3H}GTP to rhoA . The data indicate that the increase in the steady-state GTPase activity by ADP-ribosylation is caused by increasing the rate of GDP release which is suggested to be the rate limiting step of the GTPase cycle of the small GTP-binding proteins.

Gastroenterology, 1992 Feb, 102(2), 416 - 23
Clostridium difficile toxin B disrupts the barrier function of T84 monolayers; Hecht G et al.; The contribution of toxin B to Clostridium difficile-associated infection is undefined . Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood . The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin . It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence . To address these questions, we examined the effect of C . difficile toxin B on the phenotype and barrier function of T84 cell monolayers . Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84) . In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters . Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function . Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques . Flux studies confirm that the permeability defect is at the level of the tight junction . We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function . There was no evidence of cell death as determined by lactate dehydrogenase release assays.

Infect Immun, 1992 Feb, 60(2), 518 - 22
Characterization of the neurotoxin isolated from a Clostridium baratii strain implicated in infant botulism; Gimenez JA et al.; Botulism is widely known to result from ingestion of food containing botulinum neurotoxin produced in situ by certain strains of Clostridium botulinum . Infant botulism caused by C . botulinum, unlike the food-borne intoxication, is the toxicoinfectious form of botulism (S . S . Arnon, p . 331-345, in G . E . Lewis, ed., Biomedical Aspects of Botulism, 1981) . The strain of Clostridium baratii implicated in infant botulism produced a neurotoxin that was neutralized with antiserum for botulinum neurotoxin serotype F (J . D . Hall, L . M . McCroskey, B . J . Pincomb, and C . L . Hatheway, J . Clin . Microbiol . 21:654-655, 1985) . We developed a procedure to culture the toxigenic C . baratii (strain 6341) in dialysis bags and a simple purification scheme (precipitation of 900-ml culture supernatant with ammonium sulfate and two anion-exchange chromatographic steps at pH 5.5 and 8.0) that yielded up to 150 micrograms of purified neurotoxin . It is an approximately 140-kDa single-chain protein and has the following sequence of amino acid residues at the N terminus: Pro-Val-Asn-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asn-Asn-Thr-Thr-Ile- Leu . Comparison of this amino acid sequence with those of the botulinum neurotoxin serotypes A, B, and E showed 40 to 50% identical residues in comparable positions . The specific toxicity of the neurotoxin, approximately 2 x 10(6) 50% lethal doses for mice per mg of protein injected, was not enhanced significantly by mild trypsinization, although the protease cleaved the neurotoxin within a disulfide loop that generated at least two primary fragments, approximately 47 and approximately 86 kDa, that remained linked by an interchain disulfide . These two fragments resembled the light and heavy chains of the well-characterized neurotoxin serotypes A, B, C, D, E, and F produced by C . botulinum.

Indian J Med Res, 1992 Feb, 96, 12 - 5
Etiology of febrile episodes in children with acute lymphocytic leukaemia; Choudhry VP et al.; In 53 children (aged 5 months to 11 yr) with acute lymphoblastic leukemia, 68 febrile episodes were investigated for determining the etiology . Microbial organisms were isolated in 33 episodes . Bacteraemia was seen in 24 patients and in three of them Clostridium species were isolated . Escherichia coli was the commonest isolate and was seen in 11 (18.6%) febrile episodes . Other common organisms isolated were Staphylococcus aureus in nine (15.2%), and Klebsiella pneumoniae and coagulase negative Staph . in 6 (10.2%) episodes each . Pseudomonas aeruginosa and Acinetobactor were isolated in 5 (8.5%) episodes each.

FEMS Microbiol Lett, 1992 Feb 1, 70(1), 69 - 72
Cloning of a Clostridium botulinum type B toxin gene fragment encoding the N-terminus of the heavy chain; Jung HH et al.; Two lambda gt11 clones of the toxin gene of Clostridium botulinum type B were identified by the monoclonal antibody specific to the heavy chain of type B toxin . Neither of the expressed fusion proteins from the lysates of lysogenic E . coli Y1089 showed any botulinal toxic activity . One of the clones hybridized to the oligonucleotide probe which was synthesized according to the amino acid sequence of N-terminus of heavy chain . The sequence analysis revealed that highly homologous regions in N-terminus of heavy chain exist among botulinum neurotoxins (type A, B) and tetanus toxin on the amino acid sequence level.

Toxicon, 1992 Feb, 30(2), 129 - 40
Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L; Baldacini O et al.; We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L . These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin . On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay . However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct . We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins . Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L . The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.

Am J Infect Control, 1992 Feb, 20(1), 11 - 5
Lack of care giver hand contamination with endemic bacterial pathogens in a nursing home; Larson E et al.; Prevalences of Clostridium difficile and multiply resistant Staphylococcus aureus (MRSA) were determined in nursing staff and residents of a 233-bed long-term care facility . Twenty of 38 (52.6%) patients in the long-term care ward and three of 69 (4.3%) in the skilled-nursing ward were colonized with MRSA; 16 of 48 (33%) patients in the long-term care ward and seven of 52 (13%) in the nursing home ward were colonized with C . difficile . None of the 79 staff members whose hands were cultured had chronic C . difficile hand carriage and MRSA was present on only three of 79 (3.9%) . Over a 6-month period, 128,000 pairs of gloves were worn . Since C . difficile and MRSA are rarely present on washed hands of care providers, appropriate handwashing and gloving should make a significant contribution to reducing the spread of these agents in long-term care facilities.

Infect Control Hosp Epidemiol, 1992 Feb, 13(2), 98 - 103
Reduction in the incidence of Clostridium difficile-associated diarrhea in an acute care hospital and a skilled nursing facility following replacement of electronic thermometers with single-use disposables; Brooks SE et al.; OBJECTIVE: To determine if the spread of Clostridium difficile-associated diarrhea is related to the use of electronic thermometers in an acute hospital and a chronic healthcare facility . DESIGN: After finding that a significant percentage (20.8%) of electronic rectal thermometer handles were contaminated with C difficile, all electronic thermometers were replaced with disposables . A before/after trial was conducted to determine if the change to disposable thermometers would reduce the incidence of C difficile-associated diarrhea . SETTING: The study took place in a 343-bed acute hospital and a 538-bed skilled nursing facility . PATIENTS: All patients who underwent routine microbiological evaluation for nosocomially acquired diarrhea over a 1-year period were included in the study . Nosocomial diarrhea was defined as 3 or more loose stools per day for 2 consecutive days and/or abdominal findings such as pain, distension, and ileus occurring 3 or more days after admission . RESULTS: During the 6-month postintervention period, the incidence of C difficile-associated diarrhea was reduced from 2.71/1,000 patient days to 1.76/1,000 patient days in the acute hospital and from 0.41/1,000 patient days to 0.11/1,000 patient days in the skilled nursing facility . The protective effect of the intervention was statistically significant for both facilities . CONCLUSIONS: Replacement of electronic thermometers with single-use disposables significantly reduced the incidence of C difficile-associated diarrhea in both acute care and skilled nursing care facilities . Data suggest that the rectal route may be important in the transmission of C difficile in these settings.

J Clin Microbiol, 1992 Feb, 30(2), 514 - 6
Comparison of five cultural procedures for isolation of Clostridium difficile from stools; Marler LM et al.; Several procedures have been described for the culture of Clostridium difficile from stool specimens . The goal of this study was to determine the effectiveness of five of these methods for the isolation of C . difficile from feces of patients suspected of having C . difficile-associated illness . A total of 564 stool specimens were cultured by using heat shock, ethanol treatment (ET), and direct plating on Carr-Scarborough cycloserine-cefoxitin-fructose agar (CCFA) with horse blood (C/S medium), BBL CCFA medium, and Remel C . difficile agar . Cytotoxin assays were performed on all specimens . A total of 113 specimens (20%) were positive for C . difficile by one or more methods . The numbers of positive cultures by using heat shock, ET, and direct plating on C/S medium, BBL CCFA medium, and Remel C . difficile agar were 79 (70%), 89 (79%), 91 (81%), 79 (70%), and 52 (46%), respectively . We concluded that ET and direct plating on C/S medium were the most effective procedures for isolating C . difficile from stool specimens and found significant variation in the performance of modified CCFA from different manufacturers.

J Clin Microbiol, 1992 Feb, 30(2), 396 - 400
Clinical illnesses associated with isolation of dysgonic fermenter 3 from stool samples; Blum RN et al.; The clinical significance of the fastidious organism DF-3 isolated from stool cultures is unclear . We sought to improve our understanding of this organism and to further define its association with human disease . Stool cultures for DF-3 were obtained from three sources: an ongoing study of enteric pathogens in patients infected with the human immunodeficiency virus, a screening procedure in which all stool samples submitted for Clostridium difficile toxin assay were cultured for DF-3, and stool samples submitted specifically for DF-3 culture . Retrospective clinical data were obtained from chart reviews of patients with positive cultures . Antimicrobial susceptibility testing and cell wall fatty acid analysis were performed for each DF-3 isolated . Eight isolates of DF-3 were obtained over a period of 8 months . All patients either had severe underlying disease or were immunocompromised, including three patients coinfected with human immunodeficiency virus and two patients with inflammatory bowel disease . The spectrum of clinical disease ranged from chronic diarrhea with a well-defined response to therapy for DF-3 to an asymptomatic carrier state . Cell wall fatty acid analysis of these isolates demonstrated a consistent pattern with a large peak of 12-methyltetradecanoate . DF-3, a fastidious gram-negative coccobacillus, can be recovered from stool cultures of immunocompromised patients by using selective media . The presence of 12-methyltetradecanoate in cell wall fatty acid analysis assists in identification . The increased use of a selective medium-(cefoperazone-vancomycin-amphotericin B) in the evaluation of diarrhea in immunocompromised hosts, including persons with inflammatory bowel disease, may better define the association of DF-3 with human gastrointestinal disease.

Antimicrob Agents Chemother, 1992 Feb, 36(2), 331 - 8
In vitro activities of three semisynthetic amide derivatives of teicoplanin, MDL 62208, MDL 62211, and MDL 62873; Biavasco F et al.; MDL 62208, MDL 62211, and MDL 62873 are three semisynthetic amide derivatives of teicoplanin (MDL 62208 is an amide of teicoplanin aglycone, MDL 62211 is an amide of the teicoplanin A2 complex, and MDL 62873 is the corresponding derivative of peak A2-2 of the complex) . The three semisynthetic glycopeptides were evaluated for in vitro antibacterial activity in comparison with the parent drug (teicoplanin) and vancomycin . A variety of gram-positive bacteria of clinical origin, whose species were carefully determined and that included 428 staphylococci (207 methicillin susceptible and 221 methicillin resistant), 41 streptococci, 82 enterococci, 43 strains of Listeria monocytogenes, 10 JK coryneform bacteria, and 67 anaerobes belonging to the genera Clostridium, Propionibacterium, Peptostreptococcus, and Eubacterium, were tested . The only resistances to MDL 62208, MDL 62211, and MDL 62873 were encountered with vancomycin- and teicoplanin-resistant enterococci . All of the other test strains, including some teicoplanin-resistant coagulase-negative staphylococci of the species Staphylococcus haemolyticus and Staphylococcus epidermidis, were highly susceptible to the three teicoplanin amides . Only minor differences in activity were observed among MDL 62208, MDL 62211, and MDL 62873, whereas the three experimental compounds were usually found to be more potent than teicoplanin or vancomycin (especially against staphylococci, with differences mostly ranging from 2- to 16-fold) . The MBC-to-MIC ratios varied depending on the organisms, with the highest ratios usually observed for enterococci and listeriae . Overall, the MBC-to-MIC ratios yielded by the teicoplanin analogs were slightly greater than those yielded by teicoplanin or vancomycin.

Biotechnology (N Y), 1992 Feb, 10(2), 190 - 5
Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824; Mermelstein LD et al.; We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli . Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B . subtilis/C . acetobutylicum shuttle vector . Plasmid pFNK1 was used because shuttle vectors that function in E . coli were unable to electrotransform ATCC 824 unless they became deleted in the E . coli-plasmid regions . The difficulties with shuttle vectors that function in E . coli are probably due to the presence of a restriction endonuclease in ATCC 824 . This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E . coli plasmids but occurs infrequently in pFNK1 and C . acetobutylicum genes . Cloning of genes in C . acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.

J Protein Chem, 1992 Feb, 11(1), 99 - 107
Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases; Mallya SK et al.; The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class II Clostridium histolyticum collagenases (CHC) have been investigated . To facilitate this study, radioassays developed previously for the hydrolysis of these {3H}acetylated collagens by tissue collagenases have been adapted for use with the CHC . While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events . The individual kinetic parameters kcat and KM have been determined for the hydrolysis of all three collagens by both class I and class II CHC . The specific activities of these CHC toward fibrillar type I and III collagens have also been measured . In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form . The values of the kinetic parameters kcat and KM for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates . Thus, the widely held view that the CHC are more potent collagenases is not strictly correct . As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.

J Protein Chem, 1992 Feb, 11(1), 83 - 97
Identification of Clostridium histolyticum collagenase hyperreactive sites in type I, II, and III collagens: lack of correlation with local triple helical stability; French MF et al.; The class I and II Clostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens . The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus . The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments . Both fragments are next shortened by removal of a 3000 fragment . These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites . Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate . Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens . N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the alpha 1(I), alpha 2(I), alpha 1(II), and alpha 1(III) collagen chains . An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability . The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations . The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.

Diabetologia, 1992 Feb, 35(2), 109 - 15
Effect of phospholipase treatment on insulin receptor signal transduction; Zoppini G et al.; To study the role of membrane lipids in signal transduction by the insulin receptor, we have studied the effect of phospholipase C (Clostridium perfringens) and a phosphatidylinositol-specific phospholipase (Staphylococcus aureus) on insulin binding, a function of the alpha-subunit, and tyrosine kinase activity, a function of the beta-subunit in IM-9 lymphocytes and NIH 3T3 fibroblasts transfected with the human insulin receptor . Treatment of the cells with phospholipase C at concentrations up to 3.4 U/ml did not affect specific insulin binding, but reduced insulin-stimulated receptor phosphorylation by 50% . This effect of phospholipase C was observed within 10 min of treatment and occurred with no change in the basal level of phosphorylation . Pre-treatment of cells with insulin for 5 min prior to enzyme addition prevented any change in kinase activity . Insulin-stimulated phosphorylation of pp 185, the presumed endogenous substrate for the insulin receptor kinase, was also reduced following phospholipase C treatment, with an almost complete loss of insulin stimulation after exposure of cells to enzyme at concentrations as low as 0.6 U/ml . In contrast to these effects of phospholipase C on intact cells, receptor autophosphorylation was not affected in insulin receptors purified on wheat germ agglutinin-agarose from phospholipase C treated cells . Likewise, the phospholipase C effect was reduced by the addition of phosphatidylcholine, but not by the addition of the protease inhibitors, aprotinin and phenylmethylsulfonyl fluoride, to the incubation indicating its dependence on phospholipid hydrolysis . Treatment of cells with the phosphatidylinositol-specific phospholipase C did not affect any of the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)

N Z Med J, 1992 Jan 22, 105(926), 11 - 2
Antimicrobial susceptibility of anaerobic bacteria in Auckland 1987-90; Henderson G et al.; The antimicrobial susceptibility of 292 clinical isolates of anaerobic bacteria was determined by a standard agar dilution method . Metronidazole was the most active agent with only one Bacteroides fragilis, two anaerobic cocci, and Propionibacterium acnes being resistant . For B fragilis itself and other members of the B fragilis group: 35/35 (100%) and 43/44 (98%) respectively, were susceptible to amoxycillin-clavulanic acid; 47/47 (100%) and 55/56 (98%) respectively, were susceptible to cefoxitin; and 20/28 (71%) and 7/25 (28%) respectively, were susceptible to ceftriaxone . All four agents and penicillin were almost always active against anaerobic cocci and Fusobacterium species . All agents were active against clostridium isolates except for cefoxitin where only 47/57 (82%) were susceptible . These results allow comparison with isolates from other locations and may be considered when choosing an antimicrobial agent for prophylaxis or therapy of anaerobic infections.

Dtsch Med Wochenschr, 1992 Jan 17, 117(3), 91 - 5
{A lethal course in pseudomembranous enterocolitis during the parenteral administration of vancomycin and imipenem}; Arning M et al.; A 48-year-old woman required mechanical ventilation after aortic valve replacement for decompensated aortic valve stenosis when bleeding complications developed and rethoracotomy had to be performed . Acute renal failure necessitated haemodialysis . Septic fever of unknown aetiology failed to respond to oxacillin, cefotaxim and tobramycin . The endotracheal cannula and central venous catheter were changed on the 24th postoperative day and the antibiotic treatment altered to 250 mg imipenem and 125 mg vancomycin three times daily intravenously . The fever soon subsided, but recurred on the 32nd postoperative day, accompanied by increasing leucocytosis . The patient was obstipated but had no intraabdominal signs . Four days later ultrasonography demonstrated thickening of the intestinal wall and coloscopy showed typical pseudomembranous colitis . Intestinal contents were positive for Clostridium difficile toxin . Despite immediate rectal and intragastric administration of 250 mg vancomycin four times daily the patient died of pseudomembranous colitis, confirmed at autopsy . The case demonstrates that vancomycin cannot always prevent the development of pseudomembranous colitis.

J Biol Chem, 1992 Jan 15, 267(2), 897 - 900
Interaction of ferredoxin with carbon monoxide dehydrogenase from Clostridium thermoaceticum; Shanmugasundaram T et al.; Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E . (1986) Trends Biochem . Sci . 11, 14-18) . Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA . Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G . (1985) J . Biol . Chem . 260, 3970-3977) . In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex . In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide . The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex . The native CODH and cross-linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage . By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH-ferredoxin cross-linked complex . The peptide was identified with residues 229-239 of the alpha-subunit of CODH.

J Am Vet Med Assoc, 1992 Jan 15, 200(2), 214 - 7
Clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: 13 cases (1979-1982); Blackwell TE et al.; Enterotoxemia attributable to Clostridium perfringens type D in goats is difficult to diagnose because of a lack of specific clinical signs or postmortem lesions, on which to base the diagnosis . This report describes the clinical signs, postmortem lesions, and clinical responses to treatment and vaccination in 4 goat herds, in which a diagnosis of enterotoxemia was confirmed . Four clinical cases had the diagnosis confirmed on the basis of signs of diarrhea or sudden death and the isolation of C perfringens and epsilon toxin from the feces at the time of admission . The 10 necropsy cases were diagnosed on the basis of the isolation of C perfringens (not typed) or epsilon toxin from the intestinal contents of goats that died with clinical signs compatible with enterotoxemia and without lesions associated with a second serious disease . Enterocolitis was the most consistent lesion reported at necropsy in the 10 goats with enterotoxemia . Ovine enterotoxemia vaccines were of limited value in preventing enterotoxemia . These observations imply that naturally induced enterotoxemia in goats involves a different pathophysiologic mechanism than that associated with enterotoxemia in sheep.

J Biol Chem, 1992 Jan 5, 267(1), 50 - 5
ADP-ribosylation of gelsolin-actin complexes by clostridial toxins; Wille M et al.; ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied . Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin . The Km for NAD (4 microM) was identical for both substrates . C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol . In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin . The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated . Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex . ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex . When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min . According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex . This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex.

Gene, 1992 Jan 2, 110(1), 9 - 16
Cloning and characterization of a gene from Bacillus stearothermophilus var . non-diastaticus encoding a glycerol dehydrogenase; Mallinder PR et al.; A 4.1-kb EcoRI fragment which includes the gene (gldA) encoding a glycerol dehydrogenase (G1DH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus stearothermophilus var . non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to Escherichia coli glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD) mutants . Sequencing suggests that the gldA gene is likely to be monocistronic and encodes a protein of 39450 Da . The deduced amino acid composition and sequence of G1DH reveals that the protein is extremely similar to a characterized metal-dependent NAD-dependent G1DH from B . stearothermophilus RS93 . The enzyme has limited homology to the iron-activated alcohol dehydrogenase of Zymomonas mobilis and the butanol dehydrogenase of Clostridium acetobutylicum.

J Antimicrob Chemother, 1992 Jan, 29(1), 57 - 67
Multicentre study on antibiotic susceptibilities of anaerobic bacteria to cefoperazone-sulbactam and other antimicrobial agents; Clark RB et al.; The antibiotic susceptibilities of 241 anaerobic bacteria recovered from six geographic sites in North America were tested by agar dilution to cefoperazone-sulbactam and other drugs . Of the 189 Bacteroides fragilis group isolates, only one was resistant to cefoperazone-sulbactam (0.5%) or ampicillin-sulbactam (0.5%), and none was resistant to ticarcillin-clavulanate or chloramphenicol . No resistance to cefoperazone-sulbactam was observed among the other Bacteroides spp., Clostridium spp., or Peptostreptococcus spp . Resistance to cefoperazone-sulbactam is not commonly observed against anaerobic bacteria recovered from different geographical sites across North America.

Am J Med, 1992 Jan, 92(1), 53 - 60
Anaerobic bacteremia: incidence, patient characteristics, and clinical significance; Lombardi DP et al.; PURPOSE: In the 1970s, blood culture for obligate anaerobic bacteria became routine in most United States hospitals . Since then, various authorities have reported isolation of obligate anaerobes in 5% to 25% of blood cultures . Our experience suggests a much lower frequency; therefore, we retrospectively assessed the occurrence and significance of these cultures at our institutions . PATIENTS AND METHODS: Sixty-six patients at the University of Michigan Hospitals (UMH) and nine patients at the Ann Arbor Veteran's Administration Medical Center (AAVAMC) had one or more blood cultures positive for an obligate anaerobe between July 1, 1987, and December 31, 1988 . Their medical records were reviewed retrospectively . RESULTS: The proportion of positive blood cultures yielding obligate anaerobes was 3.2% at the UMH and 1.8% at the AAVAMC . The incidences of clinically significant anaerobic bacteremia at the two hospitals were 0.68 and 0.54 cases per 1,000 patient admissions . Among the 40 patients from whom significant isolates were obtained, 15 (38%) had a fatal outcome . Bacteroides and Clostridium species accounted for 90% of the isolates and all of the fatal cases . The source for anaerobic bacteremia was usually obvious; 30 of the 40 patients were given empiric antibiotic therapy for anaerobes . The gastrointestinal tract was the source in two thirds of the cases and was clearly implicated as the source of 80% of the fatal bacteremias . CONCLUSIONS: The frequency of anaerobic bacteremia in our hospitals is much lower than was suggested in several large studies during the 1970s, probably reflecting a real decline in the incidence . The clinical features of our cases are similar to those of previous studies, and the mortality is still high despite the use of antibiotics effective against anaerobes . Since most patients were thought to have anaerobic infections at the time that cultures were obtained, they were usually treated empirically . Subsequent blood cultures positive for anaerobes infrequently influenced clinical management.

J Bacteriol, 1992 Jan, 174(2), 601 - 9
Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis; Yother J et al.; Analysis of the sequence for the gene encoding PspA (pneumococcal surface protein A) of Streptococcus pneumoniae revealed the presence of four distinct domains in the mature protein . The structure of the N-terminal half of PspA was highly consistent with that of an alpha-helical coiled-coil protein . The alpha-helical domain was followed by a proline-rich domain (with two regions in which 18 of 43 and 5 of 11 of the residues are prolines) and a repeat domain consisting of 10 highly conserved 20-amino-acid repeats . A fourth domain consisting of a hydrophobic region too short to serve as a membrane anchor and a poorly charged region followed the repeats and preceded the translation stop codon . The C-terminal region of PspA did not possess features conserved among numerous other surface proteins, suggesting that PspA is attached to the cell by a mechanism unique among known surface proteins of gram-positive bacteria . The repeat domain of PspA was found to have significant homology with C-terminal repeat regions of proteins from Streptococcus mutans, Streptococcus downei, Clostridium difficile, and S . pneumoniae . Comparisons of these regions with respect to functions and homologies suggested that, through evolution, the repeat regions may have lost or gained a mechanism for attachment to the bacterial cell.

Infect Immun, 1992 Jan, 60(1), 71 - 8
Production, purification, and characterization of botulinolysin, a thiol-activated hemolysin of Clostridium botulinum; Haque A et al.; A hemolysin, botulinolysin, produced by Clostridium botulinum was purified to homogeneity and characterized . First, a strain of C . botulinum type C, strain C-203 Tox, which produced a large amount of hemolysin, was selected, and optimal culture medium and conditions for its production of hemolysin were determined . The hemolysin produced in the culture supernatant of this strain under optimal conditions was purified by a combination of ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, Sephadex G-75 gel permeation chromatography, and SP-Toyopearl 650 M cation-exchange column chromatography, with a recovery of 12% . The purified hemolysin gave a single protein band in polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulfate (SDS) . The protein in this band in PAGE with SDS was estimated to have a molecular weight of 58,000 and was immunostained with a neutralizing monoclonal antibody . In PAGE without SDS, the hemolytic activity corresponded in position to the single protein band . The pI of the hemolysin was 8.4 . Amino acid analysis of the purified hemolysin indicated the presence of four half-cystine residues per molecule . The purified hemolysin had a specific activity of 2,100 hemolytic units per microgram of protein on rabbit erythrocytes . It was activated by SH compounds, inhibited by cholesterol, and heat labile . The optimum pH for hemolysis was 6.0 to 7.0 . Rabbit, human, and guinea pig erythrocytes were the most susceptible to the hemolysin, while sheep, mouse, rat, and chicken erythrocytes were much less susceptible . The purified hemolysin had a lethal effect in mice and was cytotoxic for some cultured cells: its 50% lethal dose in mice was 310 ng, and its 50% cytotoxic dose for Vero cells was 120 ng/ml.

Infect Immun, 1992 Jan, 60(1), 102 - 10
Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli; Hunter SE et al.; The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined . Some differences between this sequence and the previously published sequence (A . S . Bhown and A . F . S . A . Habeeb, Biochem . Biophys . Res . Commun . 78:889-896, 1977) were found . A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C . perfringens type B . The gene encoded a protein with a molecular weight of 32,981 . Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified . The gene was expressed in E . coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein . Downstream of etx, two overlapping open reading frames were identified . Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001 . Southern hybridization experiments indicated that the etx gene was found only in C . perfringens types B and D, the types which produce epsilon-toxin.

Am J Gastroenterol, 1992 Jan, 87(1), 140 - 2
Clostridium cadaveris: an unusual cause of spontaneous bacterial peritonitis; Herman R et al.; Bacterial peritonitis has been known to complicate severe liver disease . Aerobic organisms are responsible for the vast majority of cases, whereas anaerobic bacteria are responsible for less than 5% of all cases reported in the literature . We now report a case of Clostridium cadaveris anaerobic bacterial peritonitis in a 58-yr-old female, an organism that to our knowledge has not been previously implicated as an infectious agent in this entity.

Curr Top Microbiol Immunol, 1992, 175, 115 - 31
Clostridium botulinum C3 ADP-ribosyltransferase; Aktories K et al.; C3 and C3-like ADP-ribosyltransferases modify the low-molecular-mass GTP-binding proteins Rho and Rac . ADP-ribosylation occurs in asparagine-41, which is located in the putative effector region of these highly conserved regulatory proteins . First studies indicate that the Rho proteins are somehow involved in the regulation of cytoskeletal proteins, e.g., microfilament proteins . Although the precise mechanism of the interaction of the C3 substrate with cytoskeletal elements is unclear, it appears that the ADP-ribosylation by C3 renders the GTP-binding protein biologically inactive . Thus C3 and/or C3-like ADP-ribosyltransferases may be useful instruments with which to study the physiological functions of its eukaryotic substrates . Moreover, those studies may help to elucidate whether these exoenzymes are of pathophysiological and pathogenetic relevance in diseases caused by clostridia producing these agents.

Farmaco, 1992 Jan, 47(1), 47 - 61
Comparative sensitivity study of Clostridium perfringens towards 16 antibiotics from different classes; Rival Y et al.; A series of 16 antibiotics from different classes (5-nitro imidazoles, beta-lactams, cyclins, macrolids, chloramphenicol) was examined for their bacteriostatic and bactericidal activities on 45 strains of Clostridium perfringens, with determination of MIC and MBC values . Several techniques of multivariate analysis were used in order to visualize differentiations in sensitivity profiles: Principal component analysis (PCA) and preferentially Correspondence factorial analysis (CFA) . This approach revealed the efficacy of antibiotics from both general and family classifications.

Prog Drug Res, 1992, 38, 19 - 28
In vitro properties of the newer quinolones; Nakanishi N et al.; Table 5 summarizes the activity of the newer quinolones against various bacteria including intracellular bacteria and the other microorganisms . In this table, the overall MIC ranges of NFLX, ENX, OFLX, and CPFX, for susceptible isolates of each bacteria are schematically presented . The newer quinolones are considered to have sufficient activity against gram-negative enteric bacteria, N . gonorrhoeae, and H . influenzae and Legionella spp . Since OFLX and CPFX show only moderate activity against staphylococci, streptococci, and P . aeruginosa, improvement is expected . As shown in Table 3, TFLX and the recent investigational quinolones such as SPFX and KB-5246 show higher and promising activity against gram-positive bacteria . However, further studies are needed with longer periods to indicate whether these newer agents will be able to stop the increase of quinolone-resistant staphylococci . Furthermore, the activity of the newer quinolones against obligate anaerobes such as clostridia and bacteroides are considered to be insufficient for clinical use . Whether it will be possible to synthesize quinolones with anti-anaerobic activity sufficiently for clinical treatment is uncertain . Although Mycobacterium tuberculosis is susceptible to the newer quinolones, other mycobacteria are somewhat less susceptible to this class of agents . In addition, the newer quinolones have adequate activity against mycoplasma, chlamydia and rickettsia . From a microbiological viewpoint the prospects for the newer quinolones would be primarily to find agents that have higher anti-staphylococcal and anti-streptococcal activity . Secondly, agents possessing superior activity against obligate anaerobes such as Bacteroides spp . and Clostridium spp . are expected to synthesize . Furthermore, it may be possible to synthesize compounds that are sufficiently active for clinical use against atypical mycoplasma, chlamydia, and rickettsia.

Microbiol Immunol, 1992, 36(3), 213 - 20
Cloning and whole nucleotide sequence of the gene for the light chain component of botulinum type E toxin from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike; Fujii N et al.; Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike . The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII . The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously . The cleavage site of L and H components of type E toxin was presumed to be Arg-422 . In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component . On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.

Antimicrob Agents Chemother, 1992 Jan, 36(1), 198 - 201
Susceptibilities of anaerobic bacteria isolated from animals with ovine foot rot to 28 antimicrobial agents; Piriz S et al.; The agar dilution method was used to determine the inhibitory activities of 28 antimicrobial agents against 35 strains of the genus Peptostreptococcus, 4 strains of the species Peptococcus niger, 20 strains of the species Megasphaera elsdenii, 7 strains from the species Acidaminococcus fermentans, 8 strains of the genus Clostridium, 11 strains of the genus Eubacterium, and 1 strain of the species Propionibacterium acidipropionici, all of which were isolated from 125 clinical cases of ovine foot rot between January 1987 and December 1988 . The three unreidopenicillins studied proved to be the most active antimicrobial agents, with a high percentage of strains being susceptible at a concentration of 64 micrograms/ml . Penicillin G, ampicillin, and the three cephalosporins studied also had good activity . Fosfomycin showed a high degree of activity among the 116 anaerobic bacteria tested.

Microbiol Immunol, 1992, 36(2), 131 - 8
Separate isolation of Clostridium difficile spores and vegetative cells from the feces of newborn infants; Miyazaki S et al.; A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces . The ratio of C . difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C . difficile . C . difficile spores alone were detected in twenty-one samples (75%) of C . difficile-positive Twenty-eight specimens . Only 10.7% (3/28) C . difficile vegetative cells alone were detected . C . difficile spores alone were detected in one of nine healthy adults . These collective results offer potential explanations for high frequent isolations of C . difficile from newborn infants without occurrence of pseudomembranous colitis.

Microbiol Immunol, 1992, 36(1), 29 - 34
Clostridium botulinum type C in healthy swine in Japan; Yamakawa K et al.; Healthy cattle and swine bred in a district of Japan were examined for the presence of Clostridium botulinum in their liver . Liver specimens were cultivated in chopped meat-glucose medium and the cultures were examined for botulinum toxin . In cattle, none of the cultures of 100 liver specimens yielded the toxin . In swine, however, C1 or C2 toxin was demonstrated in 8 of 100 liver specimens from 36 farms . One of the five farms where the carrier-state swine were present was surveyed for about 2 years to determine whether the carrier-state was transient or resident . C . botulinum type C was found in swine livers and feces, and environmental specimens at extremely high rates during the surveillances, with 76% of specimens yielding botulinum toxin following the culture . These data suggest that it is not uncommon for healthy swine to carry C . botulinum type C in the liver and that there is a close relationship between C . botulinum carrier-state in swine and the presence of this organism in their raising environments . In 20 cattle and 20 swine suffering from parturient paresis of unknown etiology no evidence for involvement of C . botulinum type C was obtained.

Can J Microbiol, 1992 Jan, 38(1), 81 - 3
Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction; McMillin DE et al.; A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile . A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C . difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains . This sensitive and specific multiplex polymerase chain reaction for C . difficile toxins may prove to be a valuable diagnostic procedure.

Antonie Van Leeuwenhoek, 1992 Jan, 61(1), 35 - 41
Purification and properties of an extra cellular xylanase enzyme of Clostridium strain SAIV; Murty MV et al.; An extracellular xylanase enzyme fraction A from a mesophilic Clostridium strain SAIV was purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration and DEAE-Sephadex A-50 ion exchange . The xylanase exhibited a molecular weight of approximately 30,000 and it was stable upto 55 degrees C with an optimum temperature of 50 degrees C . It was most stable between pH 5-7, with an optimum pH of around 6 . The Km value was 7.0 mg.xylan ml-1 and Vmax was 36 mumol.xylose liberated mg-1 min-1 . Carboxymethyl cellulose, filter paper cellulose and 4-p-nitrophenyl beta-D-xylopyranoside were not hydrolysed . The specific activity of xylanase fraction A (9.8 U mg-1) is 2-10 fold higher than the specific activity of xylanase in other mesophilic, xylanolytic, obligate anaerobic bacteria . A minor fraction of xylanase activity designated as xylanase B was also obtained supporting the view that the multiplicity of xylanases is common in microorganisms.

Mikrobiyol Bul, 1992 Jan, 26(1), 70 - 6
{Botulism: a case report}; Ural AU et al.; Botulism is an acute form of poisoning that results from ingestion of a toxin produced by Clostridium botulinum . Botulism toxin causes their major effect by blocking neuromuscular transmission in autonomic and motor nerve terminals . Guillain Barre Syndrome, Myasthenia Graves, Lambert Eaton Myasthenic Syndrome, acute poliomyelitis and diphtheria must be considered in the differential diagnosis . Electrodiagnostic studies have been shown to be of value in differentiating botulism from other paralytic diseases . Identification of the toxin in the patients serum is diagnostic . The treatment of botulism is mainly supportive . In this study we have discussed a patient who was treated in our clinic as a botulism from unknown source, the differential diagnosis from other paralytic diseases.

Avian Dis, 1992 Jan-Mar, 36(1), 59 - 62
Effects of Eimeria brunetti infection and dietary zinc on experimental induction of necrotic enteritis in broiler chickens; Baba E et al.; Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis . Inoculation with sporulated E . brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens . Feed was supplemented with zinc at 1000 ppm . Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc . Coccidial lesion scores were also highest in that group . Birds infected with E . brunetti and C . perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E . brunetti only, regardless of zinc supplementation . Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation . Zinc was tested for effects of alpha toxin production in vitro . In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin . Addition of zinc partly protected the toxin from the action of trypsin.

Eur J Clin Microbiol Infect Dis, 1992 Jan, 11(1), 40 - 3
Clostridium difficile toxin-induced reactive arthritis in a patient with chronic Reiter's syndrome; Cope A et al.; The first case of Clostridium difficile toxin-induced reactive arthritis in a patient with chronic Reiter's syndrome is described and compared with previous cases of reactive arthritis associated with this organism . This case demonstrates how distinct clinical manifestations may develop at different times in Reiter's syndrome, according to the infecting organism . Diagnostic terminology is discussed in this context . Clostridium difficile should now be considered a firmly established cause of reactive arthritis.

Proteins, 1992 Jan, 12(1), 75 - 86
Subunit assembly and active site location in the structure of glutamate dehydrogenase; Baker PJ et al.; The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227 . Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft . One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry . The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed . Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains . Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring . This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125 . Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.

Appl Environ Microbiol, 1992 Jan, 58(1), 418 - 20
Specific detection of Clostridium botulinum type B by using the polymerase chain reaction; Szabo EA et al.; The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B . Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin . Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C . botulinum type B isolates reacted with the radiolabeled internal probe . As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.

Microbiol Immunol, 1992, 36(6), 603 - 13
Role of the upstream region containing an intrinsic DNA curvature in the negative regulation of the phospholipase C gene of Clostridium perfringens; Toyonaga T et al.; The phospholipase C (alpha-toxin) gene (plc) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the plc gene were examined in Escherichia coli JM109 . When the 0.7-kb region just upstream of the putative -35 site of the gene was deleted, production of phospholipase C increased approximately 10-fold . Northern blot hybridization analysis of the plc transcript showed that the upstream region inhibited transcription from the plc promoter . Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5-6 tracts between positions -66 and -40 of the plc gene . A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plc promoter is a locus of sequence directed DNA-bending . Nested deletions of the upstream region were created from its 5' end by exonuclease III and the effects of deletions on the expression of the plc gene were examined . When the 77-bp fragment containing the two (dA)5-6 tracts were deleted, phospholipase C production increased markedly . These results indicate that the intrinsic DNA curvature upstream of the plc promoter is involved in the negative regulation of the plc gene transcription.

Microbiol Immunol, 1992, 36(6), 583 - 91
Prevalence of Clostridium botulinum type E and coexistence of C . botulinum nonproteolytic type B in the river soil of Japan; Yamakawa K et al.; Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum . Type E organism was prevalently shown throughout the whole river systems including upper part; detection rates of type E toxin in soil culture ranged from 33 to 82% . This type was also detected in soil of adjacent mountainous district . Type B and C toxins were detected at 7% and 9% of the sites examined, respectively . C . botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples . These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C . botulinum nonproteolytic type B exists in the soil of Japan.

Microbiol Immunol, 1992, 36(5), 523 - 7
An activity which restores theta toxin activity in some theta toxin-deficient mutants of Clostridium perfringens; Imagawa T et al.; Group a mutants of Clostridium perfringens are deficient in theta toxin but release a dialyzable substance ("substance A"), which restores theta toxin activity to group b mutants, into a culture supernatant; group b mutants are defective in "substance A" release . "Substance A" activity appeared in the exponentially growing phase of group a mutants and disappeared in the stationary phase . "Substance A" activity was most stable at pH 5.0 and 0 C and even increased threefold in the first 5 hr, but gradually decreased during the following 15 hr . It was quickly inactivated at neutral and higher pHs at 0 C.

J Basic Microbiol, 1992, 32(2), 99 - 105
Immunological homology among azoreductases from Clostridium and Eubacterium strains isolated from human intestinal microflora; Rafii F et al.; Azoreductases from several anaerobic intestinal bacteria have been shown to reduce azo dyes to carcinogenic aromatic amines . To evaluate the structural similarities of azoreductases from four species of Clostridium and one species of Eubacterium, a polyclonal antibody against purified Clostridium perfringens azoreductase was generated in rabbits . This antibody inhibited the azoreductase activity of all five bacteria tested . ELISA showed different degrees of binding of the antibody to various species of bacteria . In a Western blot, the antibody reacted with the purified azoreductases from all four Clostridium species and the Eubacterium species . These results demonstrate that the azoreductases from the bacteria tested share similar antigenic domains, which are probably located in the active site of the enzyme . Azoreductases from these intestinal bacteria are similar enough to be considered as a single group of enzymes with respect to their functions and antigenicity.

Arch Microbiol, 1992, 157(5), 395 - 401
Creatinine and N-methylhydantoin degradation in two newly isolated Clostridium species; Hermann M et al.; With N-methylhydantoin (NMH) as the main organic substrate, two strictly anaerobic spore forming Gram-positive bacterial strains were isolated from sewage sludge . These strains, named Clostridium sp . FS23 and Clostridium sp . FS41, totally degraded NMH, via N-carbamoylsarcosine (CS) and sarcosine as intermediates . Strain FS23 grew also with creatinine, which was converted to NMH by creatinine iminohydrolase (EC 3.5.4.21) . This enzyme was formed at high rates with all substrates tested . Cytosine and 5-fluorocytosine were not utilized as substrates by creatinine iminohydrolase preparations purified to a homogeneity of 98% . NMH amidohydrolase (NMHase) and N-carbamoylsarcosine amidohydrolase (CSHase) turned out to be inducible in both strains . Other than in aerobic organisms, NMHase from these two isolated did not require ATP for enzymatic activity . SH-group protecting agents were not necessary for stability.

Arch Microbiol, 1992, 157(3), 249 - 57
Clostridium neopropionicum sp . nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway; Tholozan JL et al.; Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters . It was the first example of propionic fermentation from ethanol . Morphologic and physiologic characterizations of the strain are presented here . This strain is described as type strain of a new species, Clostridium neopropionicum sp . nov . Whole cells of strain X4 ferment {1-13C} ethanol and CO2 to {2-13C} propionate, {1-13C} acetate and {2-13C} propanol, suggesting the absence of a randomizing pathway during the propionate formation . Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate . Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation . The same pathway may be used for the degradation of lactate or acrylate to acetate.

Zentralbl Mikrobiol, 1992, 147(1-2), 71 - 9
Antibacterial activity of N-{2-(dodecanoylmethylamine)ethyl}-alkyl dimethylammonium bromides; Ciganekova V et al.; The study contains the results of determination of antibacterial activity of the newly synthesized series of N-{2-(dodecanoylmethylamine)ethyl}-alkyldimethylammonium bromides . The efficiency of the compounds has been characterized towards three species of the genus Clostridium such as Clostridium perfringens, type A, Clostridium bifermentans, Clostridium sporogenes and one strain of the genus Lactobacillus such as Lactobacillus yamanashiensis . The antibacterial activity of the studied compounds has been tested in different time intervals . Out of seven investigated compounds, four compounds exerted the pronounced antibacterial activity on Clostridia (CMIC equals to 0.0039-0.0078 mmol/litre) and three compounds influenced the growth of Lactobacillus yamanashiensis (MIC equals to 0.004-0.006 mmol/litre) . The most effective compound was found to be n-octyl derivative . These compounds were more effective towards the tested Clostridia strains than the comparing standard disinfectant such as Ajatin.

Folia Microbiol (Praha), 1992, 37(2), 157 - 8
Effect of pH on the stability of type-C toxin of Clostridium botulinum; Halouzka J et al.; Stability of type-C botulinum toxin at pH 1.8-12.0 and during exposure to 5 and 28 degrees C for 20 and 16 h, respectively, was tested by titration on adult mice . The toxin was found in the samples kept at pH of 2.7-10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.

Acta Vet Scand, 1992, 33(4), 369 - 78
Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks; Elwinger K et al.; Two trials were conducted to study the effects of a competitive exclusion (CE) product BROILACT and the anticoccidial narasin on the incidence of necrotic enteritis (NE), the numbers of Clostridium perfringens (CP) in the caeca of broiler chicks and the performance of the birds . In trial 1 the effects of type of protein and partial replacement of a narasin containing diet with whole wheat were also studied . All groups of chicks were studied up to the point of slaughter at 43 days of age and after evisceration in a processing plant to determine slaughter yield . In trial 1, statistically significant results included the following: CE-treatment reduced total mortality, and incidence of NE, on diet containing animal but not vegetable protein . Caecal carriage of CP was also reduced, while slaughter yield increased . Narasin reduced caecal carriage of CP and increased both growth rate and slaughter yield in both trials . Whole wheat replacement improved feed conversion but reduced bird growth rate . In trial 2, both CE-treatment and narasin influenced feed intake, CE-treatment significantly only at days 22 and 44 . Narasin improved feed conversion until 5 weeks of age and CE-treatment did so until 22 days of age . In both trials, there was also an interaction effect indicating that CE-treatment increased slaughter yield for birds that were not fed narasin.

J Med, 1992, 23(3-4), 279 - 88
The natural course of Clostridium perfringens--induced pneumatosis cystoides intestinalis; Yale CE et al.; Primary pneumatosis cystoides intestinalis (PCI) is an uncommon, usually benign condition whose natural course is poorly understood and which can sometimes produce significant changes in a patient's cecum and sigmoid colon . In this study, PCI was produced by monocontaminating the peritoneal cavities of adult germfree rats with Clostridium perfringens . These animals were then observed for up to 26 weeks . PCI took up to two weeks to develop, lasted at least ten weeks in most animals, and presumably disappeared from 42% of 26 animals killed during the final 16 weeks . PCI was usually benign, but in some animals produced extensive and persistent subserosal and submucosal air cysts of the cecum and sigmoid colon without evidence of intra-abdominal sepsis . These profound segmental colonic lesions suggest a possible etiology for other segmental inflammatory bowel diseases.

Gastroenterol Clin Biol, 1992, 16(10), 777 - 81
{Bacterial translocation in Crohn disease}; Laffineur G et al.; Bacterial translocation is the passage of viable endogenous bacteria from the gastrointestinal tract to mesenteric lymph nodes and other internal organs . The aim of this work was to study bacterial translocation in patients operated on for Crohn's disease . Twenty-eight patients, mean age 29 years, not having received any antibiotics since at least 8 days, presenting with ileal (n = 12), ileo-colonic (n = 14) or colonic (n = 2) Crohn's disease were studied . In 25 out of 28 cases (89%) indication for surgery was strictures inducing an upper small bowel distension in 9 out of 25 patients . Mesenteric lymph nodes and liver biopsies, portal blood samples and peritoneum swabs were harvested after laparotomy and before gut opening . Bacterial translocation, defined as the presence of intestinal bacteria in at least one of the specimens, was present in 8 out of 28 patients . This was found in lymph nodes draining surgical territories in 7 out of 8 cases . Bacterial strains involved in translocation included E . coli (n = 5), Enterococcus (n = 3), Clostridium perfringens (n = 2), Proteus (n = 2), and Bacteroides fragilis (n = 1) . The rate of translocation differed neither according to Crohn's disease site nor with perforating or non perforating type of the disease . Five out of 9 patients operated on for strictures with proximal distension had a translocation . In conclusion, bacterial translocation was identified in 29% of patients operated on for Crohn's disease in this series . Distension of the intestine proximal to a digestive stricture could favor the occurrence of bacterial translocation in Crohn's disease.

Acta Vet Hung, 1992, 40(1-2), 71 - 4
Demonstration of Clostridium septicum infection in a goose flock; Ivanics E et al.; Clostridium septicum infection causing 5.0 to 5.2% mortality is reported for the first time in the literature from six-week-old growing geese in three flocks comprising 5,200, 5,500 and 5,900 geese, respectively . The affected birds exhibited weakness, uncoordinated movement, ataxia and, frequently, oblique position of the head and neck (torticollis) as well as signs indicative of dysequilibrium . The affected birds died within 18-24 h . Gross pathological examination revealed anaemia, hepatitis with map-like necroses of irregular outline (Fig . 1), acute enteritis, pulmonary oedema and cardiac dilatation . Light and electron-microscopic examination showed that the sinusoids of the liver were markedly dilated (Fig . 2) and filled with serous exudate and gas (Figs 2 and 3), and the hepatocytes surrounding them exhibited severe oedema (Fig . 4) . Among the hepatocytes, ciliated bacteria 7-10 mu in length and 1-3 mu in width, bounded by a well-defined cell wall and often showing signs of spore formation were observed (Figs 5 and 6) . By bacteriological examination the pathogen was isolated, its properties were studied, and the clinical entity of malignant oedema was experimentally reproduced by intramuscular injection of guinea-pigs and rabbits . The applied antibiotic (oxytetracycline) and furazolidone therapy proved effective.

Pediatriia, 1992, (7-9), 15 - 20
{Clostridium difficile and diarrhea in infants in the first half-year of life}; Cherkasskaia RS et al.; In order to study a possible etiological relationship between Clostridium and diarrhea in children of the first half year of life and to characterize the colonization of the intestine with these bacteria, bacteriological investigations of feces were carried out in neonates and babies aged 1, 4 and 14 days and 1, 3 and 6 months . The development of the children and their health status were monitored under home conditions . It has been established that the colonization of the neonates' intestine with Clostridium including C . Difficile occurs within the early times (since the 4th day of life) . Later the colonization with C . difficile becomes wavy in nature . Among 7 types of Clostridium isolated from the intestine of the children, C . difficile occurred most frequently (29.1%) . The overwhelming majority of the strains of these bacteria produced toxin whose activity did no exceed 10(-1)-10(-2) . The cytopathic effect was mostly demonstrable in 72 hours . No convincing evidence was obtained about the etiological importance of C . difficile in the development of diarrhea in the children placed under observation . It is likely that the latter one was due to the disturbance of intestinal biocenosis, that manifested by profound quantitative disorders (proliferation of the opportunistic aerobic flora, a dramatic reduction of the content of bifido- and lactic acid bacteria up to their complete absence) . At the same time a great number of children carrying C . difficile attests to a potential development of specific diarrheas (under hospital conditions and during massive antibacterial therapy).

DNA Seq, 1992, 3(3), 191 - 4
The nucleotide sequence of a putative membrane transport gene from Clostridium perfringens; Holck AL et al.; A gene from Clostridium perfringens encoding a highly hydrophobic polypeptide of M(r) = 30,698 was cloned and sequenced . The polypeptide showed similarities with the inner membrane proteins of binding protein-dependent transport systems of the gram negative enterobacteria . The sequence homology to the UgpE and MalG proteins of Escherichia coli was 27.6%, or 60.4 and 52.2%, respectively, taking into account the conservative amino acid changes . The polypeptide contained the EAA---G---------(I/V)-LP motif of the gram negative transport proteins . This motif may thus be very old, as clostridia and E . coli separated some 1.5 x 10(9) years ago . The similarities suggest a common evolutionary origin of these genes.

Nouv Rev Fr Hematol, 1992, 34(4), 345 - 6
Clostridium perfringens septicemia during the course of leukemia; Sauvage C et al.; Clostridium perfringens (CP) is an infrequent cause of septicemia in neutropenic patients . We report the case of a 17 year old man treated for acute lymphoblastic leukaemia in whom a strain of CP was isolated from blood cultures . The clinical spectrum and evolution of CP septicemia in neutropenic patients are discussed.

Arch Microbiol, 1992, 158(4), 294 - 301
2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp . nucleatum): an iron-sulfur flavoprotein; Klees AG et al.; Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg-1 . The enzyme was purified 24-fold to a specific activity of 480 nkat mg-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose . The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl2 . ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate . The enzyme is extremely sensitive towards oxygen and is inhibited by 10 microM chloramphenicol, 10 microM 2,4-dinitrophenol or 0.15 mM hydroxylamine . The pure enzyme consists of three subunits alpha (49 kDa), beta (39 kDa) and gamma (24 kDa) in approximately equal amounts . In this respect the enzyme differs from the related 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of alpha and beta but require an additional protein for activity . The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric alpha beta gamma-structure . The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each) . Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A . fermentans did not crossreact with cell free extracts or purified dehydratase from F . nucleatum . A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A . fermentans and F . nucleatum, however, showed some similarities in the beta-subunits.

Arch Microbiol, 1992, 158(2), 81 - 4
The role of tungstate and/or molybdate in the formation of aldehyde oxidoreductase in Clostridium thermoaceticum and other acetogens; immunological distances of such enzymes; White H et al.; Besides Clostridium thermoaceticum and C . formicoaceticum other resting acetogenic clostridia such as C . aceticum and C . thermoautotrophicum and to a lesser extent non-clostridial acetogens such as Butyribacterium methylotrophicum and Eubacterium limosum were able to reduce propionate to propanol at the expense of carbon monoxide or formate . Methylviologen usually increased the reduction rate . Ten microM molybdate in the growth medium decreased this capability for C . thermoaceticum but increased it or had no effect for the other organisms . Ten microM tungstate in the growth medium increased the aldehyde oxidoreductase activity in all organisms . Crude extracts of C . thermoaceticum cells grown in the presence of 10 microM or 1 mM molybdate showed by ELISA the same or even a 4 fold concentration of aldehyde oxidoreductase in the latter case . However, the enzymic activity was very low in both cases . Omission of dithionite in the growth medium diminished the antigen by a factor of about 8 . The immunological distance between the enzyme from C . thermoaceticum and C . thermoautotrophicum was rather low but very large to C . formicoaceticum and undeterminably large to the other organisms.

Ann Chir, 1992, 46(5), 453 - 7
{Surgical treatment of toxic megacolon complicating pseudomembranous colitis . Apropos of a case, review of the literature}; Panis Y et al.; Toxic megacolon complicating pseudomembranous colitis has been rarely observed . Only 36 cases have been previously reported . We present herein a new case report in which pseudomembranous colitis was secondary to prophylactic antibiotherapy with pefloxacin for hip prosthesis . Despite specific oral treatment (against Clostridium difficile) by vancomycin, toxic megacolon required urgent subtotal colectomy with ileostomy and sigmoidostomy . Postoperative course was uneventful . Analysis of the reported cases demonstrates the high overall mortality of the series (32%); the procedure of choice seems to be subtotal colectomy, which removes the septic focus, with a 12% operative mortality rate.

Microbiol Immunol, 1992, 36(3), 221 - 9
Purification and characterization of heterologous component IIs of botulinum C2 toxin; Ohishi I et al.; Botulinum C2 toxin (C2T) elaborated by certain strains of Clostridium botulinum types C and D is composed of separate and dissimilar two proteins, components I and II . Previous studies have shown that these two components of C2T produced by type C and D strains were immunologically heterologous and that C2T-producers were classified into three groups depending on the difference in molecular characteristics of the components I and II . In the present study, the heterologous component IIs of C2T were purified from three representative strains of the groups and the molecular characteristics of the components were compared . Immunological analyses by agar gel double immunodiffusion test showed that the component IIs purified from the three strains have the specific epitope(s) in addition to the common one(s) . The biological activity of C2Ts reconstituted with component I purified from a fixed strain and component II each from the three strains differed depending on the source of the component II . These results indicate that the component II of C2T produced by C . botulinum types C and D differs in molecular structure, which reflects on the difference in the biological activity of the toxin . The present study suggests that the pathophysiological activity of C2T, which possibly causes a necrotic enteritis, is dependent on the C2T-producing bacteria infected.

Appl Environ Microbiol, 1992 Jan, 58(1), 326 - 30
Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A; Garcia-Alvarado JS et al.; Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C . At 37 degrees C, all strains degraded starch and sporulated well . However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C . Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth . The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C . These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R . G . Labbe and C . L . Duncan, Can . J . Microbiol . 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.

J Med Microbiol, 1992 Jan, 36(1), 30 - 6
Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C . difficile; Martinez RD et al.; Clostridium sordellii produces two toxins, designated HT (haemorrhagic toxin) and LT (lethal toxin), that are similar to toxins A and B of C . difficile . The physicochemical properties of toxins HT and A were remarkably similar . The specific biological activities of toxin HT were almost the same as those of toxin A, and their NH2-terminal sequences shared close homology . The properties of toxins LT and B were similar, as were their NH2-terminal sequences, but toxin B was much more cytotoxic than toxin LT . Immunodiffusion analysis with specific antibodies showed that although toxins B and LT shared major antigenic determinants, each had unique epitopes . The results suggest that toxins B and LT have diverged more than toxins A and HT . Immunoblotting with antibodies to the toxins of C . difficile showed that toxins HT and LT had common antigenic determinants.

J Bacteriol, 1992 Jan, 174(2), 426 - 33
mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis; Gerischer U et al.; By using primer extension analysis, we located the transcription start point of the acetoacetate decarboxylase (adc) gene of Clostridium acetobutylicum 90 nucleotides upstream from the initiation codon with A as the first transcribed nucleotide . From this site the promoter structure TTTACT(18 bp)TATAAT was identified; it shows high homology to the consensus sequences of gram-positive bacteria and Escherichia coli . Northern blot experiments revealed a length of 850 bases for the transcript of the adc gene . It thus represents a monocistronic operon . Transcription of adc was induced by conditions necessary for the onset of solvent formation . Induction occurred long before the respective fermentation product (acetone) could be detected in the medium . Transcription of the operon containing the genes for acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (designated ctf) downstream of the adc gene but divergently transcribed is also induced by conditions necessary for the onset of solvent formation . The length of the respective RNA transcript, 4.1 kb, indicates additional coding capacity, since the genes for the two subunits of the coenzyme A transferase cover only approximately 1.5 kb . No distinct transcripts for the other open reading frames of the adc gene region, ORF1 and ORF2, could be detected . Computer analysis indicated that ORF1, which showed significant similarity to the alpha-amylase gene of Bacillus subtilis (U . Gerischer and P . Durre, J . Bacteriol . 172:6907-6918, 1990), probably is indeed a coding region . ORF2, however, does not seem to have a coding function.

Digestion, 1992, 53(3-4), 121 - 8
Search for enteric microbial pathogens in patients with ulcerative colitis; Brown WJ et al.; Microbial pathogens were sought in faeces of patients with active ulcerative colitis and again after 3 months treatment . 64 patients were examined during their first episode of ulcerative colitis and 30 with relapse of chronic disease . At presentation, bacterial pathogens were not found; 1 patient had cryptosporidiosis . In 10 patients treatment appeared to result in some loss of colonisation resistance as evidenced by colonisation with beta-haemolytic streptococci, Staphylococcus aureus, candida and Clostridium difficile . Unidentified cytotoxic activity was present in the faeces of 4 patients at presentation and 2 patients during or after treatment . We conclude that enteric infection is an uncommon finding in patients with active ulcerative colitis.

Dakar Med, 1992, 37(2), 103 - 8
{Bacteria associated with diarrheic episodes in young camels in Niger}; Bada Alambedjir R et al.; During diarrhoeic episodes of young camels in Niger, bacteria such as Salmonella enteritidis, E . Coli, Staphylococcus sp., Clostridium perfringens and other bacteria were isolated from stool samples . Bacteria roll in this pathology is discussed, and a treatment is proposed, compared with antibiogram results.

Braz J Med Biol Res, 1992, 25(5), 491 - 7
The acute phase of experimental infection with Trypanosoma cruzi is more severe in mice monoassociated with strict anaerobic bacteria; Barros MC et al.; 1 . The influence of some components of the normal human intestinal flora on the acute phase of experimental infection with strain CL of Trypanosoma cruzi was studied in 30-day-old germ-free or gnotobiotic CFW (LOB) mice monoassociated with Bacteroides fragilis, Peptostreptococcus sp or Clostridium sp by intragastric inoculation of 10(6) bacteria 10 days before the intraperitoneal infection with 5 x 10(3) trypomastigotes/g body weight . 2 . Significantly earlier parasitemia peak and mortality were observed in Bacteroides fragilis- and Clostridium-associated mice (16.75 +/- 0.96 and 15.00 +/- 1.15 days, respectively) when compared with germfree animals (18.83 +/- 1.17 days) . More precocious mortality (10.40 +/- 2.06 days) and, curiously, much lower blood parasitemia were observed in Peptostreptococcus-associated mice than in other gnotobiotic mice . 3 . The extent of cardiac tissue parasitism decreased in the following order: germfree, B . fragilis-associated, Clostridium-associated, and Peptostreptococcus-associated animals . The levels of inflammatory reaction decreased in the following order: germfree, Peptostreptococcus-associated, Clostridium-associated, and B . fragilis-associated mice . 4 . These results show that the acute phase of experimental infection with T . cruzi was more severe in mice associated with strict anaerobic bacteria when compared with germfree animals . This suggests that a normal intestinal flora may be another factor, in addition to nutritional and genetic factors, responsible for the different susceptibility of organisms of the same species infected with T . cruzi.

Arch Med Res, 1992, 23(2), 183 - 5
Effect of intestinal bacteria on the virulence of Entamoeba histolytica; Anaya-Velazquez F et al.; The effect on amebal virulence of Escherichia coli, Clostridium symbiosum and a mixture of lactobacilli was investigated . Amebas from HM1 and HK axenic strains were incubated with a single bacterial strain or lactobacilli for short, intermediate, or long periods and analyzed for their erythrophagocytosis, hemolytic activity and cytotoxicity in vitro and hepatic abscesses formation in vivo . It was found that in vitro virulence was enhanced by all studied bacteria in HK9 but not in HM1 strains, which only showed an increase in cytotoxicity . The virulence in vivo was solely increased in trophozoites of HM1 strain by all tested bacteria.

Rev Elev Med Vet Pays Trop, 1992, 45(3-4), 255 - 9
Severe heart muscle degeneration caused by Clostridium perfringens type A in camel calves (Camelus dromedarius); Wernery U et al.; Clostridium perfringens type A was isolated from different organs and intestines from three to five weeks old camel calves which have died from heart muscle necrosis . No other bacterial pathogens were isolated . Virus isolation on two different cell lines including a fetal camel skin were also negative . Mice which were injected with bacteria free filtrates prepared from intestinal contents of necropsied camel calves died after one to six hours demonstrating the presence of clostridial toxins . Our findings suggest that the cardiac muscle necrosis is caused by Clostridium perfringens type A toxins.

Matrix Suppl, 1992, 1, 116 - 26
Clostridium histolyticum collagenases: a new look at some old enzymes; Mookhtiar KA et al.; Seven collagenases denoted by the letters alpha, beta, gamma, delta, epsilon, zeta and eta have been purified to homogeneity from the culture filtrate of Clostridium histolyticum . All seven enzymes are zinc proteinases that require calcium ions for activity and have essential carboxyl, tyrosyl and lysyl residues . These enzymes can be divided into two classes on the basis of the sequence homologies in their polypeptide chains, as revealed from a comparison of their tryptic digests . This division into classes is also supported by a comparison of their specificities toward peptide substrates, their interaction with substrate-analog inhibitors, and their mode of attack of triple helical collagens . The sequence specificities of these enzymes have been studied in detail . The specificities of the two classes are similar, but complementary . Both classes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities, where the latter is thought to facilitate removal of Gly-X-Y triplets from the C-terminus of collagen fragments . The mode of attack of these collagenases on triple helical type I, II and III collagens is very similar for the enzymes within each class, but different for the two classes . The class I enzymes first hydrolyze loci near the ends of the triple helical domains of these collagen molecules, while the class II enzymes make their initial cleavages in the interior . The sites of these initial cleavages are being sequenced and preliminary results indicate that they do not resemble the tissue collagenase cleavage site with respect to either their imino acid content or distribution . The kinetic parameters for the hydrolysis of type I, II and III collagens have been measured and are similar in magnitude to those for the tissue collagenases . Synthetic peptide substrate-analog inhibitors have been prepared for both classes of collagenases and shown to be transition-state-analog inhibitors.

Antimicrob Agents Chemother, 1992 Jan, 36(1), 239 - 43
In vitro activities of three of the newer quinolones against anaerobic bacteria; Wexler HM et al.; The antimicrobial activities of three new quinolone compounds, sparfloxacin, temafloxacin, and WIN 57273, against anaerobic bacteria were determined in three separate studies . The Wadsworth agar dilution technique using brucella-laked blood agar was used throughout . The activities of other antimicrobial agents, including ciprofloxacin, imipenem, chloramphenicol, metronidazole, cefotetan, cefoxitin, and amoxicillin-clavulanic acid, were also determined . The breakpoints of the new quinolones were 2 micrograms/ml for sparfloxacin and WIN 57273 and 4 micrograms/ml for temafloxacin . WIN 57273 displayed very good activity against anaerobes, inhibiting all strains of Bacteroides fragilis group species at 2 micrograms/ml . Only two strains of Fusobacterium species were resistant (MIC, 4 micrograms/ml) . Sparfloxacin inhibited 78% of B . fragilis strains and 44% of other B . fragilis group isolates at 2 micrograms/ml . At 2 micrograms/ml, the percentages of other anaerobic species susceptible were as follows: B . gracilis, 70%; other Bacteroides species, 61%; Clostridium species, 50%; Fusobacterium species, 70%; Peptostreptococcus species, 91%; non-spore-forming gram-positive rods, 71% . Temafloxacin inhibited 91% of B . fragilis strains and 87% of other B . fragilis group species at 4 micrograms/ml . All strains of other Bacteroides species, 78% of Fusobacterium species, 80% of Clostridium species, and 90% of Peptostreptococcus species were inhibited at 4 micrograms of temafloxacin per ml.

Eur J Clin Microbiol Infect Dis, 1992 Jan, 11(1), 68 - 71
Susceptibility of anaerobic bacteria to PD 131628; Nord CE et al.; The in vitro activity of PD 131628 against anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, Bacteroides spp . and fusobacteria was determined by the agar dilution method . This activity was compared with that of ciprofloxacin, piperacillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol . PD 131628, imipenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested.

CLAO J, 1992 Jan, 18(1), 59 - 63
The collagen shield as a collagenase inhibitor and clinical indicator of collagenase activity on the ocular surface; Schiff WM et al.; Collagen shields applied to the corneas of patients with bacterial keratitis degrade rapidly, often within a few hours . Once treatment brings the infection under control, subsequently applied collagen shields degrade more slowly . In vitro models were established to evaluate the significance of these observations . Twenty-four and 72-hour collagen shields were incubated with collagenase from Clostridium histolyticum . The in vitro rate of digestion of the shields was directly proportional to the concentration of collagenase, with the rate of digestion of the 24-hour shields being greater than that of the 72-hour shields . Therefore, the rate of collagen shield degradation may be a clinically useful index of collagenase activity on the ocular surface . Ultrastructural studies of collagen shields from patients with acute bacterial keratitis revealed irregular degradation of shield matrix with no evidence of adherence of microorganisms or inflammatory cells . Co-incubation of deepithelialized rabbit corneas and collagen shields resulted in inhibition of the digestion of the rabbit corneas when the weight:weight ratio of collagen shield:rabbit cornea was increased to greater than or equal to 2:1 . Collagen shields may inhibit corneal collagen degradation in infectious ulceration and melting disorders by effectively competing for collagenase on the ocular surface.

J Neurol, 1992 Jan, 239(1), 16 - 20
Clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use; Hambleton P; The neurotoxins produced by Clostridium botulinum are the most potent acute toxins known and are the causative agents of the neuroparalytic disease botulism . The toxins act primarily at peripheral cholinergic synapses by blocking the evoked release of the neurotransmitter acetylcholine . There are seven distinct serotypes of toxin . All are polypeptides of Mr about 150 kDa that have similar structure and pharmacological action . In their most active forms the toxins exist as dichain molecules in which a heavy (H) chain is linked by disulphide bonding to a light (L) chain . The H chain is believed to be associated with the highly specific and avid binding of toxin to the motor nerve end plates and also with the process of internalisation of the toxin . The toxic activity appears to be associated with the L chain which blockades the calcium-mediated release of acetylcholine, probably by interfering at the molecular level with the mechanisms whereby neurotransmitter-containing vesicles merge with the plasmalemma . The type A toxin is now used therapeutically to treat a variety of conditions involving involuntary muscle spasm . The therapeutic toxin is a neurotoxin-haemagglutinin complex isolated from cultures of C . botulinum . A controlled manufacturing process has been developed for the therapeutic toxin which is specially formulated to give a freeze-dried product having good stability.

Infect Immun, 1992 Jan, 60(1), 219 - 30
Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread; Vazquez-Boland JA et al.; The lecithinase gene of the intracellular pathogen Listeria monocytogenes, plcB, was identified in a 5,648-bp DNA fragment which expressed lecithinase activity when cloned into Escherichia coli . This fragment is located immediately downstream of the previously identified gene mpl (prtA) . It contains five open reading frames, named actA, plcB, and ORFX, -Y, and -Z, which, together with mpl, form an operon, since a 5.7-kb-long transcript originates from a promoter located upstream of mpl (J . Mengaud, C . Geoffroy, and P . Cossart, Infect . Immun . 59:1043-1049, 1991) . A second promoter was detected in front of actA which encodes a putative membrane protein containing a region of internal repeats . plcB encodes the lecithinase, a predicted 289-amino-acid protein homologous to the phosphatidylcholine-specific phospholipases C of Bacillus cereus and Clostridium perfringens (alpha-toxin) . plcB mutants produce only small plaques on fibroblast monolayers, and an electron microscopic analysis of infected macrophages suggests that lecithinase is involved in the lysis of the two-membrane vacuoles that surround the bacteria after cell-to-cell spread . On the opposite DNA strand, downstream of the operon, three more open reading frames, ldh, ORFA, and ORFB, were found . The deduced amino acid sequence of the first one is homologous to lactate dehydrogenases . Low-stringency Southern hybridization experiments suggest that these three open reading frames lie outside of the L . monocytogenes virulence region: mpl and actA were specific for L . monocytogenes, sequences hybridizing to plcB were detected in L . ivanovii and L . seeligeri, and sequences hybridizing to ORFX, -Y, and -Z were found in L . innocua . In contrast to this, sequences hybridizing to ldh or ORFB were detected in all Listeria species (including the nonpathogenic ones).

Gastroenterology, 1992 Jan, 102(1), 35 - 40
Human colonic aspirates containing immunoglobulin A antibody to Clostridium difficile toxin A inhibit toxin A-receptor binding; Kelly CP et al.; Clostridium difficile toxin A, a 308-kilodalton protein exotoxin, is the principal causative agent of antibiotic-associated, C . difficile-induced colitis . In the current study, the prevalence of specific human serum and secretory antibody to toxin A and the possible protective effect of secretory, intestinal anti-toxin A antibody are examined . Serum (n = 35), colonic aspirates (n = 35), and duodenal aspirates (n = 20) were collected from adults at diagnostic endoscopy . Patients with evidence of colitis or a history of recent antibiotic use were excluded from the study . Specific serum immunoglobulin (Ig) A and IgG antitoxin A antibodies were detected in 60% and 57% of subjects, respectively, by enzyme-linked immunosorbent assay . Fifty-seven percent of colonic aspirates contained IgA antitoxin, whereas only 10% of duodenal aspirates were positive (P = 0.002) . Binding of toxin A to its intestinal receptor was studied using {3H}toxin A and purified rabbit ileal brush border membranes . Toxin A binding was significantly inhibited by colonic aspirates with high IgA anti-toxin A antibody levels (0.503 +/- 0.055 pmol toxin A bound per milligram of brush border membrane protein, mean +/- SE) in comparison with antitoxin A-negative aspirates (0.778 +/- 0.089 pmol; P = 0.02) and control (0.766 +/- 0.004 pmol; P = 0.03) . In the current study, a specific intestinal secretory IgA antibody response to C . difficile toxin A in humans is reported . This antibody response is more evident in the colon, the site of C . difficile infection, than in the upper intestinal tract . Our data suggest that human colonic IgA antitoxin may protect against C . difficile colitis by inhibiting the binding of toxin A to its intestinal epithelial cell receptor.

Arch Inst Pasteur Alger, 1992, 58, 205 - 24
{Production of a vaccine against enterotoxemia from Clostridium perfringens strains isolated in the field}; Cherfaoui S et al.; We have isolated eight strains of C . perfringens from cases of enterotoxaemia . Five of these strains have revealed themselves toxic with respective types (type "A":2, type "C":2, type "D":1) . In order to produce anti-enterotoxaemia vaccine, we have proceeded at the cultivation in fermenter of isolated strains and reference strains CWA 35, CWC and CWD AF . At the end of fermentation, we have evaluated the two following parameters: obtained biomass, and toxin titers . With the two classes of strains we reached an important biomass but toxins titers relatively weak comparatively to that which is usually required . It will be necessary then, to demonstrate the immunogen value of the produced vaccines by testing their efficacity.

Arch Inst Pasteur Alger, 1992, 58, 169 - 79
{Preliminary epidemiological study of carriers of Clostridium difficile}; Merad SA et al.; 171 samples have been taken from stools of sick persons in three departments of university and hospital centres of Algiers (C.H.U.A.): the department of maternity (C.H.U Parnet), the department of breast diseases (CPMC), the department of women's surgery (CPMC), 13 samples of C . difficile have been isolated with a frequency of 7.6 per 100 of cases . The rate of conveyance seems more important in adults in hospitals and often concerns sick persons enduring digestive cancer (10.25 per 100) or newborn with a frequency of 8.73 per 100.

Przegl Epidemiol, 1992, 46(4), 355 - 6
{Upper respiratory tract infections as a hypothetical source of sudden infant death syndrome (SIDS)}; Mierzejewski J et al.; A hypothesis that the common bacterial toxins are the possible cause of the Sudden Infant Death Syndrome (SIDS) was discussed . Recently, even botulinum toxin and Clostridium perfringens Type A enterotoxin are mentioned.

Przegl Epidemiol, 1992, 46(4), 321 - 4
{Isolation of Clostridium perfringens from a wound in a case of post-traumatic gas gangrene . Diagnostics remarks}; Kalwas M et al.; The authors described a case of post traumatic gas gangrene in a young man . Rapid clinical and microbiological diagnosis with isolation of Clostridium strain, and immediate therapy, including hyperbaric chamber, brought complete recovery without incapacitation.

Clin Physiol Biochem, 1992, 9(2), 78 - 86
Activation of PGE2-secretion from gastric mucosa by a type I phospholipase C is mediated by a direct release of arachidonic acid; Preclik G et al.; We investigated the effects of an exogenous Type I phospholipase C (PLC) from clostridium perfringens on arachidonic acid release and prostaglandin synthesis from gastric mucosa by determining PGE2 release from organ cultured rabbit mucosal biopsies as well as PGE2 synthesis and substrate-dependent inactivation of the prostaglandin cyclooxygenase from endogenously released arachidonic acid in mucosal homogenate . PLC dose dependently stimulated PGE2 secretion from organ cultured mucosa to 145% and 245% at 0.1 and 1.0 U/ml during a 60 minute culture period . This effect was not affected by the calmodulin antagonist N-(6-aminohexyl)-1-5-chloro-1-naphthalene-sulfonamide (W-7) or the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) . PLC could not be substituted by phorbol-12-myristate 13-acetate (PMA), an analogue of the diacylglycerol second messenger functions . During a 15 minute preincubation of mucosal homogenate at 37 degrees C, 1mM CaCl2 stimulated PGE2 synthesis from endogenous arachidonic acid about 5-fold compared to an EDTA-control . In contrast, the residual prostaglandin synthesizing capacity, determined by incubation with excess 14C-labelled arachidonic acid, was reduced by CaCl2 to 37% of the EDTA-value . Quinacrine, an inhibitor of arachidonic acid release from phosphatidylethanolamine, reduced both the stimulation of PGE2 synthesis and the inactivation of prostaglandin cyclooxygenase . Therefore we conclude, that this Ca(2+)-effect reflects activation of the Ca-dependent phospholipase A2 (PLA2) and, as a consequence, substrate-induced inactivation of the prostaglandin cyclooxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Med Dosw Mikrobiol, 1992, 44(1-2), 49 - 54
{Occurrence of Clostridium difficile in the digestive system of dogs}; Martirossian G et al.; This study was aimed at seeking strains of Clostridium difficile in feces and investigation of influence of antibiotics application on frequency of isolation and detection of toxing in vivo produced by this microorganism . Samples of feces were obtained from experimental dogs consisting of two groups . To groups I belonged 150 healthy dogs . Group II consisted of samples of feces received from four dogs before and after application of various antibiotics . Clostridium difficile was not isolated from group I dogs . From dogs belonging to group II, 28 strains were isolated . Production of toxing and classification to serological groups of the isolated strains were performed . Presence of this microorganism in feces of dogs is evident only after multiple application of antibiotics . Results of these studies suggest that dogs may constitute a reservoir of Clostridium difficile.

Arch Tierernahr, 1992, 42(1), 71 - 7
DAP-decarboxylase activity and lysine production by rumen bacteria; Styriak I et al.; The last step of pathway of lysine biosynthesis by rumen bacteria was tested . The first measurements of DAP-decarboxylase activity and of lysine production by Megasphera elsdenii, Selenomonas ruminantium, Clostridium spp., Butyrivibrio fibrisolvens and Bacteroides succinogenes as well as the first attempts to increase the lysine production by ruminal streptococci by mutation are described . The highest values were measured in Selenomonas ruminantium (DAP-decarboxylase activity = 146 micrograms DAP.min-1.mg-1 protein and lysine production was 390 micrograms.mg-1 protein) and the lowest values were ascertained in Butyrivibrio fibrisolvens (DAP-decarboxylase activity = 27 micrograms DAP.min-1.mg-1 protein and lysine production was 32 micrograms.mg-1 protein) . DAP-decarboxylase activity was increased by mutation especially in Streptococcus bovis, the lysine production in both of tested ruminal streptococci . The potential use of lysine-excreting mutants in calves in future is suggested.

Eur Surg Res, 1992, 24(6), 356 - 62
Effect of hyperbaric oxygen and surgery on experimental gas gangrene; Hirn M et al.; An experimental model of clostridial gas gangrene was developed in rats and the therapeutic value of surgical debridement alone versus a combination of surgery and hyperbaric oxygen (HBO) was assessed . The infection was produced by an intramuscular injection of Clostridium perfringens microorganisms . The mortality of untreated rats was 100% . The mortality of the rats treated only with surgery was 37.5% compared to 12.5% when HBO was added to the treatment protocol (p < 0.01) . In the group treated with HBO and surgery 82.5% of the animals healed completely and were able to walk normally, whereas the corresponding figure in the rats treated with surgery alone was 12.5% (p < 0.001) . In the present experimental setting HBO treatment was an important therapeutic adjunct to surgery reducing both mortality and morbidity.

Intensive Care Med, 1992, 18(8), 488 - 90
Clostridial sepsis with massive intravascular hemolysis: rapid diagnosis and successful treatment; Batge B et al.; A 61-year-old man developed a pyrescia accompanied by a massive intravascular hemolysis after abdominal surgery (Whipple's operation) of a pancreatic adenocarcinoma . Abdominal ultrasound and the abdominal CT-scan showed marked aerobilia and multiple liver abscesses . Laboratory tests demonstrated the presence of the Thomsen-Friedenreich cryptantigen (TCA) on the membranes of the patient's erythrocytes . The enzymatic cleavage of N-acetyl-neuraminic acid usually covering the TCA may lead to a life threatening intravascular hemolysis . Since Clostridial bacteriae typically synthesize neuraminidase, the presumptive diagnosis of Clostridial sepsis complicated by massive hemolysis was made . Immediate antibiotic therapy including penicillin G and metronidazole stopped hemolysis within a few hours and the patient servived . On the following day, microbiological examination identified Clostridium perfringens in the patient's blood cultures . Clostrial sepsis should be suspected in patients with underlying infections and/or malignant diseases, particularly of the gastrointestinal or genitourinary tract, who present with septic shock and acute intravascular hemolysis . Whereas microbiological specification of the organism is time consuming, the relatively simple agglutination test with anti-TCA peanut lectin can provide a rapid presumptive diagnosis . The immediate onset of an appropriate antimicrobial therapy is of central importance and might be life-saving.

Ann N Y Acad Sci, 1991 Dec 27, 646, 94 - 8
Cloning of an NADH-dependent butanol dehydrogenase gene from Clostridium acetobutylicum; Petersen DJ et al.; The acetone-butanol fermentation of C . acetobutylicum is characterized by the unique shift from acid to solvent production . The mechanism of the solventogenic switch involves the induction of several enzymes, including NADH-dependent butanol dehydrogenase (BDH) at the onset of solventogenesis . This enzyme is responsible for the final conversion of butyraldehyde to butanol, and is distinct from the NADPH-dependent alcohol dehydrogenase (ADH) also present in the organism . To characterize the genetic control of this gene, we have cloned and expressed it in E . coli . A lambda EMBL3 phage library of C . acetobutylicum DNA was screened via plaque hybridization using a {32P}-radiolabeled, 32-fold degenerate, 62-mer oligonucleotide probe . The probe was designed by reverse translation of the NH2-terminal amino acid sequence of purified BDH II . Southern blot experiments indicate that the phage insert was of clostridial origin and had no homology with the previously cloned NADPH-dependent ADH . Subcloning of DNA from purified positive plaques has localized the gene to a 3.5-kb EcoRI fragment from which the enzyme is well expressed . The sequence of the 25 NH2-terminal amino acids for the cloned enzyme purified from E . coli was determined and found to be identical to that for the clostridial NADH-dependent BDH II . Maxicell analysis of {35S}-radiolabeled plasmid-encoded proteins identified a species encoded by the clostridial insert with the expected Mr of 42 kD.

J Mol Biol, 1991 Dec 20, 222(4), 877 - 80
Crystallization and preliminary X-ray analysis of botulinum neurotoxin type A; Stevens RC et al.; Botulinum neurotoxin serotype A was isolated from liquid culture of Clostridium botulinum . The pure Mr approximately 150,000 neurotoxin, composed of Mr approximately 50,000 light and Mr approximately 100,000 heavy chains, has been crystallized in three different crystal morphologies; all three have the same crystal form . The most suitable crystal form for X-ray analysis are bipyrimidal and crystallize in the hexagonal space group P3(1)21 (or P3(2)21) with one dimer per asymmetric unit . The unit cell dimensions are a = b = 170.5 A, c = 161.7 A . The crystals diffract to 3 A resolution.

Biochemistry, 1991 Dec 17, 30(50), 11669 - 76
A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties; Smith ET et al.; The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2{4Fe-4S} holoprotein, and compared to synthetic C . pasteurianum and native Fds . Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C . pasteurianum Fd did not significantly increase its thermostability . The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode . Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential {Smith, E.T., & Feinberg, B.A . (1990) J . Biol . Chem . 265, 14371-14376} . An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2 . In contrast, the reduction potential of both native C . pasteurianum Fd and synthetic Fd with the C . pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent {Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M . (1991) J . Am . Chem . Soc . 113, 688-689} . On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.

Biochem Biophys Res Commun, 1991 Dec 16, 181(2), 507 - 12
Nucleotide sequence of the Clostridium thermocellum laminarinase gene; Zverlov VV et al.; The sequence presented (1022 bp) shows the Clostridium thermocellum laminarinase gene (lam1) and its flanking regions . The gene lam1 comprises an open reading frame of 726 nt, encoding a 242-aa protein with predicted Mr 27661 . The ORF startswith the translation initiation codon ATG . This ATG codon is preceded at a spacing of 7 bp by a potential ribosome binding site (GGAGGT) . A putative signal peptide was identified (the potential cleavage site is between position 27-28 aa) . The comparison of the primary protein sequence with other beta-1, 3-1, 4-glucanases showed extensive homology for Bacillus amyloliqefaciens and Bacillus subtilis glucanases (identity-46.7%; similarity-57.0%).

J Biol Chem, 1991 Dec 15, 266(35), 23824 - 8
The primary structure of the subunits of carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum; Morton TA et al.; CO dehydrogenase/acetyl-coenzyme A synthase (CODH) is the central enzyme in the pathway of acetyl-coenzyme A biosynthesis in Clostridium thermoaceticum . It catalyzes the interconversion of CO and CO2 and the synthesis of acetyl-coenzyme A from the methylated corrinoid/iron sulfur protein, CO, and coenzyme A . It is a nickel-iron-sulfur protein and contains two subunits in the form (alpha beta)3 . Reported here is the cloning and sequencing of the genes for both subunits of CODH . The gene for the alpha subunit codes for a protein with 729 amino acids and a molecular weight of 81,730, and the beta gene for a protein with 674 amino acids and a molecular weight of 72,928 . The alpha subunit follows the beta subunit by 23 bases and the genes for both subunits are preceded by a sequence which is similar to the Shine-Dalgarno sequence of Escherichia coli . No significant amino acid sequence homology has been found to any known sequence . Labeling CODH with 2,4-dinitrophenylsulfenyl chloride and isolating labeled peptide fragments demonstrated that a tryptophan, residue 418 of the alpha subunit, is protected by coenzyme A and thus may be considered a potential part of the coenzyme A site.

J Biol Chem, 1991 Dec 15, 266(35), 23714 - 23
Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin . Interpretability of contact shifts in terms of cysteine orientation; Busse SC et al.; A two-dimensional NMR study has been carried out on the four-iron clusters of a bacterial oxidized ferredoxin for the purpose of investigating the relationship between contact shift patterns and the orientation of the individual coordinated cysteines . The ferredoxin from Clostridium pasteurianum, CpFdox, was selected because of its extensive sequence homology, and likely close structural similarity, to the crystallographically characterized ferredoxin from Peptococcus aerogenes, Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L . H . (1973) J . Biol . Chem . 248, 3987-3996) . Rapid data collection rates with minimal but adequate acquisition time allowed the detection of numerous CpFdox cross-peaks from the contact-shifted and strongly relaxed coordinated cysteinyl C beta H protons in the resolved 10-20 ppm window . Relatively strong magnitude COSY cross peaks from the resolved eight cysteinyl C beta H resonance unambiguously locate the geminal C beta H partner for each residue; weaker cross-peaks locate the C alpha Hs from three of the residues . The geminal nature of the magnitude-COSY detected partners to the resolved C beta H peaks is confirmed by strong NOESY cross-peaks . The NOESY spectra, moreover, assign an additional two cysteinyl C alpha H resonances . The present results confirm some previous one-dimensional NOE assignments, revise others, and locate resonances previously undetected (Bertini, I., Briganti, F., Luchinat, C., and Scozzafara, A . (1990) Inorg . Chem . 29, 1874-1880) . A striking pairwise pseudo-symmetry in cysteinyl contact shift patterns is observed which is attributed to the previously recognized pseudo-symmetry in the crystal of PaFdox . A detailed analysis of the structural/electronic determinants of the coordinated cysteine C beta H contact shift pattern is made, and the NMR data necessary for unique interpretation are identified . It is shown that analysis of the relaxation properties of cysteine beta-methylene protons provides the stereospecific assignments necessary for comparison of shift ratios with crystallographic structural data . The available structural data on PaFdox (Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J . (1991) J . Am . Chem . Soc . 13, 2055-2064) are qualitatively but not quantitatively consistent with the observed cysteinyl contact shift pattern, with the NMR data reflecting more asymmetry than previous studies . A tentative assignment of a single pair of symmetry-related cysteines is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)

J Pediatr Surg, 1991 Dec, 26(12), 1376 - 80
Pneumatosis cystoides intestinalis in children beyond the first year of life: manifestations and management; Reynolds HL Jr et al.; Beyond infancy, pneumatosis cystoides intestinalis (PCI) is rare . Data concerning pathogenesis and treatment are limited . Our experience with 12 children was examined to define predisposing factors, presentation, treatment, and outcome . Nine children were immunosuppressed, thus identifying an important etiologic subgroup . Presentation was variable but included abdominal pain, distention, diarrhea and hematochezia . Clostridium difficile was found in 3 patients and cytomegalovirus in 1 . Radiographs showed free air in 3 . Nine were treated with antibiotics and bowel rest, 1 with bowel rest alone, 1 with oral metronidazole, and 1 with observation . PCI resolved in 7 of 9 treated with antibiotics, although 1 child with leukemia had severe hematochezia secondary to colonic ulceration and required hemicolectomy . No other patient required laparotomy . The free air resolved in 2 of 3 . There were 2 deaths, both from sepsis . One had free air on admission but no perforation was found at autopsy . Treatment recommendations remain unclear; however, C difficile and cytomegalovirus are important pathogens that should be identified and treated promptly . In symptomatic patients, bowel rest and antibiotics seem beneficial . Operative intervention should be reserved for patients with peritoneal signs, progressive deterioration, obstruction, or persistent, severe bleeding . Free air alone is not an indication for operative management in children with PCI.

Biochem Pharmacol, 1991 Dec 11, 42 Suppl, S77 - 87
Sialic acid removal modulates the myocardial and vascular activity of calcium channel ligands; Werner G et al.; Selective removal of sialic acid from isolated guinea pig left atrial strips and rabbit thoracic aortic ring segments was performed by neuraminidase prepared from Clostridium perfringens and was controlled electron microscopically . Preincubation of these organs (2 units/mL; 2 hr) resulted in enzyme mediated hydrolysis of total tissue sialic acid; 55.2% for atria and 60.9% for aorta . Contractile force of atria and arterial diameter of thoracic aorta were measured isometrically and isotonically by means of a force displacement transducer . Pretreatment of both organs with neuraminidase (2 units/mL; 2 hr) in a carbogen saturated organ bath caused a moderate left-hand shift of the cumulative concentration response curves for the dihydropyridine type calcium antagonist nisoldipine, the phenylalkylamine derivative gallopamil and the benzothiazepine diltiazem . EC50 values were significantly lower (P less than 0.05), particularly in the atrial muscle, when compared to untreated preparations . There was no effect of neuraminidase on the negative inotropic and vasodilator potency of the calcium channel modulator fendiline . Conversely, neuraminidase induced a right-hand shift in the concentration response curves shown by the pure calcium agonist (-)-S-Bay K 8644 leading to significantly higher EC50 values in both organs . Similarly, the contractile potency of calcium chloride (atria) and potassium chloride (aorta) was attenuated upon neuraminidase treatment . From the results obtained it is concluded that sialic acid removal may modulate the action of calcium channel ligands through an inhibitory effect on transmembrane calcium fluxes and/or by decreasing the external calcium availability . Whether the present results suggest a functional role for sialic acid in the regulation of calcium channels warrants further investigation.

Biochim Biophys Acta, 1991 Dec 6, 1115(2), 123 - 30
Functional studies of a glutamate dehydrogenase with known three-dimensional structure: steady-state kinetics of the forward and reverse reactions catalysed by the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum; Syed SE et al.; Steady-state kinetic properties of glutamate dehydrogenase from Clostridium symbiosum are reported . Rates with NADP(H) are over three hundred times lower than with NAD(H) under identical conditions . The 3-acetyl pyridine and 6-deamino adenine analogues of NAD+, on the other hand, are used almost as well as NAD+ itself . Amino acid specificity is very tight at both pH 7 and pH 9 . The best alternative substrate of those tested, L-alpha-amino-gamma-nitraminobutyrate, gave only 0.5% of the rate seen with glutamate . With 400 microM NAD+ a 160-fold variation of the glutamate concentration gave a linear Eadie plot apart from slight inhibition at the highest concentrations . With 40 mM L-glutamate and varied {NAD+}, the Eadie plot appeared linear between 1.6 microM and 60 microM and again between 60 microM and 2000 microM, but the slopes of the two lines differed by a factor of 8.4 . This striking pattern is not attributable to impurities in the coenzyme or to changes in the state of aggregation of the enzyme . For the high concentration range (greater than 60 microM NAD+), the presence of all four linear terms in the reciprocal form of the initial rate equation indicates a sequential mechanism . Similar measurements made for APAD+ and dnNAD+ show no sign of non-linearity in the Eadie plot over the wide concentration ranges explored . In the reductive amination direction, with NADH as coenzyme, linear reciprocal plots were obtained for all three substrates . Systematic variation of concentrations led via primary, secondary and tertiary plots to all eight possible initial-rate parameters in a linear reciprocal initial-rate equation . Compulsory-order and enzyme-substitution mechanisms appear to be excluded, and a random route to the central complex seems the only possibility compatible with the results.

Dtsch Med Wochenschr, 1991 Dec 6, 116(49), 1862 - 6
{Atraumatic Clostridium septicum infection in granulocytopenia}; Habscheid W et al.; A fatal Clostridium septicum infection occurred in three patients . Case 1 . A 55-year-old man died of septicaemia resulting from granulocytopenia of uncertain aetiology; it was associated with perforation of ileal mucosal ulcers . Autopsy revealed neutropenic enterocolitis and diffuse gas formation, especially in the brain, caused by Clostridium septicum . Case 2 . A 18-year-old boy developed a caecal invagination during imipenem-induced granulocytopenia . A fulminant postoperative Clostridium septicum infection ended fatally . At autopsy many ulcers were found at the site of invagination with gas formation involving all organs . Case 3 . Myonecrosis of the left arm, caused by Clostridium septicum, developed without external cause in a 12-year-old girl with congenital neutropenia . Despite aggressive surgical intervention she died of toxic shock . Autopsy revealed caecal mucosal ulcers as the portal of entry of Clostridium septicum.

J Bacteriol, 1991 Dec, 173(23), 7705 - 10
Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase; Gosalbes MJ et al.; Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis . A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp . The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB) . Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E . coli and culture supernatants of B . subtilis clones containing the xynD gene . Both peptides show xylanase activity in zymogram analysis . The XYND enzyme also shows alpha-L-arabinofuranosidase activity . The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O . Grepinet, M . C . Chebrou, and P . Beguin, J . Bacteriol . 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.

DICP, 1991 Dec, 25(12), 1326 - 8
Oral vancomycin-induced rash: case report and review of the literature; McCullough JM et al.; Disseminated rash and pruritus are described in an 82-year-old woman with chronic renal failure following administration of oral vancomycin hydrochloride 125 mg q6h for the treatment of Clostridium difficile colitis . Renal function was estimated to be 0.27 mL/s based on a serum creatinine of 177 mumol/L . After eight days of therapy, she developed a slightly raised maculopapular rash on her legs and torso, which spread to her abdomen and arms with continued treatment . Vancomycin was discontinued and the patient was treated symptomatically . The rash cleared and did not recur . Rechallenge with vancomycin was not initiated . No other changes in medications or initiations of new medications occurred during the time of treatment with vancomycin . The patient denied any previous immunologically mediated reactions to medications . Maculopapular rash is rare secondary to vancomycin administration, particularly after oral administration . Although clinically significant serum concentrations can be obtained in patients treated with oral vancomycin who have concomitant C . difficile colitis and renal failure, there has not been a clear correlation between these concentrations and any reported adverse sequelae . This case supports the possible occurrence of a true allergic reaction secondary to low-dose oral vancomycin administration.

Ir J Med Sci, 1991 Dec, 160(12), 385 - 6
Local beneficial effect of coumarin in experimental peritonitis; Creagh TA et al.; Coumarin, a potent immune stimulant and macrophage activator, has been used to treat brucellosis and as an immune suppressor . The effect of Coumarin and systemic antibiotics on septicaemia, survival and peritoneal contamination in experimental peritonitis was assessed . Four groups of male Sprague-Dawley rats were inoculated with Clostridium perfringens, Escherichia coli and Bacteroides fragilis . Group A received saline alone, Group B received Coumarin alone, Group C received antibiotics (Clindamycin and Cephradine) alone and Group D received both Coumarin and antibiotics . Septicaemia, confirmed by blood culture, occurred in all animals . Coumarin did not improve survival whether given alone or in combination with antibiotics . Animals given Coumarin (Groups B and D) had significantly less peritoneal soiling (54%, 0%) (P less than 0.02, P less than 0.001) than their controls (Groups A and C: 92%; 29%) . While Coumarin did not improve resistance to septicaemia it did exert a local beneficial effect.

Ann Gastroenterol Hepatol (Paris), 1991 Dec, 27(7), 343 - 8
{Current status of Clostridium difficile: recent advances in diagnostic materials and treatment of colitis and diarrheas associated with antibiotic therapy}; Dru M; Clostridium difficile infection covers several clinical pictures which have been identified only recently . The majority are based upon imbalance in the intestinal ecosystem . Major advances in recent years concerning this type of infection have involved: a) better knowledge of pathophysiological mechanisms, in particular the demonstration of a 2nd toxin, essential to the understanding of C . difficile infections; b) better codification of the management of these disorders . This is aimed today at treating the infection while maintaining the balance of the intestinal ecosystem.

Appl Environ Microbiol, 1991 Dec, 57(12), 3613 - 5
Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria; Stevens KA et al.; Nisin, produced by Lactococcus lactis subsp . lactis, has a broad spectrum of activity against gram-positive bacteria and is generally recognized as safe in the United States for use in selected pasteurized cheese spreads to control the outgrowth and toxin production of Clostridium botulinum . This study evaluated the inhibitory activity of nisin in combination with a chelating agent, disodium EDTA, against several Salmonella species and other selected gram-negative bacteria . After a 1-h exposure to 50 micrograms of nisin per ml and 20 mM disodium EDTA at 37 degrees C, a 3.2- to 6.9-log-cycle reduction in population was observed with the species tested . Treatment with disodium EDTA or nisin alone produced no significant inhibition (less than 1-log-cycle reduction) of the Salmonella and other gram-negative species tested . These results demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria . Applications involving the simultaneous treatment with nisin and chelating agents that alter the outer membrane may be of value in controlling food-borne salmonellae and other gram-negative bacteria.

Appl Environ Microbiol, 1991 Dec, 57(12), 3517 - 21
Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition; MacDonald DL et al.; Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth . We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids . Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin . When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased . Addition of dodecane and n-butanol did not affect this ratio . When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells . In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio . We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.

Appl Environ Microbiol, 1991 Dec, 57(12), 3423 - 8
Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures; Okereke A et al.; The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P . pentosaceus ATCC 43201, Lactobacillus plantarum BN, L . plantarum LB592, L . plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 degrees C was investigated by modified deferred and simultaneous antagonism methods . All the strains, except L . acidophilus N2, produced inhibition zones on lawns of C . botulinum spores at 30 degrees C . L . plantarum BN, L . lactis ATCC 11454, and P . pentosaceus ATCC 43200 and 43201 were bacteriocinogenic at 4, 10, and 15 degrees C . Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays . Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L . plantarum BN and L . lactis ATCC 11454 which had been grown at 4 and 10 degrees C . These results suggest that low levels of L . plantarum BN or L . lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards.

Microbiol Rev, 1991 Dec, 55(4), 621 - 48
Molecular genetics and pathogenesis of Clostridium perfringens; Rood JI et al.; Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals . Recently significant advances have been made in the development of C . perfringens genetics . Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C . perfringens genes and the construction of shuttle plasmids . The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated . A detailed physical map of the C . perfringens chromosome has been prepared, and numerous genes have been located on that map . Reproducible transformation methods for the introduction of plasmids into C . perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned . Now that it is possible to freely move genetic information back and forth between C . perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C . perfringens infections.

J Clin Microbiol, 1991 Dec, 29(12), 2724 - 30
Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C . difficile-associated disease; DiPersio JR et al.; A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use . Specimen centrifugation and filtration were not required . The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool) . The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii . The EIA was compared with the cytotoxin assay, culture of toxigenic C . difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C . difficile-associated disease . Results read visually and with a plate reader were similar . Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods . The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay . The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods . Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method . Of 34 patients determined to have C . difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination . Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture . The EIA demonstrated high specificity and good sensitivity for C . difficile-associated disease cases . The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.

Epidemiol Infect, 1991 Dec, 107(3), 659 - 65
Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics; Riley TV et al.; Cats and dogs being treated at two veterinary clinics were investigated for gastrointestinal carriage of Clostridium difficile using selective solid and enrichment media . Thirty-two (39.5%) of 81 stool samples yielded C . difficile . There were significant differences in isolation rates between clinics, 61.0% of animals being positive at one clinic compared to 17.5% at the other (Chi-square, P less than 0.005) . Of 29 animals receiving antibiotics, 15 (52.0%) harboured C . difficile while 11 (23.9%) of 46 animals not receiving antibiotics were positive (Chi-square, P less than 0.01) . There was no difference in carriage rate between cats (38.1%) and dogs (40.0%) . The environment at both veterinary clinics was surveyed for the presence of C . difficile . Fifteen of 20 sites at one clinic were positive compared to 6 of 14 sites at the other clinic . Both cytotoxigenic and noncytotoxigenic isolates of C . difficile were recovered from animals and environmental sites . These findings suggest that household pets may be a potentially significant reservoir of infection with C . difficile.

Epidemiol Infect, 1991 Dec, 107(3), 627 - 35
Relapse versus reinfection with Clostridium difficile; O'Neill GL et al.; Relapse of Clostridium difficile-associated diarrhoea occurs in 15-20% of patients; however, whether relapse is due to an endogenous source of the organism or reinfection from the environment remains unclear . Restriction enzyme analysis (REA) of chromosomal DNA was used to type multiple isolates from ten patients who had experienced apparent relapses . More than half the relapses were due to infection with a new strain of C . difficile . The remaining patients were infected with the same strain, but whether this strain was acquired from the environment or from endogenous sources could not be determined . Relapses with a different strain of C . difficile could occur if an individual harboured more than one strain in their gastrointestinal tract . To investigate this possibility ten other patients were assessed for carriage of multiple strains . Ten colonies from a primary culture plate from each patient were typed by REA and tested for their ability to produce cytotoxin . All isolates from the same patient were identical by both methods, indicating that multiple carriage of strains may be a rare event.

Mayo Clin Proc, 1991 Dec, 66(12), 1270 - 80
Tetracyclines, chloramphenicol, erythromycin, clindamycin, and metronidazole; Smilack JD et al.; The tetracyclines are effective in the treatment of Chlamydia, Mycoplasma pneumoniae, and rickettsial infections and also can be used for gonococcal infections in patients unable to tolerate penicillin . These drugs may cause gastrointestinal irritation, diarrhea, phototoxic dermatitis, and vestibular damage, and fatal reactions due to hepatotoxicity have occurred in pregnant women . Chloramphenicol has a broad spectrum of bacteriostatic activity, but its association with suppression of the bone marrow and aplastic anemia has relegated it to a historical role . Erythromycin is the drug of choice for the treatment of infections caused by M . pneumoniae, Legionella species, group A beta-hemolytic streptococci, and Streptococcus pneumoniae . The frequency of serious adverse effects associated with the use of erythromycin is low; dose-related epigastric distress may occur . Clindamycin is bactericidal to most nonenterococcal gram-positive aerobic bacteria and many anaerobic microorganisms . Although historically it was a frequent cause of antibiotic-associated diarrhea and colitis, clindamycin is considered an excellent alternative to beta-lactam antibiotics for treatment of many staphylococcal infections, and it has therapeutic utility in anaerobic infections and in several protozoan infections in immunosuppressed patients . Metronidazole is efficacious for treating nonpulmonary anaerobic infections, various parasitic infections (trichomoniasis, amebiasis, and giardiasis), nonspecific vaginitis, and Clostridium difficile-mediated colitis . With use of metronidazole, mild side effects such as epigastric discomfort, diarrhea, reversible neutropenia, and allergic-type cutaneous reactions may occur.

Biochem J, 1991 Dec 1, 280 ( Pt 2), 407 - 10
Kinetic mechanism of Clostridium perfringens phospholipase C . Hydrolysis of a thiophosphate analogue of lysophosphatidylcholine; Young PR et al.; The hydrolysis of S-{2-(hexadecanoyloxy)ethyl}thiophosphocholine (I), an analogue of lysophosphatidylcholine, by Clostridium perfringens phospholipase C, was followed at pH 7.5, 37 degrees C and I 1.0 (maintained with KCl), in a continuous assay, by monitoring the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) at 412 nm . Simple saturation kinetics are observed with linear mixed-type slope-intercept effects for the hydrolysis of compound (I) with variable {Ca2+} at fixed concentrations of compound (I) and a simple slope effect as {compound (I)} is varied at fixed concentrations of Ca2+ . These data are consistent with a simple ordered rapid-equilibrium mechanism in which Ca2+ binds to the enzyme first followed by substrate . The observed kinetic constants at pH 7.5, 37 degrees C and I 1.0 are K1 = 12.0 mM (Ca2+ dissociation), K2 = 36 microM {compound (I) dissociation} and Vmax . = 552 microM.min-1.mg-1 . Alkane diammonium salts inhibit the enzyme by a non-competitive mechanism that involves binding to free enzyme, E.Ca2+ and E.Ca2+.S . The use of the simple micellarized substrate under these conditions allows the determination of kinetic and inhibition constants without complications arising from enzyme-micelle interactions.

J Bacteriol, 1991 Dec, 173(24), 7956 - 62
Characterization of endoglucanase A from Clostridium cellulolyticum; Fierobe HP et al.; A construction was carried out to obtain a high level of expression in Escherichia coli of the gene celCCA, coding for the endoglucanase A from Clostridium cellulolyticum (EGCCA) . The enzyme was purified in two forms with different molecular weights, 51,000 and 44,000 . The smaller protein was probably the result of proteolysis, although great care was taken to prevent this process from occurring . Evidence was found for the loss of the conserved reiterated domains which are characteristic of C . thermocellum and C . cellulolyticum cellulases . The two forms were extensively studied, and it was demonstrated that although they had the same pH and temperature optima, they differed in their catalytic properties . The truncated protein gave the more efficient catalytic parameters on carboxymethyl cellulose and showed improved endoglucanase characteristics, whereas the intact enzyme showed truer cellulase characteristics . The possible role of clostridial reiterated domains in the hydrolytic activity toward crystalline cellulose is discussed.

J Bacteriol, 1991 Dec, 173(24), 7934 - 41
The bacteriocin lactococcin A specifically increases permeability of lactococcal cytoplasmic membranes in a voltage-independent, protein-mediated manner; van Belkum MJ et al.; Lactococcin A is a bacteriocin produced by Lactococcus lactis . Its structural gene has recently been cloned and sequenced (M . J . van Belkum, B . J . Hayema, R . E . Jeeninga, J . Kok, and G . Venema, Appl . Environ . Microbiol . 57:492-498, 1991) . Purified lactococcin A increased the permeability of the cytoplasmic membrane of L . lactis and dissipated the membrane potential . A significantly higher concentration of lactococcin A was needed to dissipate the membrane potential in an immune strain of L . lactis . Lactococcin A at low concentrations (0.029 microgram/mg of protein) inhibited secondary and phosphate-bond driven transport of amino acids in sensitive cells and caused efflux of preaccumulated amino acids . Accumulation of amino acids by immune cells was not affected by this concentration of lactococcin A . Lactococcin A also inhibited proton motive force-driven leucine uptake and leucine counterflow in membrane vesicles of the sensitive strain but not in membrane vesicles of the immune strain . These observations indicate that lactococcin A makes the membrane permeable for leucine in the presence or absence of a proton motive force and that the immunity factor(s) is membrane linked . Membrane vesicles of Clostridium acetobutylicum, Bacillus subtilis, and Escherichia coli were not affected by lactococcin A, nor were liposomes derived from phospholipids of L . lactis . These results indicate that lactococcin A acts on the cytoplasmic membrane and is very specific towards lactococci . The combined results obtained with cells, vesicles, and liposomes suggest that the specificity of lactococcin A may be mediated by a receptor protein associated with the cytoplasmic membrane.

Infect Immun, 1991 Dec, 59(12), 4338 - 42
Epitope mapping of the alpha-toxin of Clostridium perfringens; Logan AJ et al.; A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid . The toxin-binding activity of each monoclonal antibody was evaluated . The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities . In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures . Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays . Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule . This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.

J Anim Sci, 1991 Dec, 69(12), 4993 - 5000
Degradation of cellulose and forage fiber fractions by ruminal cellulolytic bacteria alone and in coculture with phenolic monomer-degrading bacteria; Varel VH et al.; We hypothesized that bacterial species capable of metabolizing phenolic monomers may act as catalysts for forage fiber breakdown by increasing microbial access to cell wall polysaccharides . Ruminal cellulolytic bacteria alone and in combination with phenolic-degrading bacteria were examined for differences in their ability to degrade fiber fractions of alfalfa or bromegrass . Electron micrographs of Fibrobacter succinogenes S85 cultured in combination with the ruminal phenolic-degrading organisms Eubacterium oxidoreducens G41 and Syntrophococcus sucromutans S195 indicated that bromegrass was degraded more extensively by the triculture than by the monoculture . The sequential detergent system was used to quantify the digestibility of fiber components from alfalfa and bromegrass . F . succinogenes incubated with the two phenolic-degrading organisms did not degrade more cell wall material than did F . succinogenes alone . However, with two other ruminal cellulolytic organisms, Clostridium longisporum B6405 and Ruminococcus albus B6403, greater (P less than .05, P less than .10, respectively) amounts of hemicellulose were degraded (72 h in vitro fermentation) from whole-plant alfalfa when E . oxidoreducens and S . sucromutants were combined with the cellulolytic species than when their monocultures were tested . Similar increases were not observed using a NDF preparation of alfalfa as the substrate . Based on these in vitro experiments, it does not seem that E . oxidoreducens and S . sucromutans play an important role in improving forage fiber degradation by cellulolytic ruminal bacteria.

Zentralbl Bakteriol, 1991 Dec, 276(1), 27 - 35
Collagenase of Clostridium perfringens type A: degradation of human complement component C1q; Wolf U et al.; The semipurified collagenases from Clostridium perfringens type A strains 2-Cli and ATCC 13124, both characterized by molecular weights of 79.4 kilodaltons, partially degraded purified human complement (C) component C1q . The following purified human serum proteins were refractory: C components C3, C4, C5, C6, C7, C8, and C9; immunoglobulin (Ig)A (from colostrum), IgG, and IgM; alpha 2-macroglobulin, haptoglobin, and C-reactive protein.

Enzyme Microb Technol, 1991 Dec, 13(12), 969 - 75
Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes; Freitag R et al.; An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described . The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein . Assay automation was achieved by utilizing the flow injection analysis (FIA) principles . The apparatus, a stopped-flow, merging-zone manifold, is described in detail . Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min . A method for simulating cultivation conditions was developed for assay optimization . Using this method, a detection limit of 1 mg l-1 together with a standard deviation of 1.5 was found . A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase . Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined . The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.

J Chromatogr, 1991 Nov 29, 587(1), 93 - 9
Purification of the coenzyme B12-containing 2-methyleneglutarate mutase from Clostridium barkeri by high-performance liquid chromatography; Michel C et al.; Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate delta-isomerase from Clostridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously . First, the mutase eluted before the delta-isomerase after incubation with the mild detergent 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent . Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order . Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (greater than 95% purity) which had a pink-orange colour (lambda max 280, 375, 470 and 532 nm) . The enzyme was active in the absence of coenzyme B12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).

FEBS Lett, 1991 Nov 18, 293(1-2), 59 - 61
The 65-kDa protein from pig heart . A new substrate for Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3); Hoffenberg SI et al.; In the pig heart sarcolemma, a 65 kDa protein is found to be ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3) . ADP-ribosylation of this protein is regulated by guanyl nucleotides and cytosol factor in a fashion similar to that for other C3 substrates . The new exoenzyme C3 substrate was partially purified . This protein is supposed to be a GTP-binding one.

Biochim Biophys Acta, 1991 Nov 15, 1080(3), 191 - 7
The partial amino acid sequence of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity; Lilley KS et al.; The amino acid sequence is reported for CNBr and tryptic peptide fragments of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum . Together with the N-terminal sequence, these make up about 75% of the total sequence . The sequence shows extensive similarity with that of the NADP(+)-dependent glutamate dehydrogenase of Escherichia coli (52% identical residues out of the 332 compared) allowing confident placing of the peptide fragments within the overall sequence . This demonstrated sequence similarity with the E . coli enzyme, despite different coenzyme specificity, is much greater than the similarity (31% identities) between the GDH's of C . symbiosum and Peptostreptococcus asaccharolyticus, both NAD(+)-linked . The evolutionary implications are discussed . In the 'fingerprint' region of the nucleotide binding fold the sequence Gly X Gly X X Ala is found, rather than Gly X Gly X X Gly . The sequence found here has previously been associated with NADP+ specificity and its finding in a strictly NAD(+)-dependent enzyme requires closer examination of the function of this structural motif.

Eur J Biochem, 1991 Nov 15, 202(1), 41 - 51
Limited proteolysis of tetanus toxin . Relation to activity and identification of cleavage sites; Krieglstein KG et al.; Tetanus toxin is synthesized by Clostridium tetani as a 151-kDa peptide chain . The primary gene product is processed post-translationally by removal of the initiating methionine residue, formation of disulfide bridges and limited proteolysis by bacterial or exogenous proteinases . The mature toxins consist of a 52-kDa light chain and a 98-kDa heavy chain, linked together by a disulfide bond . Proteolytic nicking is accompanied by increased pharmacological potency . To identify the structural alterations involved, single-chain toxin has been subjected to limited proteolysis with various enzymes . The new N-termini have been determined by Edman degradation and the C-termini by isolation of short C-terminal peptide fragments and subsequent analysis of the sequence and composition . All two-chain toxins result from proteolytic nicking within the 17-residue segment of residues 445-461 . Thus, the protease(s) of the culture broth cleave on the C-terminal side of Glu449 and partially Ala456, giving rise to two heavy chain N-termini . Trypsin and clostripain first attack the C-terminal of Arg454 and later Arg448, whereas endoproteinase Arg-C cleaves the former bond only . Chymotrypsin and endoproteinase Glu-C each split a single peptide bond, i.e . that located after Tyr452 and Glu449, respectively . Papain gives rise to a large number of cleavages within the 17-residue segment, the new C-terminus being Thr445 or Asn446 and the new N-terminus being Asp460 or Leu461 . Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864 . The original N-terminal Pro1 and C-terminal Asp1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests . The pharmacological activity of the various two-chain toxins was 5-11 times that of the single-chain toxin, as estimated from the inhibition of {3H}noradrenaline release from rat-brain homogenate . The present data on the processing and activation by limited proteolysis prove the existence of several active tetanus isotoxins . These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A., Weller, U . & Habermann, E . (1990) Eur . J . Biochem . 188, 39-45), provide the detailed protein chemical characterization of the tetanus isotoxins.

Biochemistry, 1991 Nov 12, 30(45), 10885 - 95
Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR; Blake PR et al.; The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C . The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein . However, P . furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C . One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized {Fe(III)Rd} and reduced {Fe(II)Rd} forms of P . furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies . The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls . However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons . Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra . Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca . 30% of the primary sequence . Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins . These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C . pasteurianum . However, the beta-sheet domain in P . furiosus Rd is larger than that in C . pasteurianum Rd and appears to begin at the N-terminal residue . From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed . These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.

Rev Infect Dis, 1991 Nov-Dec, 13(6), 1053 - 60
Use of the polymerase chain reaction for the specific and direct detection of Clostridium difficile in human feces; Gumerlock PH et al.; The polymerase chain reaction was used for the detection of Clostridium difficile, the etiologic agent of antibiotic-associated colitis . An upstream primer identical to a coding region (segment I) of the C . difficile 16S rRNA gene and a downstream primer complementary to a highly conserved region of eubacterial 16S rRNA served to amplify a targeted 270-base-pair fragment of genomic DNA . This technique allowed the detection of as few as 10 C . difficile organisms among 10(6) Escherichia coli bacteria . This level of sensitivity represents a 100-fold increase over that of conventional anaerobic culture . C . difficile was detected in DNA extracted directly from the stools of 23 patients with antibiotic-associated colitis and from those of four patients with diarrhea whose stools had been negative for C . difficile when assessed in a cytotoxicity assay . No amplification products were found in the stools of asymptomatic patients . When detected in stools of symptomatic patients, amplification products of C . difficile were confirmed by Southern blotting with a nonradioactive, horseradish peroxidase-catalyzed, chemiluminescent probing system in which biotin-labeled oligonucleotides were used . This system discriminates between C . difficile and similar organisms, such as Clostridium sordellii and Clostridium bifermentans . The combination of the polymerase chain reaction with enzyme-linked probing results in a faster and more sensitive assay for C . difficile than standard culture.

Br J Clin Pharmacol, 1991 Nov, 32(5), 621 - 3
Plasma metronidazole concentrations after single and repeated vaginal pessary administration; Salas-Herrera IG et al.; Metronidazole concentrations in plasma were measured by h.p.l.c . in 12 healthy female volunteers after single and repeated vaginal administration of 500 mg metronidazole pessaries . The area under the plasma concentration-time curve (AUC(0,12 h) was 8.4 +/- 3.9 micrograms ml-1 h (mean +/- s.d.) on day 1 and 20.6 +/- 7.1 micrograms ml-1 h (mean +/- s.d.) on day 5 . The peak plasma drug concentration on day 1 was 1.2 +/- 0.6 micrograms ml-1 (mean +/- s.d.) and on day 5 it was 2.0 +/- 0.7 micrograms ml-1 (mean +/- s.d.) . The plasma concentration of metronidazole at steady state was above the minimum inhibitory concentration (MIC) for anaerobic Streptococci and Clostridium tetani . These results demonstrate much lower systemic exposure than after oral administration.

Biochem J, 1991 Nov 1, 279 ( Pt 3), 787 - 92
Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains; Poole DM et al.; The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp . cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum . A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P . fluorescens subsp . cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus . The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained . The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively . Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively . The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD . The putative role of the P . fluorescens subsp . cellulosa CBD in cellulase action is discussed.

Microb Pathog, 1991 Nov, 11(5), 337 - 46
Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B; Florin I; A mutant of Chinese hamster lung fibroblasts (Don cells), resistant against Clostridium difficile toxins A and B, was isolated after mutagenization with ethylmethanesulphonate and a two-step selection with toxin B . The mutant, termed CdtR-Q, was 10(4) times more resistant to toxin B than wild-type cells and cross-resistant to toxin A (10(3) times more resistant) . The resistance was overcome by increasing the dose of toxin . The resistance has been stable after cultivation for 40 generations in the absence of toxin . The morphology of the mutant was more epithelial-like than that of the fibroblast parental cells . The plating efficiency was about half that of the wild-type, whereas the growth rate was the same . The mutant was significantly less sensitive than the wild-type to the microfilament-interacting cytochalasins B and D . It was as sensitive as the wild-type to endocytosed toxins (diphtheria, pertussis, ricin), to microtubule-interacting agents (colchicine, gossypol, nocodazole, taxol, vinblastine), and to membrane-damaging toxins with different mechanisms of action, with one exception; the mutant was more highly sensitive to the action of phospholipase C (with broad substrate-specificity) than the wild-type . The results suggest that the mutant has a normal endocytosis, and that the mutation does not affect the microtubuli . The results are consistent with a mutation affecting the microfilaments in the cytoskeleton.

Lipids, 1991 Nov, 26(11), 957 - 9
Inhibition of the Clostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle-bound ammonium and sulfonium cations; Young PR et al.; Cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides inhibit the Clostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37 degrees C, mu = 0.15 with KCl . Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but non-linear function of the mole fraction of inhibitor . Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor . Inhibition constants for cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides are 0.66 +/- 0.04 and 0.25 +/- 0.02 mM, respectively . The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K50 for dodecamethylene-bis(dimethylsulfonium) bromide, 27 microM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.

Appl Biochem Biotechnol, 1991 Nov, 31(2), 119 - 34
Cloning and expression of a Clostridium thermocellum DNA fragment that encodes a protein related to cellulosome component SL; Romaniec MP et al.; Antibodies raised against the SL subunit of the Clostridium thermocellum cellulosome were used to screen a library of C . thermocellum chromosomal DNA fragments constructed in the vector lambda gt11 . A DNA fragment that encoded a polypeptide that crossreacted with the anti-SL antibodies was isolated and its restriction map elucidated . No similarity with other previously cloned DNA fragments has been found . The anti-SL crossreacting polypeptide was isolated from recombinant Escherichia coli and found to have a mol mass of 37,000 Da and to possess low levels of CMCase and Avicelase activity . Using CMC as the substrate, a temperature optimum of 55 degrees C and a pH optimum of 6.6 were observed . These properties were compared to those of C . thermocellum SL isolated by electroelution from an SDS gel, which was also found to possess low levels of CMCase and Avicelase activities . In addition, the SL proteins produced in C . thermocellum and E . coli were able to interact positively against Avicel with an endoglucanase (Ss) purified from the C . thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-Ss antibodies.

Biol Chem Hoppe Seyler, 1991 Nov, 372(11), 999 - 1005
Purification and some properties of the tungsten-containing carboxylic acid reductase from Clostridium formicoaceticum; White H et al.; Judged by properties observed during the purification and based on the sequence of the first 25 amino acids, the enzyme from Clostridium formicoaceticum catalysing the reversible reduction of non-activated carboxylic acids to aldehydes at the expense of reduced viologens, is astonishingly different from that found by us in C . thermoaceticum . According to native and SDS gel electrophoresis the reductase is nearly homogeneous after only 26-fold purification . The specificity for various substrates and artificial electron carriers is also broad, but V of the purified aldehyde dehydrogenase activity (54 U/mg enzyme for butanal) is about 1 order of magnitude lower than that of the enzyme from C . thermoaceticum . The reductase is a dimer of two identical subunits with an Mr of 67,000 each . Increased enzyme concentrations seem to lead to higher oligomers . Per dimer 11 +/- 1 iron, 16 +/- 1 acid labile sulphur, 1.4 tungsten and after permanganate oxidation 1.6 mol pterin-6-carboxylic acid have been found.

J Emerg Med, 1991 Nov-Dec, 9(6), 431 - 6
Nontraumatic gas gangrene: case report and review of emergency therapeutics; Corey EC; Gas gangrene is a well-recognized serious life and limb threatening emergency . In certain patients the source of this infection may be nontraumatic . We present an illustrative case of nontraumatic gas gangrene caused by Clostridium Septicum and review the literature regarding its emergency management . Early recognition of nontraumatic gas gangrene is crucial to reduction of mortality and morbidity . Emergent therapy requires resuscitation, surgical consultation, pharmacological therapy, and consideration of hyperbaric oxygen therapy . Early institution of a multifactorial approach to treatment may significantly reduce the mortality and morbidity of this life threatening disease.

J Gen Microbiol, 1991 Nov, 137 ( Pt 11), 2673 - 9
Classification of medically important clostridia using restriction endonuclease site differences of PCR-amplified 16S rDNA; Gurtler V et al.; Restriction maps were constructed of enzymically amplified 16S rRNA genes (rDNA) isolated from eight Clostridium species . Using maximum parsimony, a dendrogram was constructed from these and published 16S rRNA sequence data . Two distinct clusters were identified: cluster I contained C . difficile, C . sordelli, and C . bifermentans, and showed 30 of 35 restriction sites in common; cluster II contained C . tetani, C . perfringens C . sporogenes and C . botulinum C and G, and showed 20 of 35 restriction sites in common . Further analysis of cluster I organisms revealed that of five HpaII fragments, two were found in equal amounts in all organisms, one was found in varying amounts in all organisms, and two were found, in varying amounts, only in C . sordelli and C . bifermentans . C . sordelli-specific and C . bifermentans-specific HpaII fragments were demonstrated by Southern hybridization of rDNA . One HpaII site within the rDNA was present on most alleles in C . bifermentans, present on a minority of alleles in C . sordelli and absent in C . difficile . This suggested that there were two 16S rRNA alleles with different sequences present within each of the genomes of C . bifermentans and C . sordelli.

J Clin Microbiol, 1991 Nov, 29(11), 2678 - 9
Arm abscesses caused by Clostridium botulinum; Elston HR et al.; Wound botulism is an uncommon disorder that continues to be rarely reported in the United States . A 34-year-old intravenous heroin user was admitted to the Loma Linda, Calif., Veterans Administration hospital with multiple abscesses on his forearms . His clinical course was compatible with botulism, and his culture and serum were positive for Clostridium botulinum toxin type A . Early culture and/or serum identification can lead to prompt diagnosis, treatment, and improvement in the morbidity and mortality rates of this disease.

J Clin Microbiol, 1991 Nov, 29(11), 2666 - 7
Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B; Fluit AC et al.; A total of 39 toxigenic and 20 nontoxigenic strains of Clostridium difficile were tested for the presence of either toxin A or toxin B by the polymerase chain reaction (PCR) . All toxigenic strains produced cytotoxin as assayed by using highly sensitive fetal lung fibroblasts and were positive for toxin A as well as toxin B in the PCR assay . All nontoxigenic strains failed to produce toxin and were negative in the PCR assay . This study shows that nontoxigenic strains of Clostridium difficile lack the toxin A as well as the toxin B gene.

J Clin Microbiol, 1991 Nov, 29(11), 2639 - 42
Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase; Lyerly DM et al.; Computer analysis showed that the gene encoding the latex test-reactive protein of Clostridium difficile exhibited high levels of homology with glutamate dehydrogenases from various sources . Further analysis demonstrated that the recombinant protein possessed glutamate dehydrogenase activity . Our results show that the protein that reacts in commercial latex tests for C . difficile is a glutamate dehydrogenase.

J Clin Microbiol, 1991 Nov, 29(11), 2633 - 5
Evaluation of cycloserine-cefoxitin-fructose agar and cycloserine-cefoxitin-fructose broth for recovery of Clostridium difficile from environmental sites; Clabots CR et al.; Cycloserine-cefoxitin-fructose agar (CCFA) and cycloserine-cefoxitin-fructose broth (CCFB) containing either 500 or 250 micrograms of cycloserine per ml were compared for efficacy in the isolation of Clostridium difficile from hospital ward environmental sites . A RODAC imprint technique was used to inoculate prereduced CCFA . Moistened swabs were used to inoculate prereduced CCFB from environmental sites immediately adjacent to the RODAC sample sites . CCFA (6% positive) was significantly more sensitive than CCFB (3% positive; P less than 0.005), regardless of the cycloserine concentration . When the CCFA cycloserine concentration was decreased from 500 to 250 micrograms/ml, the overall rate of positive cultures rose from 4 to 17% . Medium containing 500 micrograms of cycloserine per ml may be too inhibitory to isolate many moderately sensitive strains of C . difficile from environmental sites . Regardless of the cycloserine concentration, the CCFA RODAC imprint technique is superior to the CCFB method.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 51 - 5
Evaluation of the proposed interaction of nucleic acid with Clostridium difficile toxins A and B and the effects of nucleases on cytotoxicity; Borriello SP et al.; Both DNA and RNA were found to co-purify with Clostridium difficile toxin B but not toxin A . DNAase treatment greatly reduced the cytotoxicity of toxin B but not of toxin A . RNAase had no effect on either toxin . The effects on toxin B were shown to be due to a contaminating protease and could be inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 41 - 4
Enzyme-linked immunosorbent assay for rapid detection of toxins from Clostridium perfringens; Nagahama M et al.; An enzyme-linked immunosorbent assay (ELISA) with antibodies specific to beta, epsilon and iota ib toxins of Clostridium perfringens was developed to detect beta, epsilon and iota ib toxins, respectively . The ELISA was sensitive enough to detect as little as 1.0 ng/ml of purified beta and iota ib toxins and 0.1 ng/ml of purified epsilon toxin . By means of the ELISA method, 192 isolates of C . perfringens from food samples in Japan and Thailand, and 58 isolates from patients suffering from gas gangrene or gastroenteritis were examined . One isolate from food samples in Japan, three from food samples in Thailand and five from stools of patients with gastroenteritis were C . perfringens type D . One type B and one type C were detected from the stools of patients with gastroenteritis.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 15 - 21
Growth, sporulation and enterotoxin production by Clostridium perfringens type A in the presence of human bile salts; Heredia NL et al.; The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined . Each bile salt inhibited growth to a different degree . A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism . Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320 . A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C . perfringens strains NCTC 8239 and NCTC 8679 . The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested . No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.

Hokkaido Igaku Zasshi, 1991 Nov, 66(6), 841 - 8
{The structure and function of botulinum type C neurotoxin}; Kimura K; The structure gene for botulinum type C neurotoxin was cloned from the toxigenic bacteriophage obtained from Clostridium botulinum type C, and the whole nucleotide sequence was determined . The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000 . The signal peptide was not found after the first methionine residue . Upstream of the ATG codon, sequences predicted as a Shine-Dalgarno and a promoter were found . When the deduced amino acid sequence of type C toxin was compared with those of type A and D botulinum toxins and tetanus toxin, type C toxin shared about 52% identity with type D toxin, but shared only about 33% identity with type A and tetanus toxins . The structure and function of type C toxin were estimated from the results of epitope map with monoclonal antibodies and DNA thermal stability map.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 615 - 22
ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility; Stasia MJ et al.; A 24-kDa G protein, ADP-ribosylable by exoenzyme C3 from Clostridium botulinum and therefore related to the rho family, was found to be abundantly present in human and bovine neutrophils, and preferentially located in cytosol . In human myeloid HL60 cells, the amount of C3 substrate increased during differentiation of the HL60 cells into granulocytes . The effect of exoenzyme C3 on different functions of bovine neutrophils, namely generation of O-2, degranulation and chemotaxis, has been tested, using electropermeabilized cells . Exoenzyme C3 hardly affected the respiratory burst and the degranulation . In contrast, it efficiently inhibited the spontaneous and chemoattractant-induced motility of the cells and disorganized the actin microfilament assembly.

FEBS Lett, 1991 Oct 21, 291(2), 336 - 40
Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac; Koch G et al.; Mastoparan, which has been shown to active G proteins, inhibits the ADP-ribosylation of 20 kDa human platelet membrane proteins catalyzed by Clostridium botulinum exoenzyme C3 half-maximally and maximally (90%) at 20 and 100 microM concentrations, respectively . Inhibition of ADP-ribosylation was enhanced by GTP-gamma S . Mastoparan increased GTP hydrolysis by porcine brain rho protein and stimulated GTP binding in a concentration dependent manner . The data suggest that mastoparan not only interacts with heterotrimeric G proteins but also with low molecular mass GTP-binding proteins of the rho/rac family.

FEBS Lett, 1991 Oct 21, 291(2), 185 - 8
Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components; Tokatlidis K et al.; The function of the non-catalytic, duplicated segment found in C . thermocellum cellulases was investigated . Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross-reacted with a variety of cellulosome components ranging between 50 and 100 kDa . 125I-labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit . 125I-labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein . The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.

Biochem Biophys Res Commun, 1991 Oct 15, 180(1), 416 - 22
Utilization of methoxylated aromatic compounds by the acetogen Clostridium thermoaceticum: expression and specificity of the co-dependent O-demethylating activity; Daniel SL et al.; The aromatic CO-dependent O-demethylating activity of Clostridium thermoaceticum was evaluated . Secondary aromatic substituent groups (-OH, -CO2H, -CH2OH, and -OCH3) were critical to O demethylation . O-demethylating activities and specificities were similar from cells grown at the expense of different methoxylated aromatic compounds; all O-methyl-grown cells catalyzed the same sequential O demethylation of multi-methoxylated compounds, suggesting that a broad specificity O demethylase was involved in O demethylation . In cell-fractionation studies, CO-dependent O demethylation was catalyzed by membrane-associated components.

J Biol Chem, 1991 Oct 15, 266(29), 19312 - 9
Clostridium botulinum C3 ADP-ribosyltransferase gene . Cloning, sequencing, and expression of a functional protein in Escherichia coli; Nemoto Y et al.; C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells . The enzyme was purified from a culture filtrate of C . botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation . Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction . A 2.1-kilobase pair HincII fragment of C . botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment . The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids . Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium . Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E . coli, exported into the periplasm and released into the culture medium.

FEMS Microbiol Lett, 1991 Oct 15, 67(3), 347 - 9
Lithotrophic growth and hydrogen metabolism by Clostridium magnum; Bomar M et al.; Clostridium magnum, originally described as a non-autotrophic homoacetogenic bacterium, was found to be able to grow with H2/CO2, formate, or methanol with stoichiometric acetate formation, provided that the growth medium contained at least 0.025% (w/v) yeast extract . Hydrogen was also formed as a byproduct of glucose fermentation, and was consumed again after glucose consumption . Hydrogen formation from glucose was independent of growth conditions and reached similar maximal concentrations in mineral media with or without ammonia added as well as in non-growing cultures or in the presence of carbon monoxide.

Lakartidningen . 1991 Oct 9;88(41):3374, 3377, 3379.
{The Norrköping study . Cephalosporins are often the implicating factors in Clostridium difficile infections}; Eriksson S et al.; In a retrospective study carried out at Norrkoping Central Hospital, the incidence of Clostridium difficile-associated diarrhoea and colitis was found to be correlated to in-patient consumption (in terms of defined daily doses) of the implicated anti-microbial agents . The third generation cephalosporin, cefotaxime, was implicated 38 times more often than small spectrum penicillins . In general, the cephalosporins were predominantly responsible, accounting for 46 per cent (67/147) of the episodes but only 12 per cent of overall consumption of antibiotics at the hospital . These findings are in accord with data previously published in the nationwide report by the Medical Product Agency, Uppsala.

Biochemistry, 1991 Oct 8, 30(40), 9697 - 704
Primary structure of hydrogenase I from Clostridium pasteurianum; Meyer J et al.; Peptides obtained by cleavage of Clostridium pasteurianum hydrogenase I have been sequenced . The data allowed design of oligonucleotide probes which were used to clone a 2310-bp Sau3A fragment containing the hydrogenase encoding gene . The latter has been sequenced and was found to translate into a protein composed of 574 amino acids (Mr = 63,836), including 22 cysteines . C . pasteurianum hydrogenase is homologous to, but longer than, the large subunit of Desulfovibrio vulgaris (Hildenborough) {Fe} hydrogenase . It includes an additional N-terminal domain of ca . 110 amino acids which contains eight cysteine residues and which therefore could accommodate two of its postulated four {4Fe-4S} clusters . C . pasteurianum hydrogenase is most similar in length, cysteine positions, and sequence altogether to the translation product of a putative hydrogenase encoding gene from D . vulgaris (Hildenborough) . Comparisons of the available {Fe} hydrogenase sequences show that these enzymes constitute a structurally rather homogeneous family . While they differ in the length of their N-termini and in the number of their {4Fe-4S} clusters, they are highly similar in their C-terminal halves, which are postulated to harbor the hydrogen-activating H cluster . Five conserved cysteine residues occurring in this domain are likely ligands of the H cluster . Possible ligation by other residues, and in particular by methionine, is discussed . The comparisons carried out here show that the H clusters most probably possess a common structural framework in all {Fe} hydrogenases . On the basis of the available data on these proteins and on the current developments in iron-sulfur chemistry, the H clusters possibly contain six to eight iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Oct 8, 30(40), 9665 - 75
Optically detected triplet-state magnetic resonance studies of the DNA complexes of the bisquinoline analogue of echinomycin; Alfredson TV et al.; The polymeric DNA and model duplex oligonucleotide complexes of the bisquinoline analogue of echinomycin (2QN) have been studied by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoline chromophores of the drug used as intrinsic probes . Plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of 2QN which was ascribed to the presence of a major and minor conformation of the drug in aqueous solutions (referred to as the red and blue forms of 2QN, respectively, in this report) . ODMR results, in conjunction with findings from low-temperature phosphorescence investigations, indicate that the quinoline chromophores of the major (red) form of 2QN are involved in aromatic stacking interactions in complexes with the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, Clostridium perfringens, and calf thymus as evidenced by red shifts in the phosphorescence 0,0-band of the drug, reductions in the phosphorescence lifetime and zero-field splitting (zfs) D and E parameters, and polarity reversals of the ODMR slow passage signals upon complex formation between the analogue and DNA . The polarity reversals, which reflect shifts in the triplet-state sublevel populations induced by complex formation, apparently result from changes in the triplet sublevel decay constants upon binding to the natural DNAs . The 2QN complexes of the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) {abbreviated as poly{d(G-C)2}} and poly(dA-dT).poly(dA-dT) {abbreviated as poly(dA-dT).poly(dA-dT) {abbreviated as poly{d(A-T)2}, the homopolymer duplexes poly(dG).poly(dC) {abbreviated as poly(dG.dC)} and poly(dA).poly(dT) {abbreviated as poly(dA.dT)}, and the self-complementary oligonucleotides d(ACGT)2, d(TCGA)2, and d(ACGTACGT)2 were also investigated . The extent of reduction of the zfs D parameter (delta D) for the major form of 2QN upon complex formation with the polymeric DNAs was found to scale linearly with the standard free energy of the drug-DNA interaction (delta G degrees) calculated from previously reported binding studies for these targets {Fox, K . R., et al . (1980) Biochem . J . 191, 729-740} . This relationship between spectroscopic and thermodynamic properties of the 2QN-polynucleotide complexes is a consequence of the effects of base stacking interactions on the electronic states of the intercalator, which were postulated to arise from second-order shifts of the ground-state and the triplet-state energies of the complex on the basis of a modification of the solvent effect theory of van Egmond et al . {(1975) Chem . Phys . Lett . 34, 423-426}.

Scand J Gastroenterol, 1991 Oct, 26(10), 1000 - 6
Phospholipase C from Clostridium perfringens stimulates formation and release of platelet-activating factor (PAF-acether) in cultured intestinal epithelial cells (INT 407); Gustafson C et al.; This study demonstrates the ability of phospholipase C from Clostridium perfringens to stimulate the generation of platelet-activating factor (PAF-acether) in cultured intestinal epithelial cells (INT 407) . Cells were exposed to phospholipase C for up to 60 min, and the content of PAF-acether within the cells and in the extracellular medium was determined . Phospholipase C caused a time-dependent formation of PAF-acether within the cells and also release of PAF-acether to the medium . In contrast, phospholipase C did not affect the cellular acetylhydrolase activity or the ability of the cells to metabolize extracellularly added 14C-PAF-acether . These findings suggest the possibility that intestinal epithelial cells, when stimulated with a naturally occurring intestinal bacterial toxin, generate and release PAF-acether . The possibility that this might contribute to the pathophysiology of inflammatory bowel disease is discussed.

Am J Gastroenterol, 1991 Oct, 86(10), 1461 - 5
The chemotactic response of human granulocytes to Clostridium difficile toxin A is age dependent; Triadafilopoulos G et al.; Elderly patients are at high risk for developing diarrhea and colitis as a complication of antimicrobial therapy . Clostridium difficile, the causative agent of antibiotic-associated diarrhea and colitis produces an enterotoxin (toxin A) and a cytotoxin (toxin B) . Of these two exotoxins, toxin A appears to be largely responsible for the inflammatory phenomena of C . difficile colitis, because it produces secretion, pronounced granulocytic infiltration, and epithelial cell necrosis and ulceration in ligated ileal loops of experimental animals . We have recently demonstrated that the inflammatory effects of C . difficile toxin A in the intestine may be related to its ability to mobilize intracellular calcium and elicit a chemotactic response by human granulocytes . In this study, in order to explain why the elderly are at greater risk for developing antibiotic-associated colitis, we investigated the effects of toxin A on activation of granulocytes from healthy elderly and young subjects . Highly purified toxin A and the chemotactic factor N-formyl-Met-Leu-Phe (FMLP) at concentrations of 10(-7) M both elicited a significant (p less than 0.001) and comparable chemotactic and chemokinetic response in human granulocytes from both age groups . A significantly (p less than 0.001) increased chemotactic effect in elderly subjects compared with young subjects was elicited by toxin A and not by FMLP . These findings suggest that the enhanced intestinal inflammatory effects of C . difficile in the elderly, compared with the young, may be related to the ability of its enterotoxin to elicit a more pronounced chemotactic response by granulocytes.

South Med J, 1991 Oct, 84(10), 1263 - 5
Life-threatening complications of empiric ceftriaxone therapy for 'seronegative Lyme disease'; Nadelman RB et al.; Lyme disease, now the most common tick-borne illness in the United States, has recently received much media attention, due in part to its potentially serious sequelae in untreated patients . Because a rare patient with late illness may lack antibodies to the etiologic agent, Borrelia burgdorferi, physicians may be tempted to give empiric antibiotics for illnesses that may not be Lyme disease . We have described a patient who, despite negative laboratory evidence for late Lyme disease, was treated for 3 weeks with intravenous ceftriaxone and sustained serious complications, including granulocytopenia, fever, hepatitis, and Clostridium difficile-associated diarrhea . We caution physicians to weight carefully the risks of empiric treatment for ill-defined medical problems, and to recognize the hazards of even "safe" medications.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8288 - 92
Ultraviolet irradiation of DNA complexed with alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers; Nicholson WL et al.; UV irradiation of complexes of DNA and an alpha/beta-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased . The yields of pyrimidine dimers and spore photoproduct were less than 0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m2; in the absence of SASP the yields were reversed-4.5% and 0.3%, respectively . Complexes of DNA with alpha/beta-type SASP from Bacillus cereus, Bacillus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation . However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro . These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of alpha/beta-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form . The yields of spore photoproduct in vitro were significantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores . It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.

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