Clostridia

J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 3089 - 97
Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions; Gurtler V; To develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon {A, 15-1407 and B, 907-1407 (16S-16S); C, 1392-507 and D, 907-507 (16S-23S)} were enzymically amplified from 24 strains . When region A was hybridized to HindIII-digested genomic DNA isolated from C . difficile strains, all of the variable length restriction fragments hybridized . When region B was hybridized to HindIII-digested genomic DNA isolated from C . difficile strains, a set of variable length restriction fragments (Group II) hybridized predominantly . When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained . When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains . From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain . When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C . difficile strains ranging in size from 852-1210 bp . After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1993 Nov 30, 134(1), 107 - 11
Sequence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824; Walter KA et al.; The genes encoding both Clostridium acetobutylicum ATCC 824 butyrate synthesis pathway enzymes, phosphotransbutyrylase (ptb) and butyrate kinase (buk), were sequenced . The genes are immediately adjacent on the chromosome, with ptb preceding buk . A single transcription start point (tsp) was identified 57 bp upstream from the ptb start codon by primer extension analysis . The ptb and buk genes appear to form an operon . A putative Rho-independent terminator structure was identified 26 bp downstream from buk.

Biochim Biophys Acta, 1993 Nov 28, 1158(3), 333 - 8
Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes; Nishiki T et al.; The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9) . The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine . 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides . The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity . The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety . The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

Biochim Biophys Acta, 1993 Nov 21, 1153(1), 89 - 96
Membrane disorganization induced by perfringolysin O (theta-toxin) of Clostridium perfringens--effect of toxin binding and self-assembly on liposomes; Iwamoto M et al.; theta-Toxin (perfringolysin O) of Clostridium perfringens binds to membrane cholesterol with high (Kd approximately 10(-9) M) and low (Kd approximately 10(-7) M) affinities and causes membrane lysis of intact cells and liposomes . In order to understand the lytic process at the molecular level, the lysis of liposomes was investigated in comparison with that of intact cells . The toxin dose required to cause 50% lysis (RD50) of phosphatidylcholine/phosphatidylglycerol (82:18, mol/mol) liposomes containing 36-40 mol% cholesterol was 300-1400-times higher than the RD50 value for sheep or human erythrocytes when samples with the same cholesterol concentration were compared . However, the average number of toxin molecules bound per liposome vesicle at 50% lysis was estimated as 10-18 from the RD50 values, close to the number on erythrocytes at 50% lysis, suggesting that the number of toxin molecules adsorbed per vesicle is important for lysis . As to the toxin dose required for membrane lysis, no significant difference was observed between liposomes containing both high- and low-affinity toxin-binding sites and those containing only low-affinity sites, suggesting that theta-toxin molecules bound to low-affinity sites can assemble and cause membrane lysis as well as those bound to high-affinity sites . theta-Toxin assembles on liposomal membranes, as on erythrocytes, in a high-molecular-weight polymeric form as judged from sedimentation patterns in sucrose density-gradient centrifugation . The high-molecular-weight polymers were detected only under conditions where cell or liposome lysis occurred . At low toxin doses, slower sedimenting toxin oligomers and monomers were predominant on liposomal membranes . These results indicate that toxin assembly on membranes is essential for liposome lysis as it is for cell lysis and that assembly occurs on membranes without membrane proteins.

Biochim Biophys Acta, 1993 Nov 21, 1153(1), 53 - 8
Ionic factors regulating the interaction of Gardnerella vaginalis hemolysin with red blood cells; Cauci S et al.; We have studied the hemolytic properties of an exotoxin released by Gardnerella vaginalis (Gvh) . We found that hemolysis induced by Gvh is modulated by the composition of the isotonic buffer in which the red cells are suspended . In particular, low pH enhances its lytic activity, whereas low ionic strength and divalent cations diminish it . The inhibitory effects of reduced salt concentration and divalent cations occur despite normal binding of the toxin to the cells . This suggests that some post-binding step is impaired . The toxin is also able to damage cholesterol-containing lipid vesicles, and even on these model membranes it is more active at low pH . From this point of view, Gvh has some similarity to Clostridium perfringens theta-toxin, a membrane-damaging toxin belonging to the family of 'thiol-activated' cytolysins produced by Gram-positive bacteria.

J Biol Chem, 1993 Nov 15, 268(32), 23792 - 8
Single-stranded DNA binding activity of C1-tetrahydrofolate synthase enzymes; Wahls WP et al.; In eukaryotes C1-5,6,7,8-tetrahydrofolate (THF) synthase is a trifunctional enzyme that catalyzes the interconversion of reduced forms of folate to supply activated one-carbon units required for a variety of metabolic pathways . The enzymatic activities include 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) . In bacteria separate, monofunctional or bifunctional polypeptides catalyze the same reactions . We have purified C1-THF synthase from the fission yeast Schizosaccharomyces pombe and found its physical and enzymatic properties similar to those of other eukaryotic C1-THF synthase enzymes . Unexpectedly, the S . pombe enzyme bound strongly (Keq = 100 pM) to single-stranded DNA, but not to double-stranded DNA or to RNA . The binding was sequence-independent, apparently not cooperative, and not detectably inhibited by C1-THF synthase substrates or cofactors . Trifunctional cytoplasmic enzyme from Saccharomyces cerevisiae and monofunctional (synthetase) enzyme from Clostridium acidiurici also bound tightly to single-stranded DNA, while bifunctional (dehydrogenase and cyclohydrolase) enzyme from Escherichia coli did not, suggesting that single-stranded DNA binding is a conserved function of the synthetase domain of C1-THF synthase enzymes.

FEMS Microbiol Lett, 1993 Nov 15, 114(1), 53 - 60
Cloning, nucleotide sequence and structural analysis of the Clostridium acetobutylicum dnaJ gene; Behrens S et al.; The complete dnaJ gene of Clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5' end of the gene as a probe . Nucleotide sequencing of a positively reacting 2.2-kb HincII fragment, contained in the recombinant plasmid pKG4, revealed that the reading frame of the dnaJ gene of C . acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated M(r) of 40376 and an isoelectric point of 9.54 . The deduced amino acid sequence showed high similarity to the DnaJ proteins of other bacteria (e.g . Escherichia coli, Bacillus subtilis) as well as of an archaeon (Methanosarcina mazei) and to the corresponding proteins of eukaryotes (Saccharomyces cerevisiae, Homo sapiens) . The areas of similarity included a conserved N-terminal domain of about 70 amino acids, a glycine-rich region of about 30 residues, and a central domain containing four repeats of a CXXCXGXG motif, whereas the C-terminal domain was less conserved . Northern (RNA) blot analysis indicated that dnaJ is induced by heat shock and that it is part of the dnaK operon of C . acetobutylicum . The 5' end (901 bp) of another gene (orfB), downstream of dnaJ and not heat-inducible, showed no significant similarity to other sequences available in EMBL and GenBank databases.

FEBS Lett, 1993 Nov 8, 334(1), 32 - 6
ADP-ribosylation of Rho proteins inhibits sperm motility; Hinsch KD et al.; The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins . They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme) . The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells . Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility . These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

J Biol Chem, 1993 Nov 5, 268(31), 23215 - 8
NAD-binding site of the C3-like ADP-ribosyltransferase from Clostridium limosum; Jung M et al.; Treatment of the Rho-ADP-ribosylating C3-like transferase from Clostridium limosum by ultraviolet irradiation in the presence of {carbonyl-14C}NAD incorporated 1 mol of label/mol of exoenzyme . Concomitantly, the transferase and NAD glycohydrolase activity was impaired . A peptide containing the radiolabel was obtained by proteolysis with either staphylococcal protease V8 or trypsin . Their amino acid sequences were Ala/Asp-Gly-Tyr-Ile-Glu-Pro-Ile-Ser-Thr-Phe-Lys-Gly-Gln-Leu-X-Val-Leu-Le u-Pro- Arg and Gly-Gln-Leu-X-Val-Leu-Leu-Pro-Arg, respectively . These sequences correspond with regions Ala-160 through Arg-179 and Gly-171 through Arg-179, respectively, of the very similar Clostridium botulinum C3 transferase, with X being Glu in the unlabeled enzyme . This identifies the glutamic acid residue that corresponds to Glu-174 of C . botulinum C3 transferase as part of the NAD-binding site of the catalytic center of the C . limosum exoenzyme.

Protein Eng, 1993 Nov, 6(8), 947 - 52
Properties conferred on Clostridium thermocellum endoglucanase CelC by grafting the duplicated segment of endoglucanase CelD; Tokatlidis K et al.; The DNA sequence encoding the duplicated 22 amino acid segment of Clostridium thermocellum endoglucanase CelD was fused to the 3'-terminus of the celC gene encoding C.thermocellum endoglucanase CelC . The presence of the duplicated segment endowed CelC with the capacity to form cytoplasmic inclusion bodies containing active enzyme when the hybrid gene was expressed in Escherichia coli . Inclusion body formation prevented proteolytic cleavage of the duplicated segment . The intact hybrid protein CelC-Cel'D was purified from inclusion bodies and characterized . In contrast to CelC, CelC-Cel'D was able to bind to CipA, a protein acting as a scaffolding component of the C.thermocellum cellulase complex (cellulosome) . However, the catalytic properties of CelC-Cel'D were similar to those of CelC . These results suggest that foreign proteins tagged with the duplicated segment could be incorporated into the cellulosome in order to modify the enzymatic properties of the complex . The formation of inclusion bodies by proteins carrying the duplicated segment may also prove a convenient means of purifying cloned gene products that are sensitive to proteolytic degradation.

Enferm Infecc Microbiol Clin, 1993 Nov, 11(9), 479 - 81
{Yield of detection of Clostridium difficile toxin versus stool culture in the study of nosocomial diarrhea}; Rabasa M et al.; BACKGROUND: The aim of the study was to determine whether the detection of Clostridium difficile toxin in stools may be more profitable than conventional stool cultures for the etiologic study of nosocomial diarrhea and to analyze what risk factors favor the development of nosocomial diarrhea by C . difficile . METHODS: The presence of enteropathogens and A and B toxins of C . difficile were investigated (by monoclonal antibody enzymoimmunoassay) in stools of patients with nosocomial diarrhea . A series of patients simultaneously admitted without diarrhea were selected as the control group . RESULTS: During a 6 month period 92 patients with nosocomial diarrhea and 82 controls without diarrhea were studied . The C . difficile toxin was detected in 8 of these 174 patients (4.6%) . Eight point seven percent of the nosocomial diarrheas were related with C . difficile while only 1% were due to an enteropathogen (Salmonella enteritidis) . C . difficile toxin was not detected in any patient who did not have diarrhea . In comparison with the patients with diarrhea due to other causes, the patients with diarrhea by C . difficile had more frequently received antibiotics over the previous 7 days (57 vs 88%) and had been hospitalized for a longer time (> or = 7 days) (58 vs 88%) (p < 0.05) . CONCLUSIONS: In the author's institution infection by Clostridium difficile is the most frequent cause of nosocomial infectious diarrhea, especially in patients admitted for a prolonged time or who receive antibiotics . The routine investigation of enteropathogens in the cases of nosocomial diarrhea does not seem justified while the detection of the A and B toxins of C . difficile may be more profitable.

Appl Environ Microbiol, 1993 Nov, 59(11), 3825 - 31
Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain; Zhou Y et al.; Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C . botulinum type E neurotoxin . Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C . butyricum and C . botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures . The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA . All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome . Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains . The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome . Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene . The toxin gene was not transferred between C . botulinum and C . butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures . Nontoxigenic C . butyricum or C . botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrob Agents Chemother, 1993 Nov, 37(11), 2509 - 13
In vitro activity of Bay Y3118 against anaerobic bacteria; Wexler HM et al.; The antimicrobial activity of a new quinolone, Bay Y3118, was determined against 326 strains of anaerobic bacteria and compared with the activities of ampicillin-sulbactam, cefotetan, clindamycin, imipenem, metronidazole, and sparfloxacin . The National Committee for Clinical Laboratory Standards-approved Wadsworth agar dilution technique with Brucella-laked blood agar was used throughout the study . Breakpoints used to determine the percent susceptible were 2 micrograms/ml for Bay Y3118 and sparfloxacin, 4 micrograms/ml for clindamycin, 8 micrograms/ml for imipenem, 16 micrograms/ml for metronidazole and ampicillin-sulbactam, and 32 micrograms/ml for cefotetan . Species tested included Bacteroides fragilis (57 strains), other B . fragilis group species (79 strains), Bacteroides gracilis (10 strains), other Bacteroides spp . (9 strains), Prevotella spp . (30 strains), Porphyromonas spp . (9 strains), Fusobacterium spp . (36 strains), Bilophila wadsworthia (14 strains), Clostridium spp . (36 strains), Peptostreptococcus spp . (20 strains), and gram-positive non-spore-forming rods (26 strains) . Bay Y3118 inhibited all but 1 of 326 anaerobic bacteria tested at the breakpoint level or lower.

AIDS, 1993 Nov, 7(11), 1441 - 7
Risk factors for Clostridium difficile-associated diarrhoea in HIV-infected patients; Hutin Y et al.; OBJECTIVE: To identify risk factors associated with a first episode of Clostridium difficile-associated diarrhoea (CDAD) in patients with HIV infection . DESIGN: A case-control study . SETTING: University teaching hospital HIV inpatient unit . PATIENTS AND METHODS: Nineteen HIV-infected patients with CDAD, defined as diarrhoea with positive stool culture for Clostridium difficile (CD) and positive stool cytotoxin B assay, were compared with 38 randomly selected controls (HIV-infected patients hospitalized on the ward on the day the matched case was diagnosed) . CD isolates were phenotyped by electrophoretic protein patterns . RESULTS: The incidence of CDAD among HIV-infected patients was 4.1/100 of patient-admissions . On univariate analysis, cases were more likely to have used clindamycin {11 out of 19 compared with four out of 38; odds ratio (OR) 19; 95% confidence interval (CI), 2-160; P = 0.0007}, and pyrimethamine (14 out of 19 compared with 13 out of 38; OR, 4.8; 95% CI, 1.4-16, P = 0.02) in the month before diagnosis, and to have had cerebral toxoplasmosis (12 out of 19 compared with 13 out of 38; OR, 2.8; 95% CI, 0.9-8.6; P = 0.09) . There was also a significant increase of the risk of CDAD as duration of hospitalization in the ward increased (chi 2 for trend, P = 0.007) . Multivariate models associated two risk factors with CDAD: clindamycin use (OR, 42; 95% CI, 2-813; P = 0.01), and prolonged hospitalization in the ward (OR, 3.6 per week in the ward; 95% CI, 1-13, P = 0.048) . Of 18 available CD isolates, 15 (83%) had identical electrophoretic protein pattern . CONCLUSIONS: Clindamycin use and prolonged hospitalization in the ward were the main risk factors associated with CDAD in this study . These observations, together with the occurrence of one major phenotype of CD, suggest nosocomial transmission of CD in the ward.

Appl Biochem Biotechnol, 1993 Nov, 43(2), 147 - 51
Cellulase Ss (CelS) is synonymous with the major cellobiohydrolase (subunit S8) from the cellulosome of Clostridium thermocellum; Morag E et al.; The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved . The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component Ss (CelS) from the cellulosome of ATCC strain 27405 . This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.

J Clin Microbiol, 1993 Nov, 31(11), 2861 - 5
Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases; Whittier S et al.; Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.) . The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C . difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens . Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay . However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs . The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay . Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%) . These data indicate that the four EIAs all have specific shortcomings . When using these EIAs, testing strategies that take these shortcomings into consideration should be developed.

Australas Radiol, 1993 Nov, 37(4), 399 - 400
Fatal Clostridium septicum myonecrosis; Hiew CY et al.; A 20 year old leukaemic patient with neutropaenia secondary to chemotherapy, who developed overwhelming sepsis, myonecrosis, vascular occlusion and necrotizing enterocolitis due to Clostridium septicum infection is described . Plain abdominal radiographs and a computed tomography scan of the abdomen and pelvis showed gas in the retroperitoneal soft tissues . Clostridium septicum septicaemia has a recognized association with malignancy and neutropaenia and has a high mortality if not diagnosed and treated early . Computed tomography scanning of the abdomen, pelvis and head is advised in any patient with a positive C . septicum blood culture.

Aktuelle Radiol, 1993 Nov, 3(6), 370 - 2
{Generalized clostridial infection in a 60-year-old patient with massive soft tissue emphysema on thoracic radiography}; Rand T et al.; We report a case of a 60-year-old patient with progressive soft tissue emphysema caused by infection with clostridium septicum . In contrast to a rather linear spread of air in non infectious soft tissue-emphysema, in this case a mainly vesicular spread of air in the soft tissue is noted on plain films . Together with the clinical history, this finding may indicate an infectious cause . The radiological interpretation is an important step in the diagnostical workup.

Am J Emerg Med, 1993 Nov, 11(6), 622 - 5
Gas gangrene from subcutaneous insulin administration; Chin RL et al.; A case of gas gangrene that caused intractable shoulder pain refractory to narcotics in an immunocompromised host is presented . Gas gangrene has been associated with severe trauma involving penetrating wounds, compound fractures, extensive soft-tissue injury, intramuscular injection of epinephrine, and interruption of arterial blood supply . This case describes an elderly insulin-dependent diabetic woman who developed gas gangrene in her arm and leg at the site of her subcutaneous insulin injections . The responsible organism was Clostridium septicum . Emergency medicine physicians must consider gas gangrene Clostridium infection in immunocompromised individuals without evidence of trauma who present with localized and intractable pain.

J Bacteriol, 1993 Nov, 175(22), 7260 - 8
Comparative analysis of C3 and botulinal neurotoxin genes and their environment in Clostridium botulinum types C and D; Hauser D et al.; The C3 exoenzyme gene is located on a bacteriophage in Clostridium botulinum types C and D (M . R . Popoff, D . Hauser, P . Boquet, M . W . Eklund, and D . M . Gill, Infect . Immun . 59:3673-3679, 1991) . A derivative CN phage from phage C of C . botulinum Stockholm (C-St) (K . Oguma, H . Iida, and K . Inoue, Jpn . J . Microbiol . 19:167-172, 1975), isolated as neurotoxin negative, also does not produce exoenzyme C3 . The botulinal neurotoxin C1 gene is present on the CN phage but contains a stop mutation in the DNA region encoding the N-terminal part of the heavy chain (codon 553) . The putative truncated botulinal neurotoxin C1 protein was not recovered in a C . botulinum strain harboring the CN phage . We found that the C3 gene is localized on a 21.5-kbp DNA fragment flanked by the core motif 5'-AAGGAG-3' in DNAs of phage C of C . botulinum 468 (C-468), C-St phage, and phage D of C . botulinum 1873 (D-1873) . The 21.5-kbp DNA fragment is deleted in CN phage DNA, and the motif 5'-AAGGAG-3' is present only in one copy at the deletion junction, but the deletion in the CN phage could be nonspecific, since this phage was obtained by nitrosoguanidine treatment . These findings could indicate that the C3 gene is localized on a 21.5-kbp mobile element . C . botulinum type C strain 003-9 produces a C3 exoenzyme (Y . Nemoto, T . Namba, S . Kozaki, and S . Narumiya, J . Biol . Chem . 266:19312-19319, 1991), and Staphylococcus aureus E1 produces a related C3 enzyme which is named epidernmal cell differentiation inhibitor (S . Inoue, M . Sugai, Y . Murooka, S . Y . Paik, Y . M . Hong, H . Oghai, and H . Suginaka, Biochem . Biophys . Res . Comm . 174:459-464, 1991) and which shares 80.6 and 56.6% similarity, respectively with the C3 enzymes from C-468 or C-St and D-1873 phages athe amino acid level . The features of the putative 21.5-kbp transposon were not found in C . botulinum 003-9 and S . aureus E1, as determined by analysis of the C3 and epidermal cell differentiation inhibitor gene-flanking DNA regions . These data suggest a common ancestral origin and divergent evolution of the C3 genes in these three groups of bacterial strains and dissemination of a 21.5-kbp element carrying the C3 gene C-468, C-St, and D-1873 phages.

J Bacteriol, 1993 Nov, 175(21), 7119 - 22
The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases; Takagi M et al.; We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE) . The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method . The HBD had specific interactions with endoglucanases (EngB and EngD) from C . cellulovorans . These results indicated that the HBD was an endoglucanase binding site of CbpA.

J Bacteriol, 1993 Nov, 175(21), 6959 - 69
Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis; Fischer RJ et al.; A DNA region of Clostridium acetobutylicum contiguous with the adc operon has been cloned and sequenced . Structural genes encoding the acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (ctfB and ctfA) and an alcohol/aldehyde dehydrogenase (adhE) could be identified . These three genes together with a small open reading frame (ORF) of unknown function (upstream of adhE) formed an operon (sol operon), as shown by mRNA analyses . The complete sol operon was transcriptionally induced or derepressed before the onset of solventogenesis, thus confirming earlier results of Northern hybridizations with a ctfB gene probe (U . Gerischer and P . Durre, J . Bacteriol . 174:426-433, 1992) . Upstream of the sol operon, we identified two putative promoters that were located in regions with possible stem-loop structures formed by several inverted repeats . The distal promoter P1 showed only minor transcription initiation in solventogenic C . acetobutylicum cells but was recognized in Escherichia coli, presumably because of its high similarity to the sigma 70 consensus sequence . The adhE-proximal promoter P2 directed the major transcription start point in solventogenic C . acetobutylicum but was not recognized in E . coli . The clostridial AdhE showed high similarity to a novel family (type III) of alcohol dehydrogenases . Two other ORFs (ORF 5 and ORF 6) were found on the cloned DNA region that showed no significant similarity to sequences in various available data bases . mRNA studies revealed that ORF 5 formed a monocistronic operon and showed increased expression before onset of solventogenesis.

Res Microbiol, 1993 Nov-Dec, 144(9), 741 - 53
Quantitative investigations into the elimination of in vitro-obtained spores of the non-pathogenic Clostridium butyricum strain CNRZ 528, and their persistence in organs of different species following intravenous spore administration; Fabricius EM et al.; Before the Clostridium tumour assay can be applied to the diagnosis of cancer, we sought to investigate--within the framework of a biopharmaceutical safety test--the organ persistence of test spores of Clostridium butyricum CNRZ 528 . We found that non-pathogenic spores obtained in vitro, like pathogenic native spores, escape phagocytosis in various organs up until about 2 years, as tested by anaerobic cultures . The elimination of spores depended on the species of animal, the spore dose and the organs investigated . In rabbits, one week after injection, we recovered clostridial spores from blood and spleen cultures more rarely than from liver and lung . The half-life of blood clearance in patients was one day or, at half the spore dose, two days . That deep tissues of healthy animals are not normally sterile became evident in rabbits after sporadic isolation and characterization of non-administered saccharolytic and proteolytic clostridial species . During a 10-year observation period, the rate of obtainment of viable spores by in vitro cultures lessened; however, for administration of the spores in clinical phase I and phase II studies, the spore quality was acceptable.

J Emerg Med, 1993 Nov-Dec, 11(6), 677 - 83
Tetanus prophylaxis following ocular injuries; Benson WH et al.; The administration of prophylaxis against tetanus following a corneal abrasion is routinely performed in many acute care facilities, despite a lack of support in the literature for its necessity . The risk of developing clinical tetanus from three different types of injuries to the eye was evaluated in an animal model . Clinical tetanus was induced in unimmunized mice by injecting Clostridium tetani organisms or toxin into the anterior chamber . Immunized mice injected intracamerally did not develop signs of tetanus . Tetanus was not induced by topical inoculation of either live organisms or toxin following corneal epithelial debridement or stromal scarification of unimmunized and immunized mice . The results of this study support the administration of prophylaxis against tetanus following perforating ocular injuries . However, our results do not support its routine use following uncomplicated corneal abrasions or other types of nonperforating ocular injuries.

Eur J Epidemiol, 1993 Nov, 9(6), 671 - 3
A case of infant botulism associated with honey feeding in Italy; Fenicia L et al.; A case of infant botulism in a 9 week-old female is described . A strain of C . botulinum type B was isolated from the feces of the baby . The epidemiologic study detected in a sample of home canned honey Clostridium botulinum spores of the same serotype that was isolated from the patient . The honey had been used only to sweeten the pacifier of the baby . This is the first case of infant botulism in Europe linked conclusively to honey.

Eur J Clin Microbiol Infect Dis, 1993 Nov, 12(11), 882 - 6
Comparison of four methods in the diagnosis of Clostridium difficile disease; Mattia AR et al.; Nine hundred forty-five stool specimens from patients suspected of having Clostridium difficile disease were examined using a cell culture cytotoxicity assay (CTA), two enzyme immunoassay (EIA) kits (Cytoclone for toxins A and B; VIDAS for toxin A) and a latex agglutination assay (CDT) . One hundred nineteen specimens had positive titers (> or = 90) in the CTA; clinical review of 16 discordant samples and 49 controls supported the significance of 90 as the positive cut-off titer . The performance of the two EIAs and the latex assay was assessed relative to CTA titers of the samples . Sensitivity was < or = 50% for all three assays for the 24 specimens with CTA titers of 90, but it reached 97-100% for the two EIAs and 84% for the latex assay at titers of > or = 2,250 . The Cytoclone EIA exhibited higher sensitivity at the lower positive titers . Overall, specificity of the methods ranged from 96.7% (CDT latex assay) to 99.1% (Cytoclone EIA).

Zygote, 1993 Nov, 1(4), 325 - 31
A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs; Mabuchi I et al.; Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins . C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation . No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining . When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows . C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs . By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation . Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow . This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis . These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.

Mol Microbiol, 1993 Nov, 10(3), 627 - 34
Activation and mechanism of Clostridium septicum alpha toxin; Ballard J et al.; Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48 kDa . The 48 kDa toxin was found to be an inactive protoxin (ATpro) which could be activated via a carboxy-terminal cleavage with trypsin . The cleavage site was located approximately 4 kDa from the carboxy-terminus . Proteolytically activated ATpro had a specific activity of approximately 1.5 x 10(6) haemolytic units mg-1 . The trypsin-activated toxin (ATact) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of M(r) > 210,000 on erythrocyte membranes . ATpro did not exhibit these properties . ATact formed pores with a diameter of at least 1.3-1.6 nm . We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.

Eur J Pharmacol, 1993 Oct 26, 243(3), 213 - 9
Differential effects of Mandevilla velutina compounds on paw oedema induced by phospholipase A2 and phospholipase C; Neves PC et al.; This study compares the effect of Mandevilla velutina compounds with some anti-inflammatory drugs against phospholipase A2- and phospholipase C-induced rat hindpaw oedema . Injection of phospholipase A2 (Naja naja, 2.5-20 U/paw) and phospholipase C (Clostridium perfringens, 0.03-0.05 U/paw) caused a dose-and-time-related increase in paw oedema . Compounds MV 8608 (55 mumol/kg) and MV 8612 (32 mumol/kg, i.p.) inhibited phospholipase A2-induced paw oedema without interfering with phospholipase C-induced oedema . Local injection of both M . velutina compounds also partially attenuated the oedema evoked by phospholipases A2 and C . Dexamethasone (1.3 mumol/kg, p.o.) suppressed only phospholipase A2-induced paw oedema, while indomethacin (11 mumol/kg, p.o.) attenuated only the early phase of phospholipase C-induced oedema . By contrast, phenidone (616 mumol/kg, i.p.) inhibited only phospholipase C-induced oedema, while cyproheptadine (31 mumol/kg) and pyrilamine (100 mumol/kg, p.o.) inhibited only phospholipase A2 oedema . Treatment of animals with compound 48/80 markedly suppressed phospholipase A2-induced paw oedema and to a lesser degree the oedema caused by phospholipase C . Our results indicate that there are marked differences regarding the mechanisms underlying the paw oedema responses caused by phospholipase A2 and phospholipase C . In addition, our data show that M . velutina compounds cause potent and long-lasting inhibition of the pro-inflammatory action of phospholipase A2, an effect which may account for their reported anti-inflammatory activities.

FEBS Lett, 1993 Oct 18, 332(3), 268 - 72
Selective interaction of ferricyanide with cluster I of Clostridium pasteurianum 2{Fe4S4} ferredoxin; Bertini I et al.; Treatment of Clostridium pasteurianum ferredoxin (CpFd) with stoichiometric amounts of potassium ferricyanide results in the specific conversion of cluster I into a Fe3S4+ species while leaving cluster II unaltered . Ferricyanide-treated CpFd derivative has been purified and characterized through biochemical and spectroscopical techniques . The cluster conversion process is reversible and reconstitution of native CpFd has been afforded under appropriate conditions.

Eur J Biochem, 1993 Oct 15, 217(2), 791 - 8
Components of glycine reductase from Eubacterium acidaminophilum . Cloning, sequencing and identification of the genes for thioredoxin reductase, thioredoxin and selenoprotein PA; Lubbers M et al.; The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced . The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA) . The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains . The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC . The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA . The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase . Thioredoxin reductase and thioredoxin of E . acidaminophilum could be successfully expressed in E . coli.

Eur J Biochem, 1993 Oct 15, 217(2), 557 - 65
Purification and characterization of endoglucanase C from Clostridium cellulolyticum . Catalytic comparison with endoglucanase A; Fierobe HP et al.; An Escherichia coli clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum . This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa . N-terminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity . The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences) . These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles . Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different . Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA . The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention . The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA . It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters . CelCCC and CelCCA were found not to act synergistically on the tested substrates.

Gastroenterology, 1993 Oct, 105(4), 1045 - 9
Effects of intrasphincteric botulinum toxin on the lower esophageal sphincter in piglets; Pasricha PJ et al.; BACKGROUND: The toxin of Clostridium botulinum (BoTx) inhibits the release of acetylcholine from nerve terminals and causes paralysis of skeletal muscle . The present study examined the hypothesis that BoTx may have a similar effect on gastrointestinal smooth muscle . METHODS: Baseline lower esophageal sphincter (LES) pressures were obtained in five piglets, and normal saline was injected endoscopically into the LES . One week later, LES pressure was measured again, followed by injection of BoTx into the LES . After another week, LES pressure was measured again . RESULTS: Compared with a baseline LES pressure of 8.2 +/- 1.5 mm Hg, LES pressure decreased to 3.2 +/- 1.0 mm Hg after BoTx injection, a reduction of about 60% (P < 0.01) . By contrast, LES pressure did not change significantly after normal saline injection . The animals showed no evidence of toxicity . Data from other experiments showed that after injection with toxin, the LES responds normally to bethanechol and pentagastrin but displays a paradoxical response to edrophonium and cholecystokinin . CONCLUSIONS: BoTx is a potent inhibitor of resting LES tone . Its relatively specific anticholinergic effect may help clarify the role of cholinergic and noncholinergic pathways in the regulation of gastrointestinal sphincters.

Surg Clin North Am, 1993 Oct, 73(5), 1063 - 74
Pseudomembranous colitis; Counihan TC et al.; Pseudomembranous colitis is an inflammatory disease of the colon and rectum characterized by the development of elevated mucosal plaques . It usually is associated with antibiotic therapy and is caused by elaboration of toxin from the anaerobic bacterium, Clostridium difficile . The hallmark of treatment is orally administered vancomycin or metronidazole . The mortality rate is high in patients whose condition is not diagnosed and appropriately treated . Emergency surgery occasionally is needed for complications, including colonic perforation and toxic colitis.

Proteins, 1993 Oct, 17(2), 152 - 60
Influence of protein flexibility on the redox potential of rubredoxin: energy minimization studies; Shenoy VS et al.; A theoretical investigation of the protein contribution to the redox potential of the iron-sulfur protein rubredoxin is presented . Structures of the oxidized and reduced forms of the protein were obtained by energy minimizing the oxidized crystal structure of Clostridium pasteurianum rubredoxin with appropriate charges and parameters . By including 102 crystal waters, structures close to the original crystal structure were obtained (rms difference of 1.16 A), even with extensive minimization, thus allowing accurate calculations of comparative energies . Our calculations indicate an energy change of about -60 kcal/mol (2.58 eV) in the protein alone upon reduction . This energy change was due to both the change in charge of the redox site and the subsequent relaxation of the protein . An energy minimization procedure for the relaxation gives rms differences between the oxidized and reduced states of about 0.2 A . The changes were small and occurred in both the backbone and sidechain mainly near the Fe-S center but contributed about -16 kcal/mol (0.69 eV) to the total protein contribution . Although the neglect of certain effects such as electronic polarization may make the relaxation energies calculated an upper limit, the results indicate that protein relaxation contributes substantially to the redox potential.

J Pediatr Surg, 1993 Oct, 28(10), 1221 - 5; discussion 1225-6
Retinoic acid enhances killing of neuroblastoma cells by Newcastle disease virus; Reichard KW et al.; Newcastle disease virus (NDV), an avian pathogen, selectively replicates in and kills neuroblastoma (NB) cells, but not normal fibroblasts in vitro and in vivo in nude mice . NDV cytotoxicity towards NB cells is enhanced by N-myc oncogene amplification . To further define the antineoplastic effects of NDV, we examined NDV's interaction with NB cells following short-term exposure to the differentiating agent, all-trans retinoic acid (RA), and to neuraminidase . The human NB cell line IMR-32, after treatment with 50 mumol/L RA, became eight times more sensitive to NDV in a cytotoxicity assay . A time course study to determine the optimal incubation period of IMR-32 cells with RA indicated that a fourfold increase in sensitivity towards NDV killing occurred after only 8 hours of RA incubation prior to addition of virus . Maximal sensitivity was achieved at 24 hours of RA incubation and remained constant for longer incubation periods (up to 72 hours) . The sensitization of IMR-32 NB cells to NDV was constant for RA doses between 3 mumol/L and 50 mumol/L . Plaque formation, which indicates replication, virus spread and cytotoxicity by a single infectious virus particle, was also enhanced by RA . This effect does not appear to require N-myc amplification in the target NB cells since RA had similar effects upon the high N-myc (IMR-32) and the low N-myc expressing cells (SK-N-SH) . Enhanced sialylation has been shown by others to mediate the growth inhibitory effects of RA on a variety of tumor lines . Removal of sialic acid from the IMR-32 NB cell surface using Clostridium neuraminidase (2.7 mg/mL) inhibited 75% of NDV plaque formation . These results demonstrate that NDV killing of two NB cell lines is enhanced using clinically achievable levels of RA and that sialylation of the NB cell surface is important for virus binding and cytotoxicity.

J Wildl Dis, 1993 Oct, 29(4), 533 - 9
Seasonal prevalence of Clostridium botulinum type C in sediments of a northern California wetland; Sandler RJ et al.; The prevalence of Clostridium botulinum type C (% of positive sediment samples) was determined in 10 marshes at Sacramento National Wildlife Refuge (SNWR), located in the Central Valley of California (USA), where avian botulism epizootics occur regularly . Fifty-two percent of 2,200 sediment samples collected over an 18-mo period contained C . botulinum type C (both neurotoxic and aneurotoxic) which was present throughout the year in all 10 marshes . The prevalence of C . botulinum type C was similar in marshes with either high or low botulism losses in the previous 5 yr . Marshes with avian botulism mortality during the study had similar prevalences as marshes with no mortality . However, the prevalence of C . botulinum type C was higher in marshes that remained flooded all year (permanent) compared with marshes that were drained in the spring and reflooded in the fall (seasonal) . The prevalence of C . botulinum type C declined in seasonal marshes during the dry period . Similar declines did not occur in the permanently flooded marshes.

J Gen Microbiol, 1993 Oct, 139 ( Pt 10), 2399 - 407
Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius; Koivula TT et al.; Two starch-degrading enzymes produced by Bacillus acidocaldarius (renamed as Alicyclobacillus acidocaldarius) were identified . According to SDS-PAGE, the apparent molecular masses of the enzymes were 90 and 160 kDa . Eight peptide fragments and the N-terminal end of the 90 kDa polypeptide were sequenced . An oligonucleotide, based on the amino acid sequence of a peptide fragment of the 90 kDa protein, was used to screen a lambda gt10 bank of B . acidocaldarius, and the region encoding the 90 kDa protein was cloned . Unexpectedly, the ORF continued upstream of the N terminus of the 90 kDa protein . The entire ORF was 1301 amino acids (aa) long (calculated molecular mass 140 kDa) and it was preceded by a putative ribosomal binding site and a promoter . Computer analysis showed that the 1301 aa protein was closely related to an alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum . We suggest that the starch-degrading 160 kDa protein of B . acidocaldarius is an alpha-amylase-pullulanase, and the 90 kDa protein is a cleavage product of the 160 kDa protein . Another ORF, apparently in the same transcription unit, was found downstream from the amylase gene . It encoded a protein that was closely related to the maltose-binding protein of Escherichia coli.

Anal Biochem, 1993 Oct, 214(1), 222 - 6
Synthesis of N alpha-{3H}acetyl-L-lysine chloromethyl ketone and its use in the fluorographic detection of proteases; Nishikata M; Tritiated N alpha-acetyl-L-lysine chloromethyl ketone (ALCK) was synthesized on a laboratory scale for use as an active-site-directed affinity label in the fluorographic detection of proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The synthesis involved acetylation of N epsilon-benzyloxycarbonyl-L-lysine chloromethyl ketone with {3H}acetic anhydride just before the removal of the benzyloxycarbonyl group . By this method, {3H}ALCK with a specific activity of 250 mCi/mmol was obtained as a crystal . Trypsin, thrombin, plasmin, papain, and clostripain were inactivated by ALCK according to first-order kinetics . For fluorographic detection of proteases, enzyme samples were allowed to react with {3H}ALCK and then resolved by SDS-PAGE . Proteases that reacted with {3H}ALCK could be detected with a sensitivity equivalent to or higher than that of Coomassie brilliant blue R-250 staining . A trypsin-like protease in Pronase, clostripain as a contaminant in a commercial preparation of Clostridium histolyticum collagenase, and cysteine proteases in Porphyromonas gingivalis could be detected.

FEMS Microbiol Lett, 1993 Oct 1, 113(1), 87 - 92
Towards a phylogeny of the clostridia based on 16S rRNA sequences; Lawson PA et al.; The 16S rRNA sequences of 17 species of the genus Clostridium were determined by direct sequencing of their PCR amplified genes . The sequences were aligned with those from other known clostridial species and representative low G + C Gram-positive relatives, and a phylogenetic tree was constructed . It was evident from the comparative sequence analysis that the genus Clostridium as presently constituted is phylogenetically extremely heterogeneous . This study corroborates and extends earlier findings in showing that many non-sporeforming bacteria are phylogenetically closely intermixed with Clostridium species . The taxonomic implications of the phylogenetic findings are discussed.

FEMS Microbiol Lett, 1993 Oct 1, 113(1), 23 - 8
Clostridium bifermentans serovar malaysia: characterization of putative mosquito larvicidal proteins; Nicolas L et al.; The toxicity of Clostridium bifermentans serovar malaysia to mosquito larvae is due to protein toxins, belonging to a novel class of insecticidal toxins . Toxic extracts contains three major proteins of 66, 18 and 16 kDa . The 18-kDa and 16-kDa proteins are probably involved in toxicity . They are synthesised during sporulation, concomitant with activity . They are absent from non-toxic strains of C . bifermentans and are present at very low levels in non-toxic C . bifermentans serovar malaysia cultures produced at 42 degrees C . The 66-kDa protein is present throughout the growth phases of C . bifermentans serovar malaysia, and an immunologically related 66-kDa protein is present in non-toxic C . bifermentans strains.

Epidemiol Infect, 1993 Oct, 111(2), 257 - 64
A molecular characterization of Clostridium difficile isolates from humans, animals and their environments; O'Neill G et al.; It is generally accepted that most patients with Clostridium difficile-associated diarrhoea acquire the organism from the environment . Recently we demonstrated that household pets may constitute a significant reservoir of C . difficile through gastrointestinal carriage in up to 39% of cats and dogs . These findings suggested that direct transmission from household pets, or contamination of the environment by them, may be a factor in the pathogenesis of C . difficile-associated diarrhoea . To investigate this possibility, we examined isolates of C . difficile from humans, pets and the environment by restriction enzyme analysis (REA) and restriction fragment length polymorphism (RFLP) typing using enhanced chemiluminescence . Both REA and RFLP typing methods used Hind III digests of chromosomal DNA . A total of 116 isolates of C . difficile from pets (26), veterinary clinic environmental sites (33), humans (37) and hospital environmental sites (20) was examined . REA was far more discriminatory than RFLP typing and for all isolates there were 34 REA types versus 6 RFLP types . There was good correlation between the REA types found in isolates from pets and from the veterinary clinic environment, and between isolates from humans and from those found in the hospital environment . There was, however, no correlation between REA type of C . difficile found in pets and isolates of human origin . We conclude that there may still be a risk of humans acquiring C . difficile from domestic pets as these findings may be the result of geographical variation.

Cardiovasc Surg, 1993 Oct, 1(5), 494 - 8
Culture of intraluminal thrombus during abdominal aortic aneurysm resection: significant contamination is rare; Steed DL et al.; The significance of positive bacterial cultures from intraluminal thrombus in patients undergoing repair of abdominal aortic aneurysm remains controversial . Over the last 4 years, thrombus was cultured during aneurysm repair in 116 patients . All patients received cephalosporin antibiotic before and for 48 h after operation . Although none of the aneurysms appeared to be clinically infected, six patients (5.2%) had positive cultures . Four groups were identified based on the bacteria cultured: group I, coagulase-negative staphylococci, light growth (three patients); group II, coagulase-negative staphylococci, light growth and 'Streptococcus viridans' (one patient); group III, Bacillus sp., heavy growth (Gram-negative stain) (one patient); group IV, Clostridium perfringens, occasional growth (one patient) . One of the six patients died during resection; the other five are alive without graft infection at 5-24 (mean 12) months after operation . The absence of graft infection suggests that positive cultures were not clinically significant or were adequately covered by the antibiotic prophylaxis . The incidence of positive cultures was lower than previously reported . Routine culture of aneurysm thrombus in the absence of clinical infection is probably not cost-effective.

Eur J Clin Microbiol Infect Dis, 1993 Oct, 12(10), 784 - 6
In vitro activity of DMG-Mino and DMG-DM Dot, two new glycylcyclines, against anaerobic bacteria; Nord CE et al.; The in vitro activity of DMG-Mino and DMG-DM Dot against 350 anaerobic bacterial strains including anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, other Bacteroides species and fusobacteria was determined by the agar dilution method . Their activity was compared with that of minocycline, doxycycline, piperacillin, cefoxitin, imipenem, clindamycin and metronidazole . DMG-Mino and DMG-DM Dot and imipenem were the most active agents tested . DMG-Mino and DMG-DM Dot had in vitro activity superior to that of minocycline and doxycycline.

Biochemistry, 1993 Sep 28, 32(38), 9881 - 7
Effect of replacing conserved proline residues on the EPR and NMR properties of Clostridium pasteurianum 2{4Fe-4S} ferredoxin; Gaillard J et al.; Most of {4Fe-4S} proteins bind their metallic center by four cysteine residues, three clustered in a single stretch of seven amino acids and a remote fourth generally followed by a proline residue . Two such prolines in Clostridium pasteurianum 2{4Fe-4S} ferredoxin have been substituted by different amino acids and the resulting molecular variants studied with EPR and NMR spectroscopies . The isolated EPR contributions of the {4Fe-4S}+ clusters do not change much in all variants . The exact positions or the number of features composing the fully reduced EPR spectra built by the two interacting {4Fe-4S}+ S = 1/2 systems vary slightly but, in none of the proteins in which either proline 19 or 48 were substituted, do they indicate a major difference either in the folding of the ferredoxin or in the electronic structure of its clusters . A subset of paramagnetically shifted NMR signals is significantly affected by these replacements at both redox levels . The corresponding protons belong to two cysteines liganding the cluster close to the substitution . These data, combined with the presently available three-dimensional information, form the basis for partial assignments of the most shifted resonances in the NMR spectra of such proteins . The positions of intermediate lines in the NMR spectra of semireduced ferredoxins depend on the difference between the redox potentials of the two clusters; this difference is sensitive to the substitutions of either conserved proline residue by lysine.

J Mol Biol, 1993 Sep 20, 233(2), 325 - 7
Crystallization and preliminary X-ray analysis of the catalytic domain of endoglucanase from Clostridium cellulolyticum; Roig V et al.; The catalytic domain of an endoglucanase belonging to family A (CelCCA) from an anaerobic bacterium (Clostridium cellulolyticum) has been crystallized in a form suitable for X-ray diffraction analysis . The crystals have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique . The crystals, which diffract to 2.0 A resolution, belong to the orthorhombic space group P2(1)2(1)2(1) and have the following cell constants: a = 52.4 A, b = 76.2 A and c = 113.5 A.

Nature, 1993 Sep 9, 365(6442), 160 - 3
Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25; Blasi J et al.; Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum . Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles . Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25 . Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro . Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators . In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity . These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25 . Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.

Gene, 1993 Sep 6, 131(1), 107 - 12
Cloning and sequence analysis of the genes encoding phosphotransbutyrylase and butyrate kinase from Clostridium acetobutylicum NCIMB 8052; Oultram JD et al.; An 8.1-kb fragment of chromosomal DNA from Clostridium acetobutylicum NCIMB 8052 (formerly NCIB 8052) has been cloned into plasmid pAT153 and shown to allow the growth of Escherichia coli LJ32 (F+ atoC2c atoD32 fadR) on butyrate as the sole source of carbon and energy . Deletion analysis delineated a 3.9-kb subfragment capable of complementation . The nucleotide sequence of this fragment was determined and it was shown to encode three complete, and two incomplete open reading frames (ORFs) . Based on enzymic studies of recombinant clones, two of these ORFs were shown to encode phosphotransbutyrylase and butyrate kinase . The above enzymes are involved in the acidogenic phase of fermentation in C . acetobutylicum . The fragment also carries an incomplete ORF encoding a polypeptide exhibiting substantial similarity to dihydropteroate synthase.

J Clin Microbiol, 1993 Sep, 31(9), 2402 - 9
Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay; Doellgast GJ et al.; A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum . The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay . In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E . Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution . Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase . Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C . botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins . ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed . This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.

J Clin Microbiol, 1993 Sep, 31(9), 2255 - 62
Gene probes for identification of the botulinal neurotoxin gene and specific identification of neurotoxin types B, E, and F; Campbell KD et al.; A polymerase chain reaction method was developed for the specific detection of the botulinum neurotoxin (BoNT) gene of Clostridium botulinum . Degenerate oligonucleotide primers, designed from the nucleotide sequence of the heavy chain of the BoNT gene, amplified a specific fragment of approximately 1.1 kb from strains of C . botulinum toxin types A, B, E, F, and G and neurotoxin-producing strains of Clostridium barati and Clostridium butyricum, but no fragment was obtained from nontoxigenic strains . The fragments amplified from several strains of C . botulinum types B, E, and F were cloned in Escherichia coli and their nucleotide sequences were determined . Sequences within this region were used to design oligonucleotide probes specific for BoNT type B (BoNT/B), BoNT/E, and BoNT/F genes . An additional probe was designed for the detection of the BoNT/F gene of C . barati, which differed in sequence from BoNT/F genes of both proteolytic and nonproteolytic strains of C . botulinum.

Gastroenterol Clin North Am, 1993 Sep, 22(3), 623 - 37
Clostridium difficile colitis and diarrhea; Pothoulakis C et al.; Clostridium difficile is now regarded as the most prevalent nosocomial pathogen, infecting as many as a quarter of hospitalized patients . The pathophysiology of infection with this unusual enteric pathogen involves alteration of the normal enteric flora by antibiotics, ingestion of spores, and colonization by C . difficile . The organism then releases potent exotoxins that produce an inflammatory colitis and diarrhea . A spectrum of host responses occurs, ranging from the asymptomatic carrier state to life-threatening pseudomembranous colitis . Effective therapy with vancomycin or metronidazole is available, but relapses occur in 15% to 20% of patients and may necessitate multiple courses of therapy.

Gastroenterol Clin North Am, 1993 Sep, 22(3), 517 - 33
Approach to acute diarrhea in the elderly; Bennett RG et al.; Diarrhea is a common problem among the elderly that can have catastrophic results . Atherosclerosis predisposes older adults to morbid sequelae from dehydration resulting from diarrhea . Deaths related to diarrheal illnesses are recognized among older adults living in the community as well as among those confined to nursing homes . Outbreaks have most often been associated with excess deaths from diarrhea among nursing-home patients . Although most cases of dehydration from diarrhea result from gastrointestinal infections, noninfectious causes of diarrhea related to prescription of laxatives, side effects of medications, and use of enteral feedings are common . Clostridium difficile infection is particularly common among older adults in hospitals and nursing homes, and relapsing disease in these groups may be more frequent than among younger adults . The approach to an elderly patient with diarrhea is to ensure proper hydration using available oral rehydration solutions, proceed with diagnostic tests likely to yield a positive result, avoid the use of harmful antiperistaltic drugs, and provide adequate follow-up of the nutritional state.

FEMS Microbiol Lett, 1993 Sep 1, 112(2), 223 - 7
Transcript mapping of the rubredoxin gene from Clostridium pasteurianum; Mathieu I et al.; The transcripts of the rubredoxin gene from C . pasteurianum have been shown to have a size of approx . 230 bases by Northern blotting techniques . The transcription start has been located within 41 bases upstream of the initiator codon . The data demonstrate that the rubredoxin gene is monocistronic . Two of the three open reading frames occurring upstream of the rubredoxin gene are adjacent and appear to encode a thioredoxin (or glutaredoxin) reductase and a thioredoxin (or glutaredoxin).

Infect Immun, 1993 Sep, 61(9), 3711 - 8
Evidence for coupling of Clostridium perfringens alpha-toxin-induced hemolysis to stimulated phosphatidic acid formation in rabbit erythrocytes; Sakurai J et al.; When rabbit erythrocytes were exposed to low concentrations of Clostridium perfringens alpha-toxin, hot-cold hemolysis was observed . The toxin induced production of phosphatidic acid (PA) in a dose-dependent manner when incubated with erythrocytes at 37 degrees C . When erythrocyte membranes were incubated with the toxin and {gamma-32P}ATP in the presence or absence of ethanol, {32P}PA formation was maximal within 30 s, then sharply decreased, and began again after 5 min of incubation . Ethanol had no effect on the early appearance (at approximately 5 min) of PA formation induced by the toxin but significantly inhibited formation of PA over 10 min of incubation . Treatment of erythrocyte membranes with alpha-toxin resulted in the biphasic formation of 1,2-diacylglycerol and PA as well as an increase of inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-4,5-bisphosphate (PIP2) within 30 s . Neomycin inhibited the toxin-induced increase in turbidity of egg yolk suspensions but did not inhibit the toxin-induced hemolysis of intact erythrocytes . On the other hand, neomycin inhibited the toxin-induced hemolysis of saponin-treated erythrocytes . In addition, neomycin inhibited PA formation induced by the toxin in erythrocyte membranes . IP3 was released by incubation of PIP2 with erythrocyte membranes but not by incubation of PIP2 with the toxin . The toxin stimulated the membrane-induced release of IP3 from PIP2 . These data suggest that the toxin-induced hemolysis is dependent on the action of phospholipase C in erythrocyte membranes.

Gastroenterology, 1993 Sep, 105(3), 701 - 7
Ketotifen inhibits Clostridium difficile toxin A-induced enteritis in rat ileum; Pothoulakis C et al.; BACKGROUND: Clostridium difficile toxin A is the principal mediator of inflammatory enterocolitis in experimental animals . The purpose of this study was to explore the effect of ketotifen, an anti-inflammatory drug, on toxin A-induced enterotoxicity in rat ileum . METHODS: The effects of intragastric administration of ketotifen on secretion, mannitol permeability, histological damage, and mucosal levels of leukotriene B4, leukotriene C4, and platelet activating factor in toxin A-exposed rat ileal loops were measured in vivo . The effects of ketotifen on toxin A-mediated release of rat mast cell protease II (rat mucosa mast cell product) release were also measured in rat ileal explants in vitro . The effect of ketotifen on neutrophil migration in vitro was also evaluated . RESULTS: Ketotifen pretreatment inhibited toxin A-associated intestinal secretion by 42.5% and mannitol permeability by 56.3% and reduced epithelial cell inflammation and necrosis . These effects were associated with reduced levels of leukotriene B4 by 65.8%, leukotriene C4 by 88.8%, platelet activating factor by 77.8%, and inhibition of rat mast cell protease II by 58.4% . In addition, pretreatment of neutrophils with ketotifen inhibited neutrophil migration in vitro . CONCLUSIONS: The protective effect of ketotifen in this animal model was associated with significant inhibition of release of mast cells and neutrophil derived mediators, supporting their involvement in C . difficile enteritis.

J Bacteriol, 1993 Sep, 175(18), 5762 - 8
Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A; Goldstein MA et al.; Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD) . The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector . The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose . Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides . It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose) . At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton . Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g . The measured dissociation constant was in the 1 microM range for all cellulose samples . The results suggest that the CBD binds specifically to crystalline cellulose . Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD . The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose . This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety . This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.

Dis Colon Rectum, 1993 Sep, 36(9), 844 - 9
Quantitative cultures of the mucosal-associated bacteria in the mechanically prepared colon and rectum; Bleday R et al.; Little is known about the mucosal microflora of the colon and rectum at the time of elective surgery . Our objective was to determine the concentrations of anaerobic and aerobic bacteria associated with the mucosa of the mechanically prepared large bowel . Ten patients were studied after a standard polyethylene glycol-electrolyte lavage preparation . No patient had taken antibiotics in the preceding four weeks . Sterile wire brushes passed through the colonoscope during advancement were used to culture the rectal, transverse colon, and cecal mucosa . Total anaerobic, aerobic, Gram-positive, and enteric bacterial counts were determined along with specific cultures for Bacteroides fragilis, Clostridium difficile, Escherichia coli, Pseudomonas aeruginosa, enterococcus, and staphylococcus species . The results showed that there was a significant increase (P < 0.01) in aerobes, anaerobes, enterics, Gram positives, B . fragilis, and E . coli mucosal counts with proximal progression . Aerobes showed a steady gradient, while anaerobes demonstrated an increase from the rectum to the transverse colon but no change between the transverse colon and cecum . We conclude that, in the prepared bowel, there is an increase in the mucosal bacterial counts in the more proximal portions of the bowel . The results may serve as a baseline for future studies on the mucosal-associated bacteria of the large intestine.

Ann Hematol, 1993 Sep, 67(3), 145 - 7
Investigation of the pathogenesis of massive hemolysis in a case of Clostridium perfringens septicemia; Hubl W et al.; Massive hemolysis is a rare, usually fatal complication of Clostridium perfringens septicemia . Of all toxins produced by the bacterium, phospholipase C (PLC) is believed to be the most likely cause of hemolysis . An influence of neuraminidase has often been suspected . In the present study, a case of C . perfringens septicemia with acute massive intravascular hemolysis is described . It led to death within 4 h of admission to the hospital . While the course of events was comparable to previously reported cases, we succeeded in gaining deeper insight into the pathogenesis by monitoring serum anti-T titer and quantifying serum PLC activity during the course of the disease . We excluded an effect of neuraminidase by a negative direct antiglobulin test, a negative anti-T lectin test, and a steady serum anti-T titer of 1 in 32 . Serum PLC activity, on the other hand, showed a nearly fivefold increase (6.0 to 27.3 U/l), which is consistent with the hypothesized dominant role of this enzyme.

Infect Immun, 1993 Sep, 61(9), 3988 - 93
Clostridium difficile toxin A elicits Ca(2+)-independent cytotoxic effects in cultured normal rat intestinal crypt cells; Fiorentini C et al.; In rat intestinal crypt cells, Clostridium difficile toxin A induces (i) early cytoskeletal alterations involving the whole population and (ii) late effects in 30 to 40% of the cells, consisting mainly of surface blebbing and nuclear fragmentation . All these effects were Ca2+ independent and were not abolished by protein synthesis inhibitors.

Infect Immun, 1993 Sep, 61(9), 3958 - 65
Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus; Hunter SE et al.; Oligonucleotide probes designed on the basis of the N-terminal sequence of Clostridium perfringens beta-toxin were used to isolate the encoding gene (cpb) . The nucleotide sequence of cpb was determined, and on the basis of DNA hybridization experiments it was shown that the gene is found only in type B and C strains of C . perfringens . The deduced amino acid sequence of the beta-toxin revealed homology with the alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus . The beta-toxin purified from C . perfringens appeared to exist in monomeric and multimeric forms . Recombinant beta-toxin, produced in Escherichia coli, appeared to be mainly in the multimeric form.

Res Microbiol, 1993 Sep, 144(7), 547 - 56
Sequence of the gene coding for the neurotoxin of Clostridium botulinum type A associated with infant botulism: comparison with other clostridial neurotoxins; Willems A et al.; The neurotoxin gene from a strain of Clostridium botulinum type A causing infant botulism was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated using primers designed to conserved regions of published botulinal toxin (BoNT) sequences . Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated that the toxin gene encodes a protein of 1,296 amino acid residues . Comparative alignment of the derived infant BoNT/A sequence with those of other published neurotoxins revealed highest sequence relatedness with BoNT/A of classical food-borne botulism . The sequence identity between infant and classical BoNT/A was 94.9% for the light chain (corresponding to 23 amino acid changes) and 87.1% for the heavy chain (corresponding to 109 amino acid changes).

Vaccine, 1993 Sep, 11(12), 1253 - 8
A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens protects mice against experimental gas gangrene; Williamson ED et al.; Fragments of the alpha-toxin of Clostridium perfringens have been produced using genetic manipulation techniques . Antibody which cross-reacted with the alpha-toxin was induced after immunization with fragments representing the N- (Cpa1-249) and C-terminal (Cpa247-370) domains of the toxin . Smaller fragments of the alpha-toxin did not induce cross-reacting antibody . Anti-Cpa1-249 serum neutralized phospholipase C activity but not haemolytic activity of the toxin . Anti-Cpa247-370 serum neutralized both the phospholipase C and haemolytic activities . Only immunization with Cpa247-370 induced protection against the lethal effects of the toxin . Immunization with Cpa247-370 also provided protection in a mouse model against at least 10 LD100 doses of C . perfringens type A . This result confirms the essential role of this toxin in the pathogenesis of gas gangrene.

J Appl Bacteriol, 1993 Sep, 75(3), 234 - 9
Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples; Fach P et al.; A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism . A two primer set and an oligonucleotide detection probe were used to specifically detect Cl . botulinum type A neurotoxin gene (BoNT/A) . After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization . This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction . The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10(3) bacteria per g according to the type of food samples . No cross-reactions were observed with the other Cl . botulinum toxinotypes and other bacteria found routinely in food . This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl . botulinum type A in food samples.

Zentralbl Veterinarmed A, 1993 Sep, 40(7), 525 - 32
An outbreak of enterotoxaemia in suckling camels; el Sanousi SM et al.; An outbreak of enterotoxaemia was observed for the first time in suckling camels in Saudi Arabia . The animals were weak, diarrhoeic and succumbed quickly to exertion . The main pathological findings were those of acute catarrhal enteritis and acute myocardial degeneration . Clostridium perfringens was isolated from the enteric lesions; Aeromonas hydrophila was also identified . The properties of both isolates were studied.

Ann Pharmacother, 1993 Sep, 27(9), 1082 - 9
Review of in vitro activity, pharmacokinetic characteristics, safety, and clinical efficacy of cefprozil, a new oral cephalosporin; Barriere SL; OBJECTIVE: To review the pharmacokinetics, microbiology, clinical efficacy, safety, and tolerance of cefprozil, a new, broad-spectrum oral cephalosporin . DATA SOURCES: Published clinical trials and microbiologic, pharmacokinetic, and safety data were identified by MEDLINE; additional references were derived from bibliographies of these articles; microbiologic data on file were provided by Bristol-Myers Squibb . STUDY SELECTION: Only published comparative clinical trial reports are included in the review of clinical efficacy . Noncomparative clinical data pertaining to uses of cefprozil not approved by the Food and Drug Administration are not included . DATA SYNTHESIS: Data are presented on the in vitro microbiologic activity of cefprozil against 10,152 bacterial isolates, including most of the clinically important streptococci (e.g., Streptococcus pyogenes, Streptococcus pneumoniae), beta-lactamase-positive and -negative Staphylococcus aureus and Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, Proteus mirabilis, Clostridium difficile, and numerous other gram-negative aerobes and anaerobes . In clinical trials, cefprozil appears to be at least as effective as commonly used comparison agents such as cefaclor, cefixime, and amoxicillin/clavulanic acid . Additionally, cefprozil is better tolerated than the latter two agents, especially with regard to gastrointestinal adverse effects . CONCLUSIONS: Cefprozil is a broad-spectrum cephalosporin that provides coverage against both gram-negative and -positive bacteria that may cause otitis media, pharyngitis/tonsillitis, skin and skin-structure infections, secondary bacterial infection of acute bronchitis, and acute bacterial exacerbations of chronic bronchitis . The beta-lactamase stability of cefprozil appears to exceed that of other oral cephalosporins for some important pathogens . Cefprozil is used primarily for second-line treatment as less-expensive, first-line generic alternatives generally are available . Cefprozil demonstrates clinical advantages over many other orally administered beta-lactam antibiotics in terms of antimicrobial spectrum, a once- or twice-daily dosing regimen, and/or reduced incidence of adverse effects.

Appl Environ Microbiol, 1993 Sep, 59(9), 3011 - 20
Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction; Szabo EA et al.; The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes . Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins . Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment . Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains . Each assay was specific for the intended target . The PCR reliably identified multiple strains having the same neurotoxin type . The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type . As little as 10 fg of DNA (approximately three clostridial cells) was detected . C . botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations . This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.

Support Care Cancer, 1993 Sep, 1(5), 250 - 5
Anaerobic bacteremia in a cancer center; Noriega LM et al.; Seventy-five episodes of clinically relevant anaerobic bacterial bacteremia observed in cancer patients were reviewed . Gastrointestinal (22.7%), hematological (22.7%) and female genital tract (18.6%) cancers were the most common underlying malignant diseases . Among 84 strains of strict anaerobic bacteria recovered in the 75 patients, gram-negative rods were isolated in 49 patients (58.3%), gram-positive rods in 29 patients (34.5%) and gram-positive cocci in 6 patients (8%) . Bacteroides spp . and Clostridium spp . were the most frequent pathogens (85.7%) . Twenty-one episodes of bacteremia were polymicrobial, aerobic gram-positive cocci being the most frequently associated pathogens . When identified, the primary sites were the gastrointestinal tract (40%), the female genital tract (17.3%), skin and soft tissue (14.6%), the oropharynx (12%) and the lower respiratory tract (6.7%) . The source remained unknown in 7 cases (9.3%) . The overall survival (evaluated 10 days after the occurrence of bacteremia) was 82.5% . There was no difference in mortality between patients with monomicrobial and polymicrobial bacteremia . Pulmonary complications were more frequent in patients with fatal outcome in comparison to patients who survived . The mortality rate of the patients adequately treated was 10.3% compared to 41% for the patients not treated or treated inadequately (P = 0.016, chi 2).

J Am Geriatr Soc, 1993 Sep, 41(9), 940 - 6
Clostridium difficile colonization in residents of long-term care facilities: prevalence and risk factors; Walker KJ et al.; OBJECTIVE: To determine the period prevalence of Clostridium difficile disease and asymptomatic carriage in the residents of long-term care facilities (LTCF) and to characterize the risk factors for colonization or associated disease . DESIGN: Period prevalence survey . SETTING: Two long-term care facilities in St . Paul, MN . PARTICIPANTS: Specimens were collected from 225 LTCF residents . MEASUREMENTS: The dependent variable was the culture result for C . difficile, which was isolated and identified using selective culture media and a commercial anaerobe identification kit . Tissue culture assay was used to detect the ability of each C . difficile isolate to produce toxin . Independent variables (including gender, age, race, current medical diagnoses, severity of underlying disease, case mix, current clinical symptoms, current medications, antibiotic use within 4 weeks prior to specimen procurement, and other pertinent history) were obtained from the current medical record of each participant . RESULTS: Of 225 stool cultures that were obtained, 16 (7.1%) were positive for C . difficile . None of the residents with a positive culture was symptomatic . History of nosocomial infection and the use of antibiotics in general, cephalosporins, trimethoprim/sulfamethoxazole (TMP/SMX), and histamine-2 blockers were significantly associated with positive C . difficile culture (P < or = 0.05) by univariate analyses . Trends towards significance (0.05 < 0.10) were noted for narcotic use, previous hospitalization, LTCF, and non-insulin-dependent diabetes mellitus . Logistic regression analysis revealed significant, independent predictors of positive culture: antibiotic use in general (P = 0.028; relative risk = 3.31), histamine-2 antagonist use (P = 0.038; relative risk = 3.27), cephalosporin use (P = 0.038; relative risk = 4.66), and TMP/SMX use (P = 0.007; relative risk = 8.45) . CONCLUSIONS: The use of antibiotics, particularly cephalosporins and TMP/SMX, is a significant risk factor for asymptomatic carriage of C . difficile in long-term care facilities . The use of H-2 blockers was also a significant risk factor for carriage; however, this finding has not been reported previously and should be confirmed by independent studies . These medications should be used judiciously in the LTCF population . When diarrheal diseases are encountered in LTCF residents, a high index of suspicion for C . difficile infection should be maintained and the appropriate diagnostic and therapeutic measures taken.

Mol Microbiol, 1993 Sep, 9(6), 1143 - 55
Pore-forming and haemolytic properties of the Gardnerella vaginalis cytolysin; Cauci S et al.; The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders . This 59 kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 x 10(6) HU mg-1 being obtained . The toxin-induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm . The damage is inhibited by osmotic protectants and shows a sigmoidal dose-response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion . The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol-1 has been derived . Lipid membranes can be very efficient inhibitors of Gvh-haemolysis, being able to bind the toxin quite avidly . The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer . The functional properties of Gvh have been compared with those of Clostridium perfringens thetatoxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram-positive and Gram-negative bacteria, respectively . The toxin shares several features with the family of the so-called 'sulphydryl-activated' cytolysins produced by Gram-positive bacteria, although Gvh does not truly belong to this family, being deactivated by beta-mercaptoethanol and being antigenically distinct from them . We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.

Mol Microbiol, 1993 Sep, 9(5), 1019 - 25
Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme; Croux C et al.; Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall . These enzymes show both substrate and bond specificities . The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action . The bond specificity allows their classification as muramidases (lysozymes), glucosaminidases, amidases, and endopeptidases . To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments . This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose . Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls . Since the parental LYC muramidase was choline-independent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved . The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae . The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.

Zentralbl Bakteriol, 1993 Sep, 280(1-2), 93 - 106
Isolation of a sialic acid-specific surface haemagglutinin of Helicobacter pylori strain NCTC 11637; Lelwala-Guruge J et al.; A deionized water extract of Helicobacter pylori NCTC 11637 contained haemagglutinin activity that was (i) soluble (i.e., not associated with particulate material sedimented by centrifugation at 100,000 x g for 1 h), (ii) stable to lyophilization, (iii) heat-labile, (iv) chymotrypsin-sensitive, (v) inhibited by fetuin, orosomucoid, and NANLac, but not by asialofetuin and (vi) inactive against guinea pig erythrocytes incubated with Clostridium perfringens neuraminidase, but active against untreated guinea pig erythrocytes . The data support the idea that the haemagglutinin is a protein which recognizes the alpha-(2-3) structure of sialylated glycoconjugates . Fractionation of the extract by isoelectric focusing and by gel filtration with Sephacryl S-400 indicated that the haemagglutinin has a pI of 3.7 and consist of high molecular-weight-protein aggregates . SDS-PAGE analysis of the preparation purified by gel filtration showed 3 protein bands at ca . 64 kD, 56 kD and 20 kD . Electron microscopy of H . pylori incubated with gold-labelled fetuin indicated that the haemagglutinin was associated with loosely adherent material on the bacterial surface, and that the purified haemagglutinin did not reveal a fimbrial structure . The ability to bind to sialoglycoconjugates on the erythrocyte membrane suggests that the haemagglutinin may be an important colonization factor enabling H . pylori to bind to similar saccharide structures on epithelial cells.

Nature, 1993 Aug 26, 364(6440), 827 - 30
Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles; Schmid MF et al.; The seven botulinum neurotoxin (NT) serotypes produced by strains of Clostridium botulinum inhibit neurotransmitter release from synaptic vesicles . Neurotoxin is synthesized as a roughly 150K single-chain protein . Proteolysis produces two fragments, the 50K L-chain and 100K H-chain, that remain linked by a disulphide bond . Intoxication involves membrane attachment by the C-terminal half of the H-chain, endocytotic/lysosomal internalization, vesicle channel formation mediated by the 50K N-terminal half of the H-chain at low pH, and finally blockade of synaptic vesicle fusion after the L-chain reaches the cytosol . We report here the visualization of the neurotoxin-membrane complex by electron cryomicroscopy and image processing . Three-dimensional reconstructions show the neurotoxin bound to the exterior of ganglioside/PC lipid vesicles and show channels entirely perforating the vesicle wall . Each channel appears to arise from the interaction of four neurotoxin molecules.

Biochem J, 1993 Aug 15, 294 ( Pt 1), 215 - 8
13C-n.m.r . of the cyanylated apoflavodoxin and flavodoxin from Clostridium pasteurianum; Doherty GM et al.; The thiol group of the flavodoxin from Clostridium pasteurianum has been cyanylated in a single step using {cyanato-13C}2-nitro-5-thiocyanatobenzoic acid . This chemical modification increases the dissociation constant of the apoflavodoxin-FMN complex 10-fold from 0.33 +/- 0.15 nM to 2.9 +/- 1.3 nM . The thiocyanate carbons of the cyanylated cysteine residue of apoflavodoxin and flavodoxin had chemical shift values of 114.7 and 112.3 p.p.m . respectively . From these chemical shifts we conclude that the binding of FMN by the cyanylated apoflavodoxin causes a decrease in the polarity and/or hydrogen bonding capacity of the environment of the thiocyanate group . We compare these results with those obtained from similar studies on the cyanylated apoflavodoxin and flavodoxin from Megasphaera elsdenii and we discuss how FMN binding and cyanylation perturb the structures of apoflavodoxins from Megasphaera elsdenii and Clostridium pasteurianum.

Eur J Pharmacol, 1993 Aug 15, 246(3), 293 - 7
Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils; Just I et al.; The gelsolin-actin complex was ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin in lysates of human platelets and human neutrophils . When {32P}orthophosphate-labelled human neutrophils were treated with C . botulinum C2 toxin, {32P}ADP-ribosylated gelsolin-actin was precipitated with anti-gelsolin antibody . Stimulation of neutrophils by formyl-methionine-leucine-phenylalanine decreased the interaction of gelsolin with ADP-ribosylated actin in toxin-treated cells . The data indicate that the gelsolin-actin complex is a pathophysiological substrate for actin-ADP-ribosylating toxins.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3705 - 9
The different binding modes of Hoechst 33258 to DNA studied by electric linear dichroism; Bailly C et al.; The binding mode of the bisbenzimidazole derivative Hoechst 33258 to a series of DNAs and polynucleotides has been investigated by electric linear dichroism . Positive reduced dichroisms were measured for the poly(dA-dT).poly(dA-dT)- and poly(dA).poly(dT)-Hoechst complexes in agreement with a deep penetration of the drug into the minor groove . Similarly, the drug displays positive reduced dichroism in the presence of the DNAs from calf thymus, Clostridium perfringens and Coliphage T4 . Conversely, negative reduced dichroisms were obtained when Hoechst 33258 was bound to poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dG).poly(dC) as well as with the GC-rich DNA from Micrococcus lysodeikticus indicating that in this case minor groove binding cannot occur . Substitution of guanosines for inosines induces a reversal of the reduced dichroism from negative to positive . Therefore, as anticipated it is the 2-amino group of guanines protruding in this groove which prevents Hoechst 33258 from getting access to the minor groove of GC sequences . The ELD data obtained with the GC-rich biopolymers are consistent with an intercalative binding . Competition experiments performed with the intercalating drug proflavine lend credence to the involvement of an intercalative binding rather than to an external or major groove binding of Hoechst 33258 at GC sequences.

Biochim Biophys Acta, 1993 Aug 7, 1164(3), 305 - 10
Purification and properties of a flavodoxin from the heterocystous cyanobacterium Anabaena sphaerica; Yakunin AF et al.; A flavodoxin was purified to homogeneity from the nitrogen-fixing heterocystous cyanobacterium Anabaena sphaerica grown under iron-limited conditions . The protein has a molecular mass of 21 kDa, and its spectral properties and amino-acid composition are very close to that of flavodoxins from other cyanobacteria . A . sphaerica flavodoxin supported the activities of A . sphaerica NADP reductase and Clostridium butyricum hydrogenase in reconstituted systems with illuminated plant chloroplasts as reductant . With the use of polyclonal anti-flavodoxin antiserum it was found that nitrogen-fixing cultures of A . sphaerica grown under iron-sufficient conditions contain low but significant amounts of flavodoxin (0.2-0.6 micrograms/mg crude extract protein) which increased dramatically (to 8-15 micrograms/mg crude extract protein) after the iron concentration in the medium was decreased to below 1 microM Fe . The flavodoxin content of both iron-limited and iron-sufficient . A . sphaerica was also shown to depend upon the growth phase of the (batch) cultures with a maximum at early exponential phase, coinciding with maximal in-vivo nitrogenase activity . These results suggest that A . sphaerica flavodoxin not only substitutes for ferredoxin under iron-limiting conditions, but also fulfills some specific role under iron-sufficient conditions.

J Biol Chem, 1993 Aug 5, 268(22), 16332 - 44
Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis; Mathupala SP et al.; The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined . The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780 . Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis . The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme . The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes . Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme . These findings substantiate amylopullulanase as a new type of amylosaccharidase.

Kansenshogaku Zasshi, 1993 Aug, 67(8), 724 - 9
{Identification of enterotoxin-producing Clostridium perfringens by the polymerase chain reaction}; Kato N et al.; Polymerase chain reaction was applied to identify enterotoxin-producing Clostridium perfringens by amplifying a segment of the C . perfringens enterotoxin gene . All of the four enterotoxin-positive reference strains tested were PCR positive while an enterotoxin-negative strain was PCR positive . All 17 clostridial strains (16 species) other than C . perfringens were PCR negative . With clinical strains isolated from various clinical specimens in Japan, Korea, and Thailand, all three enterotoxin-positive isolates were PCR positive and all 82 enterotoxin-negative isolates were PCR negative . PCR results for amplifying a region containing the initiation codon of the C . perfringens enterotoxin gene also demonstrated complete agreement with enterotoxin producibility . These results suggested that the PCR assay is a rapid and simple test for identifying the enterotoxin-producing C . perfringens without using any cultures and spore treatments.

Clin Infect Dis, 1993 Aug, 17(2), 231 - 7
Evaluation of therapy with hyperbaric oxygen for experimental infection with Clostridium perfringens; Stevens DL et al.; The effects of inoculum size and treatment delays on the efficacy of hyperbaric oxygen (HBO) were evaluated in a murine model of Clostridium perfringens myositis in which the infection was treated with an HBO regimen identical to that used for humans . The efficacies of treatment with penicillin, metronidazole, or clindamycin--alone or in combination with HBO--were also assessed . Survival was inversely related to the size of the bacterial inoculum used for challenge, and delays in treatment markedly reduced the efficacies of all single and combination regimens . When animals were challenged with > 10(8) colony-forming units, survival was significantly higher among those treated with clindamycin or metronidazole than among those treated with penicillin . HBO alone did not improve survival at any inoculum tested . However, when administered early, HBO plus metronidazole or penicillin demonstrated significant additive efficacies in animals challenged with > or = 10(9) organisms . Clindamycin was more effective at the higher inocula than penicillin, metronidazole, or HBO, and its superior efficacy was not further enhanced by adjunctive therapy with HBO.

Biokhimiia, 1993 Aug, 58(8), 1213 - 20
{Purification and some properties of Clostridium thermocellum endoglucanase, formed by a recombinant Escherichia coli strain}; Mosolova TP et al.; The endoglucanase (EG5) has been isolated from the recombinant strain of E . coli TG1 carrying a plasmid with C . thermocellum F7 chromosomal DNA insertion . Using high performance ion-exchange chromatography and chromatofocusing, the enzyme was 98-fold purified with a 27% yield . The enzyme has a molecular mass of 35 kDa (SDS-PAGE data) and is represented by three isoforms with pI 4.4-4.8 (isoelectrofocusing data) . The activity of EG5 was determined by chromatography on carboxymethylcellulose, amorphous cellulose, xylan, lichenan and avicel . The optimal conditions for these substrates hydrolysis are: pH 6.0-6.5, 80 degrees C (60 degrees C for avicel).

Hematol Oncol Clin North Am, 1993 Aug, 7(4), 865 - 85
Approach to management of fever and infection in patients with primary bone marrow failure and hemoglobinopathies; Weinberger M; The characteristic spectrum of infections in patients with aplastic anemia, chronic neutropenic diseases, sickle cell disease, thalassemia, and other hemoglobinopathies are described . The major risk factor for infection in patients with bone marrow failure is the degree of neutropenia and monocytopenia . In patients with aplastic anemia, invasive fungal infections emerge as the major causes of mortality . Life-threatening infections are rare in patients with chronic neutropenic diseases; however, necrotizing enterocolitis due to Clostridium species may be an exception . Bacterial infections, predominantly with encapsulated bacteria, are the most common cause of death in patients with sickle cell disease, especially those who are younger than 5 years of age . Patients with thalassemia and other hemoglobinopathies are particularly susceptible to life-threatening infections with Yersinia enterocolitica as a result of iron overload or of the chelating therapy with desferrioxamine.

J Bacteriol, 1993 Aug, 175(16), 5097 - 105
Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii; Ismaiel AA et al.; Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species . In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii . An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C . beijerinckii . When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated . The ADHs from the two strains had similar structural and kinetic properties . Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000 . The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected . They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols . The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group . ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates . There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria . However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre . Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.

Clin Orthop, 1993 Aug, (293), 265 - 8
Clostridium perfringens infection of an anterior iliac crest bone graft donor site . A case report; Miller SD et al.; Postoperative infection of elective surgical wounds with Clostridium species has been linked to gastrointestinal tract lesions . A 55-year-old man with a history of peptic ulcer disease was treated by open reduction and internal fixation with autogeneic cancellous bone grafting from an anterior iliac crest donor site for nonunion of the clavicle . A mild serosanguinous drainage from the Penrose drain site at the iliac crest had ceased on postoperative Day 11; the patient complained of pain and a brownish drainage on postoperative Day 15 . The infection was documented with cultures positive for Clostridium perfringens . Aggressive emergent surgical and antibiotic therapy resulted in complete clinical recovery . The wound infection did not advance to severe tissue damage and myonecrosis . This case represents the first reported infection of an iliac crest bone graft site with C . perfringens.

Infect Immun, 1993 Aug, 61(8), 3429 - 39
Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli; Czeczulin JR et al.; A complete copy of the gene (cpe) encoding Clostridium perfringens enterotoxin (CPE), an important virulence factor involved in C . perfringens food poisoning and other gastrointestinal illnesses, has been cloned, sequenced, and expressed in Escherichia coli . The cpe gene was shown to encode a 319-amino-acid polypeptide with a deduced molecular weight of 35,317 . There was no consensus sequence for a typical signal peptide present in the 5' region of cpe . Cell lysates from recombinant cpe-positive E . coli were shown by quantitative immunoblot analysis to contain moderate amounts of CPE, and this recombinant CPE was equal to native CPE in cytotoxicity for mammalian Vero cells . CPE expression in recombinant E . coli appeared to be largely driven from a clostridial promoter . Immunoblot analysis also demonstrated very low levels of CPE in vegetative cell lysates of enterotoxin-positive C . perfringens . However, when the same C . perfringens strain was induced to sporulate, much stronger CPE expression was detected in these sporulating cells than in either vegetative C . perfringens cells or recombinant E . coli . Collectively, these results strongly suggest that sporulation is not essential for cpe expression, but sporulation does facilitate high-level cpe expression.

Indian J Med Sci, 1993 Aug, 47(8), 201 - 3
Anaerobic infection in chronic maxillary sinusitis; Greval RS et al.; 1) Ten per cent of cases of chronic maxillary sinusitis in a series of 50 cases treated in the ENT department of Dayanand Medical College, Ludhiana, were caused by anaerobic infection . 2) The causative anaerobic micro-organisms encountered were Peptostreptococcus and Clostridium species . 3) Treatment should, therefore, include metronidazole, especially in those cases refractory to conventional antibiotic therapy.

Wei Sheng Wu Xue Bao, 1993 Aug, 33(4), 280 - 4
{The isolation and identification of a Clostridium botulinum serotype A strain}; Liu S et al.; 319 soil specimens were collected from different places of China for isolating Clostridium botulinum . A strain of Clostridium botulinum was isolated from a culture of soil specimens in Ruoergai of Sichuan Province, the strain was called As-3 . The As-3 was identified as Clostridium botulinum serotype A according to its biological properties, biochemical serological and toxicological characteristics and DNA determination . Its DNA G + C mol is 24.9% . The toxin produced by As-3 strain can only be neutralized by type A antiserum.

Am J Infect Control, 1993 Aug, 21(4), 183 - 8
An epidemiologic study of nosocomial infections in a pediatric long-term care facility; Vermaat JH et al.; OBJECTIVE: To determine the incidence of hospital-acquired (nosocomial) infection in pediatric long-term care facilities . DESIGN: Prospective cohort . SETTING: An 87-bed pediatric long-term care facility . PATIENTS: All patients receiving long-term care at Bloorview Children's Hospital during the study period . RESULTS: Infection developed in 40.1% of patients (n = 456) . The nosocomial infection rate per 1000 patient days (mean, 7.84) varied substantially, from 1.66 in May 1988 to 16.37 day in April, 1989 . The proportional frequencies of infections were as follows: respiratory, 41.6% (37.0% upper, 4.6% lower); urinary tract, 31.0%; skin, 15.6% (gastric tube site 5.0%, other 10.6%), eyes, 6.4%; gastrointestinal, 3.5%; and other, 1.5% . Of those infections for which an organism was recovered (48.5%), pathogens included Escherichia coli (22.5%), Enterococcus (14.8%), Staphylococcus (14.8%), Streptococcus (11.2%), Klebsiella (10.5%), Pseudomonas (10.1%), Proteus (4.3%), yeast (4.3%), Salmonella (0.7%), Clostridium difficile (0.4%), and other (6.2%) . CONCLUSIONS: The incidence and nature of infections in pediatric long-term care facilities differs from those in acute care facilities . Physicians should become familiar with the infection rates in the populations whom they treat . Control requires compliance with currently recognized effective strategies as well as innovative practical approaches to respiratory disease . Behavioral problems related to frequent clean, intermittent catheterization in young adults need to be addressed.

Eur J Clin Microbiol Infect Dis, 1993 Aug, 12(8), 640 - 2
In vitro activity of the new quinolone BAY y 3118 against anaerobic bacteria; Nord CE et al.; The in vitro activity of BAY y 3118 against anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, other Bacteroides spp . and fusobacteria was determined by an agar dilution method . This activity was compared with that of ciprofloxacin, ofloxacin, piperacillin, cefoxitin, imipenem, clindamycin and metronidazole . BAY y 3118, imipenem, clindamycin and metronidazole were the most active agents tested . The in vitro activity of BAY y 3118 against anaerobic bacteria was superior to that of ciprofloxacin and ofloxacin.

Clin Investig, 1993 Aug, 71(8), 595 - 9
Clinical significance of anaerobic bacteremias in a general hospital . A prospective study from 1988 to 1992; Gomez J et al.; A prospective study was designed to investigate anaerobic bacteremias and evaluate their incidence and significance in a general hospital . One or more blood cultures positive for anaerobic microorganisms were analyzed from each of a total of 61 patients hospitalized between January 1988 and April 1992, in accordance with an established protocol . The clinical repercussions of bacteremia were also analyzed . Two percent of blood cultures were positive for anaerobes, with an incidence of 0.6 cases per 1000 hospitalized patients . The most frequently isolated anaerobes were Bacteroides fragilis and Clostridium perfringens . Intraabdominal disease was the route of entry in 50% of the patients . A death rate of 37.3% was mostly attributed to B . fragilis . Hospitalization in the surgical department, nosocomial acquisition, previous surgery, critical initial clinical status and the presence of complications were significantly associated with increased death rates . No significant differences were found in the clinical course between patients whose antibiotic treatment was judged adequate and those for whom it was considered inadequate . The frequency and incidence of anaerobic bacteremia was low in our hospital . The well-known clinical and epidemiological characteristics of these infections facilitates their prompt diagnosis and empirical treatment with antibiotics of proven effectiveness against anaerobes.

Arch Dis Child, 1993 Aug, 69(2), 239 - 40
Clostridium difficile after haemolytic uraemic syndrome; Burgner DP et al.; Six children are described who developed diarrhoea associated with Clostridium difficile during the course of haemolytic uraemic syndrome . The significance of this infection is discussed within the context of the pathophysiology of haemolytic uraemic syndrome.

Arch Dis Child, 1993 Aug, 69(2), 221 - 4
Prolonged carriage of Clostridium difficile in Hirschsprung's disease; Hardy SP et al.; The role of Clostridium difficile in the aetiology of diarrhoea in children with Hirschsprung's disease was investigated in a prospective longitudinal study . In 64 children with Hirschsprung's disease no significant difference was found in the isolation rate of C difficile in patients with diarrhoea (32%) and without diarrhoea (26%) . Comparable isolation rates were found in 47 control children with and without diarrhoea (27% and 16% respectively) . The number of strains producing toxin B was similar in the four groups of children . In contrast to the disappearance of C difficile by 12 months of age in the control groups of children, C difficile could be repeatedly isolated from a proportion of children with Hirschsprung's disease over 12 months of age . These findings help to reconcile the existing contradictory reports on the incidence of C difficile in Hirschsprung's disease associated enterocolitis.

Antimicrob Agents Chemother, 1993 Aug, 37(8), 1649 - 54
Susceptibilities of 428 gram-positive and -negative anaerobic bacteria to Bay y3118 compared with their susceptibilities to ciprofloxacin, clindamycin, metronidazole, piperacillin, piperacillin-tazobactam, and cefoxitin; Pankuch GA et al.; The susceptibilities of 428 gram-negative and gram-positive anaerobes (including selected cefoxitin-resistant strains) to Bay y3118 (a new fluoroquinolone), ciprofloxacin, clindamycin, metronidazole, cefoxitin, piperacillin, and piperacillin-tazobactam were tested . Organisms comprised 115 Bacteroides fragilis group, 116 non-B . fragilis Bacteroides, Prevotella, and Porphyromonas spp., 40 fusobacteria, 58 peptostreptococci, 48 gram-positive non-spore-forming rods, and 51 clostridia . beta-Lactamase production was demonstrated in 87% of the gram-negative rods but in none of the gram-positive organisms . Overall, Bay y3118 was the most active agent, with all organisms inhibited at an MIC of < or = 2.0 micrograms/ml (MICs for 50% {MIC50} and 90% {MIC90} of strains tested, 0.125 and 0.5 microgram/ml, respectively) . By contrast, ciprofloxacin was much less active, with only 42% of strains susceptible at a breakpoint of 2.0 micrograms/ml (MIC50, 4.0 micrograms/ml; MIC90, 16.0 micrograms/ml) . Metronidazole was active against all gram-negative rods, but 7% of peptostreptococci, 83% of gram-positive non-spore-forming rods, and 4% of non-Clostridium perfringens, non-Clostridium difficile clostridia were resistant to this agent (MICs, > 16.0 micrograms/ml) . Clindamycin was active against 94% of Bacteroides, Prevotella, and Porphyromonas spp., 91% of peptostreptococci, and 100% of gram-positive non-spore-forming rods, but was active against only 70% of fusobacteria and 53% of clostridia . Cefoxitin was active against > or = 90% of all groups except the B . fragilis group and non-Propionibacterium acnes gram-positive non-spore-forming rods (both 85%) and C . difficile (20%) . Significant enhancement of piperacillin by tazobactam was seen in all beta-lactamase-positive strains (99% susceptible; MIC90, 8.0 micrograms/ml), and all beta-lactamase-negative strains were susceptible to piperacillin (MIC90, 8.0 micrograms/ml) . Clinical studies are required to delineate the role of Bay y3118 in the treatment of anaerobic infections.

J Clin Microbiol, 1993 Aug, 31(8), 2208 - 11
Comparison of typing methods for Clostridium difficile isolates; Wolfhagen MJ et al.; A simple discriminative typing method for Clostridium difficile has been developed . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis . Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.

Bioorg Med Chem, 1993 Aug, 1(2), 147 - 9
X-Neu5Ac: a novel substrate for chromogenic assay of neuraminidase activity in bacterial expression systems; Fujii I et al.; A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-alpha-D-glycero-D-galacto-2-nonulopyranosidon ic acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity . Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5'-dibromo-4,-4'-dichloroindigo 3 . Preliminary kinetic studies indicate that this compound is a good substrate (Km 0.89 x 10(-3) M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme . These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.

Biochemistry, 1993 Jul 20, 32(28), 7104 - 15
X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-A resolution; Kim J et al.; The crystal structure of the nitrogenase molybdenum-iron (MoFe) protein from Clostridium pasteurianum (Cp1) has been determined at 3.0-A resolution by a combination of isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging . The structure of Cp1, including the two types of metal centers associated with the protein (the FeMo-cofactor and the P-cluster pair), is similar to that previously described for the MoFe-protein from Azotobacter vinelandii (Av1) . Unique features of the Cp1 structure arise from the presence of an approximately 50-residue insertion in the alpha subunit and an approximately 50-residue deletion in the beta subunit . As a consequence, the FeMo-cofactor is more buried in Cp1 than in Av1, since the insertion is located on the surface above the FeMo-cofactor . The location of this insertion near the putative nitrogenase iron protein binding site provides a structural basis for the observation that the nitrogenase proteins from C . pasteurianum have low activity with complementary nitrogenase proteins isolated from other organisms . Mechanistic implications of the Cp1 structure for substrate entry/product release, substrate binding to the FeMo-cofactor, and electron- and proton-transfer reactions of nitrogenase are discussed.

Biochim Biophys Acta, 1993 Jul 18, 1174(1), 83 - 6
Organization of potential alternative nitrogenase genes from Clostridium pasteurianum; Zinoni F et al.; A 3.3 kb HindIII genomic DNA fragment from Clostridium pasteurianum ATCC 6013 which hybridized to the anfDGK genes for the Fe-only 'alternative' nitrogenase from Azotobacter vinelandii was cloned . Open reading frames (ORFs D, G, and K) with high sequence identity to anfD, anfG, and part of anfK were located in the nucleotide sequence obtained for 2494 bp of this fragment . In C . pasteurianum, ORFD maps approximately 1.8 kb downstream of nifH3 and is transcribed in the same direction . There was no evidence for additional copies of ORFDGK-like sequences in the genome of C . pasteurianum, other than those encoding the Mo-nitrogenase . Physiological and biochemical studies suggest that a nitrogenase not requiring molybdenum may occur in C . pasteurianum . This enzyme is probably encoded by nifH3 and ORFs D, G, and K identified here.

Biochim Biophys Acta, 1993 Jul 18, 1174(1), 108 - 10
Cloning and sequencing of the gene encoding the {2Fe-2S} ferredoxin from Clostridium pasteurianum; Meyer J; A 1348 basepair EcoRI fragment of genomic DNA containing the {2Fe-2S} ferredoxin gene from Clostridium pasteurianum has been cloned and sequenced . The translated protein sequence is identical to the sequence of the protein as previously determined by Edman chemistry . Two potential open reading frames occur upstream and downstream of the ferredoxin gene . However, the features surrounding the latter gene strongly suggest that it constitutes a separate transcriptional unit . The sequence data provide no evidence that this ferredoxin is involved in nitrogen fixation.

Biochem Biophys Res Commun, 1993 Jul 15, 194(1), 104 - 11
Mutated forms of a {2Fe-2S} ferredoxin with serine ligands to the iron-sulfur cluster; Fujinaga J et al.; The {2Fe-2S} ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56 and 60 . Residues 24, 56 and 60 have been separately mutated into serine . The modified ferredoxins have been purified and were all found to contain a {2Fe-2S}-type cluster . The electronic absorption and EPR spectra of the C24S protein were only slightly different from those of the native one . In contrast, the C56S and C60S ferredoxins displayed spectroscopic features witnessing the presence of an oxygen ligand to the iron-sulfur cluster: the UV-visible absorption bands were shifted to higher energy by ca . 20 nm, and the high field components of the EPR spectra were shifted from gx = 1.92 and gy = 1.95 to gx = 1.88 and gy = 1.92, respectively.

Eur J Biochem, 1993 Jul 15, 215(2), 421 - 9
Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum; Scherf U et al.; 4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein . The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1 . The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1 . The equilibrium of the reversible dehydration was determined from both sides as K = {crotonyl-CoA}/{4-hydroxybutyryl-CoA} = 4.2 +/- 0.3 . Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA . The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure . Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters . Exposure to air resulted initially in a slight activation followed by irreversible inactivation . Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow . Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III) . The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen . During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved . A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA . In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA . Finally isomerisation yields crotonyl-CoA . 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases . Contrary to the latter enzyme {e.g . (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase} which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity . Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.

Eur J Biochem, 1993 Jul 15, 215(2), 341 - 9
L-serine and L-threonine dehydratase from Clostridium propionicum . Two enzymes with different prosthetic groups; Hofmeister AE et al.; L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate {Grabowski, R . and Buckel, W . (1991) Eur . J . Biochem . 199, 89-94} . It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability . Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase . These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids . L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa) . The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM . L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine . L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes . Incubation with NaB3H4 specifically labelled the enzyme . Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate . L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity . The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM) . Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air . Fe2+ specifically reactivated the enzyme . L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P . asaccharolyticus . Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical . In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.

FEBS Lett, 1993 Jul 12, 326(1-3), 281 - 4
Identification of a cysteine involved in the interaction between carbon monoxide dehydrogenase and corrinoid/Fe-S protein from Clostridium thermoaceticum; Shanmugasundaram T et al.; In Clostridium thermoaceticum, the synthesis of acetyl-CoA from methyl tetrahydrofolate occurs via a series of enzymatic reactions involving methyl transferase, corrinoid/Fe-S protein (corrinoid), carbon monoxide dehydrogenase (CODH) and ferredoxin . We have investigated the possibility of one or more of these proteins existing as multi-enzyme complexes in vivo with higher catalytic activity . A protein complex consisting of CODH and corrinoid was isolated from the cell-free extracts of Clostridium thermoaceticum . The acetyl-CoA synthesis was found to be approximately 1.8-fold higher with the complex than that observed with the isolated protein components . HPLC gel filtration analyses of the native and DTE reduced complex suggested that the CODH:corrinoid complex is held together primarily by an inter disulfide bond . By differential labeling of thiols with {14C}N-ethylmaleimide it was found that Cys-506 of the alpha subunit of CODH was involved in the disulfide linkage with the corrinoid of the complex.

FEBS Lett, 1993 Jul 5, 325(3), 196 - 200
Electron transport components of the parasitic protozoon Giardia lamblia; Ellis JE et al.; The energy metabolism of the intestinal parasite, Giardia lamblia, involves the iron-sulphur protein, pyruvate:ferredoxin oxidoreductase . Cell fractionation studies showed that this enzyme is associated with the membranes . NADH and NADPH dehydrogenases were found in both the membrane and cytosolic fractions . EPR spectroscopic studies showed the presence of iron-sulphur clusters in the membrane fraction and in the cytosolic fraction, non-sedimentable at 6 x 10(6) g.min . An acidic, soluble protein fraction was separated from the cytosol . It had an EPR spectrum in the reduced state, characteristic of the 2{4Fe-4S} type of ferredoxin, with g-factors at 2.04 . 1.93 and 1.89, and the midpoint redox potential was estimated to be -360 mV . This species is probably a ferredoxin, like those of anaerobic bacteria such as Clostridium and Desulfovibrio spp . and also that of Entamoeba histolytica . The protein was readily and irreversibly oxidized to give {3Fe-4S} clusters.

J Biol Chem, 1993 Jul 5, 268(19), 14096 - 102
Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1,4-glucanase; Wang Q et al.; A new mechanism-based inactivator of beta-1,4-glucanases, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside, was synthesized and used to trap the intermediate formed during catalysis by endoglucanase C (CelC) from Clostridium thermocellum . Ion spray mass spectrometry confirmed the 1:1 stoichiometry of the incorporation of the inactivator into the enzyme . Inactivation followed the required pseudo first-order kinetic behavior and kinetic parameters for the process were determined . Although the intermediate trapped was relatively stable (t1/2 = 25 h), turnover was facilitated by transglycosylation following the addition of phenyl-beta-D-thiocellobioside and cellobiose, thus demonstrating the catalytic competence of the trapped intermediate . The nucleophilic amino acid residue involved was identified as Glu280 by labeling the enzyme with tritiated inactivator, cleaving it into peptides and sequencing the radiolabeled peptide . Ion spray mass spectrometric analysis of the peptide confirmed the sequence and the mode of attachment of the sugar to the peptide . Alignment of all known related beta-1,4-glucanases showed that Glu280 is strictly conserved in family A enzymes, consistent with its key role in catalysis.

Infect Immun, 1993 Jul, 61(7), 2912 - 8
Purification and characterization of alpha-toxin produced by Clostridium novyi type A; Ball DW et al.; Our study describes the production, purification, and properties of alpha-toxin from Clostridium novyi type A 19402 . The bacterium produced maximal amounts of alpha-toxin when grown at 37 degrees C for 72 h in dialysis flask cultures containing brain heart infusion supplemented with 0.75% Tween 80 and 2% glycerol . The alpha-toxin was purified by precipitation with polyethylene glycol 6000, followed by chromatography on Q-Sepharose, phenyl-agarose, and Mono-Q . By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the toxin exhibited a single band with an M(r) of 200,000 . The toxin also exhibited a single immunoprecipitin arc by crossed immunoelectrophoresis with antiserum against crude toxin . It was stable when stored at 4 degrees C and also following exposure to buffers with pHs in the range of 4 to 7 . The toxin had a minimum lethal dose in mice of 5 to 10 ng, caused rounding of a variety of cells in tissue culture, and was negative in the rabbit ileal loop assay . The cytotoxic activity was inhibited by agents that affect receptor-mediated processes, and the toxin was less active on a CHO mutant cell line that is defective in endosomal acidification . The analysis of the amino acid composition revealed an unusually high proline content . The N-terminal sequence is Met-Leu-Ile-Thr-Arg-Glu-Gln-Leu-Met-Lys.

J Biochem (Tokyo), 1993 Jul, 114(1), 122 - 5
Both medullasin and human leukocyte elastase are essentially devoid of elastinolytic activity; Aoki Y et al.; Elastinolytic activity of medullasin was investigated precisely and compared with that of human leukocyte elastase, because the structure of medullasin is quite similar to that of human neutrophil elastase, which was reported to have elastinolytic activity . When elastinolytic activity of medullasin and human leukocyte elastase was determined by employing unstained elastin fibers and measuring the increase in 280-nm absorbance of the supernatant, elastinolytic activity amounting to several percent of that of porcine pancreas elastase was apparently observed . However, the susceptibility of elastin preparations to these proteases was proportional to their hydroxyproline content . Both medullasin and human leukocyte elastase digested collagen fibers obtained from bovine Achilles tendon to the same extent as collagenase from Clostridium histolyticum . When elastinolytic activity was determined by employing elastin fibers stained with orcein, both proteases showed negligible elastinolytic activity . The activity remained negligible even when the pH or ionic strength of the reaction mixture was altered . These results indicate that medullasin and human leukocyte elastase are essentially devoid of elastinolytic activity, and that apparent elastinolytic activity observed when unstained elastin fibers were employed as the substrate is due to the digestion of collagen fibers mingled with elastin preparations.

Int J Food Microbiol, 1993 Jul, 19(2), 109 - 22
Modeling lag phase of nonproteolytic Clostridium botulinum toxigenesis in cooked turkey and chicken breast as affected by temperature, sodium lactate, sodium chloride and spore inoculum; Meng J et al.; The length of the lag phase (LP) of toxigenesis in commercially cooked turkey meat stored under vacuum was determined as affected by 0, 1.2, 2 and 3% sodium lactate (L), 0, 1 and 2% NaCl (S), spore (pool of nonproteolytic B and E strains: B2, B17, B197, B706, E211, E250, E KA-2 and E Beluga) inoculum (I) of 10(-2) to 10(4), storage temperature (T) of 4, 8, 12, 16, 20 and 30 degrees C and their interactions . The time from inoculation to the detection of first toxic sample was defined as LP . Using regression analysis the following model predictive of LP of C . botulinum toxigenesis in the cooked turkey breast was derived: Log(1/LP) = -2.2877 -0.1235(S) -0.2174(L) +0.4391(square root of T) +0.0204(square root of T) (I) . The model explained 94.5% of the variation in results, in which square root of T was the most influential factor (65%), followed by L (21.2%), interaction of I and square root of T (4.9%) and S (3.4%) . The model predicted LPs longer than those observed in 28.3% of the comparisons, but only in 1% of the comparisons when the lower limit of the 90% confidence interval of LP was used . Similar trends on the effect of L on C . botulinum were observed in an inoculated chicken meat study . This study demonstrated quantitatively that increasing L and S concentrations and lowering of T had a beneficial effect on delaying toxigenesis.

J Clin Microbiol, 1993 Jul, 31(7), 1870 - 5
Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems; Clabots CR et al.; A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed . REA typing was performed by two extraction methods for 1,965 C . difficile isolates obtained from patients with symptomatic C . difficile disease, asymptomatic patients who were C . difficile culture positive, and environmental surfaces . This isolate collection yielded 206 unique REA types, which were organized into 75 groups . A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis . The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained . Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains . Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type . REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods . REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C . difficile strains and is suitable for large-scale epidemiologic studies.

Nippon Jibiinkoka Gakkai Kaiho, 1993 Jul, 96(7), 1079 - 85
{Three patients with gas gangrene of the head and neck}; Ohi K et al.; Three patients with non-clostridial gas gangrene of the neck are reported . Patient 1 was a 57-year-old man, patient 2 a 63-year-old woman, and patient 3 a 44-year-old man . All three were treated by thorough debridement and precise administration of antibiotics . We also discuss 26 cases (including our 3) of gas gangrene of the head and neck, reported in Japan from 1975 to 1992, from which the following data were obtained: 1 . The 26 patients consisted of 17 males and 9 females . 2 . They ranged in age from 2 to 88 years, with a mean 56.5 years . 3 . Causes included acute pharyngolaryngeal inflammation (46%), dental disease (27%), trauma (8%) and unknown etiology (19%) . 4 . As a result of bacteriological assessment, the condition was found to be attributable to Clostridium in only 2 patients, and in the remainder the condition was non-clostridial . 5 . The mortality rate was 15% . The patients who died were at least 80 years old, and their prognosis had been poor . 6 . CT was useful for diagnosis and treatment.

J Appl Bacteriol, 1993 Jul, 75(1), 55 - 7
In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics; Devriese LA et al.; Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves . All were resistant to 64 micrograms ml-1 of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC90 0.25 micrograms ml-1), avilamycin (MIC90 0.5 micrograms ml-1) and salinomycin (MIC90 < or = 0.12 micrograms ml-1) . Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated . Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl . perfringens isolates from the same animal host species.

Diagn Microbiol Infect Dis, 1993 Jul, 17(1), 7 - 12
Comparison of vidas Clostridium difficile toxin-A assay and premier C . difficile toxin-A assay to cytotoxin-B tissue culture assay for the detection of toxins of C . difficile; Knapp CC et al.; Damage to the intestinal mucosa by Clostridium difficile (CD) is toxin mediated . Two enzyme immunoassays (EIAs) for toxin-A detection, the automated Vitek immunodiagnostic assay system CDA (Vidas CDA), and the Premier toxin A (Premier) were tested for their ability to detect toxin A in 301 stool samples and compared with an in-house tissue culture assay for toxin B (TCA) . Of these 301 samples, 49 were TCA positive and 252 were TCA negative . Agreement between Vidas CDA and TCA on the initial run was 85% (255 of 301) and increased to 94% (278 of 296) when discordant samples were retested from available frozen specimens . Corresponding levels of agreement for Premier were 91% (272 of 301) and 98% (284 of 288), respectively . If tissue culture positivity at any titer was used as the sole criterion for positivity of the specimen, agreement with positive TCA before and after repeat testing was 57% (26 of 49) and 74% (34 of 46) for Vidas CDA and 65% (32 of 49) and 95% (36 of 38) for Premier . Agreement with negative TCA titers was good: 90% for Vidas CDA and 95% for Premier, and 98% for Vidas CDA and 99% for Premier after repeat testing . Predictive values positive and negative after repeat testing were, respectively, 88% and 96% for Vidas CDA, and 95% and 99% for Premier . Results for the automated and manual EIA methods for detection of C . difficile toxin A were obtained in 2.5 h as compared with 36-48 h for tissue culture.

Clin Microbiol Rev, 1993 Jul, 6(3), 251 - 65
Clostridium difficile: clinical disease and diagnosis; Knoop FC et al.; Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis . Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978 . Since clinical and pathological features of C . difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens . The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions.

Appl Environ Microbiol, 1993 Jul, 59(7), 2339 - 42
Colony immunoblot assay of botulinal toxin; Goodnough MC et al.; Botulinal neurotoxin in and around colonies of Clostridium botulinum types A, B, and E and of toxigenic Clostridium butyricum was detected by an enzyme-linked immunoassay procedure whereby the toxin was transferred from the agar medium to a nitrocellulose support and the immobilized toxin was probed with type-specific antibodies . The method identified the toxin types of the colonies grown from a mixed inoculum of C . botulinum serotypes . The specificity of the antitoxins for type A and B toxins was improved by adsorption of the antitoxins with the antigens of heterologous type cultures.

Clin Infect Dis, 1993 Jul, 17(1), 109 - 13
Clostridium difficile infection associated with antineoplastic chemotherapy: a review; Anand A et al.; Colitis and infection due to Clostridium difficile have been reported in patients receiving antineoplastic chemotherapy for cancer without prior antibiotic treatment . Chemotherapeutic agents can alter the normal bowel flora and cause extensive intestinal inflammatory changes, potentiating both the growth of C . difficile and its production of toxin . This review includes all 23 known reported cases of C . difficile infection associated with antineoplastic chemotherapy and examines the pathogenesis, clinical features, and management of this condition . Chemotherapy-associated C . difficile colitis has been documented in association with a variety of neoplasms . Various classes of antineoplastic agents have been incriminated, methotrexate most commonly . A spectrum of illness ranging from mild to fulminant has been reported . Symptoms, management, and outcome have appeared to be no different than for antibiotic-associated cases, but the available data are limited . Chemotherapy-associated infection with C . difficile may be underreported because it is not suspected and/or because frequent concomitant use of antibiotics masks its true incidence . C . difficile infection should be kept in mind whenever a patient undergoing antineoplastic chemotherapy develops diarrhea . Prompt, appropriate diagnostic testing and early treatment may avert morbidity and death.

J Clin Microbiol, 1993 Jul, 31(7), 1845 - 9
Cloning of a DNA sequence unique to Clostridium botulinum group I by selective hybridization; McKinney MW et al.; Nucleic acid sequences were isolated from a strain of Clostridium botulinum type A by a selective hybridization method known as deletion enrichment . Nontoxigenic C . sporogenes was used to produce a C . botulinum type A sequence-enriched library . A probe, pCBM44, which showed specific hybridization to a 4.0-kb HindIII fragment present in all of the C . botulinum type A strains tested was isolated, and there was no hybridization to any strains of C . sporogenes . Upon further investigation, pCBM44 was found to hybridize to all of the group I proteolytic C . botulinum strains tested (toxin types A, B, and F) but not to hybridize to groups II, III, and IV (toxin types B, C, D, or E) . The probe did not cross-react with nine other Clostridium spp . Such a probe, which differentiates between nontoxigenic C . sporogenes and neurotoxigenic C . botulinum group I strains, should prove extremely useful.

Mol Gen Genet, 1993 Jul, 240(1), 140 - 5
The heterodimeric protease clostripain from Clostridium histolyticum is encoded by a single gene; Dargatz H et al.; Clostripain (EC 3.4.22.8) is a heterodimeric cysteine endopeptidase with strict specificity for Arg-Xaa peptidyl bonds . It is secreted by Clostridium histolyticum strains . For the first time we present evidence that both polypeptide chains of native clostripain are encoded by a single gene . DNA sequencing of two overlapping genomic DNA fragments revealed a single open reading frame (ORF) of 1581 nucleotides encoding a polypeptide of 526 amino acid residues . The ORF is preceded by canonical transcription signals and both chains of the clostripain heterodimer are completely represented by the deduced coding sequence . Most interestingly, the sequences coding for the light and the heavy chain are joined by a DNA stretch coding for a linker nonapeptide that is preceded by the C-terminal arginyl residue of the light chain and also ends with an arginyl residue . Heterologous expression of the gene in Escherichia coli yielded an enzyme capable of hydrolyzing the clostripain substrates N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-carbobenzoxy-L-arginine p-nitroanilide (Z-Arg-pNA).

J Bacteriol, 1993 Jul, 175(14), 4298 - 306
Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes; Goldfine H et al.; Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus . Recent work indicates that the L . monocytogenes enzyme plays a role during infections of mammalian cells (J.-A . Vazquez-Boland, C . Kocks, S . Dramsi, H . Ohayon, C . Geoffroy, J . Mengaud, and P . Cossart, Infect . Immun . 60:219-230, 1992) . The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate . With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI) . Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4 . When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed . The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0 . PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L . monocytogenes and by the homologous enzyme from B . cereus . The water-soluble hydrolysis product was identified as inositol-1-phosphate . For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed . 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC . The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE . I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B . cereus which is strongly inhibited by halides . Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L . monocytogenes and B . cereus.

Appl Environ Microbiol, 1993 Jul, 59(7), 2311 - 6
Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp . strain 41M-1; Nakamura S et al.; An alkaliphilic Bacillus sp . strain, 41M-1, isolated from soil produced multiple xylanases extracellularly . One of these xylanases was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography . The moleculr mass of this enzyme (xylanase J) was 36 kDa, and the isoelectric point was pH 5.3 . Xylanase J was most active at pH 9.0 . The optimum temperature for the activity at pH 9.0 was around 50 degrees C . The enzyme was stable up to 55 degrees C at pH 9.0 for 30 min . Xylanase J was completely inhibited by the Hg2+ion and N-bromosuccinimide . The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase . The apparent Km and Vmax values on xylan were 3.3 mg/ml and 1,100 micromol-1 mg-1, respectively . Xylanase J showed high sequence homology with the xylanases from Bacillus pumilus and Clostridium acetobutylicum in the N-terminal region . Xylanase J acted on neither crystalline cellulose nor carboxymethyl cellulose, indicating a possible application of the enzyme in biobleaching processes.

Angiologia, 1993 Jul-Aug, 45(4), 135 - 6
{Pseudomembranous colitis after surgery for a ruptured abdominal aortic aneurysm}; Lozano Sanchez F et al.; We present a rare postoperative complication after surgical procedures for rupture of abdominal aortic aneurysms . The disease, a pseudomembranous colitis, was early recognized (by evidence of clostridium difficile after a coprocultive) and satisfactorily treated with vancomycin . From the literature review we found only a similar case but results were absolutely different from our case.

Infection, 1993 Jul-Aug, 21(4), 241 - 4
Anaerobic bacteremia: a retrospective four-year analysis in general medicine and cancer patients; Kornowski R et al.; Anaerobic bacteremia was studied in 32 medical patients (mean age 72 years) in a four-year retrospective analysis . Malignancy was the most common underlying disease and probable portal of entry . The gastrointestinal tract was affected most often, followed by the respiratory and urinary tracts . Bacteremia occurred either following invasive (non surgical) procedures or spontaneously . The clinical course ranged from asymptomatic bacteremia, to mild febrile illness, to sepsis and septic shock (two, 12, 16 and two patients, respectively) . The case fatality rate was 25% . The causative organisms were Clostridium and Bacteroides species . All organisms isolated were susceptible to chloramphenicol . Early diagnosis and prompt treatment may reduce mortality in cases of anaerobic sepsis.

Appl Biochem Biotechnol, 1993 Jul, 42(1), 9 - 18
Purification and properties of Clostridium thermocellum endoglucanase 5 produced in Escherichia coli; Mosolova TP et al.; Endoglucanase 5 (EG5) has been isolated from the strain of E . coli TG1 harboring recombinant plasmid pCU108, which contains the cel5 gene of C . thermocellum . The enzyme has been produced with 98-fold purification and a final yield of 27% by using subsequent twofold high performance ion-exchange chromatography on Mono Q and high performance chromatofocusing on Mono P . The protein has a mol mass of 35 kDa and includes 3 multiple forms with pI 4.4-4.8 as evidenced by analytical gel isoelectrofocusing . EG5 cleaves CMC (Km = 0.097 g/L, Vmax = 8.2 mg/min.mg of protein), amorphous cellulose, xylan, lichenan as a substrate with an optimum temperature of 80 degrees C and pH 6.0 and Avicel (Km = 18.2 g/L, Vmax = 0.035 mg/min.mg of protein) with an optimum temperature of 60 degrees C and pH 6.0 . Cellobiose in concentrations up to 200 micrograms/mL do not inhibit the hydrolysis of CMC by EG5, but 10-30 micrograms/mL of glucose significantly decrease the activity of this enzyme . The stimulating role of calcium chloride and concentration of protein in the system has been demonstrated for Avicel hydrolysis by EG5.

Toxicon, 1993 Jul, 31(7), 845 - 52
Dithiothreitol generates an activated 250,000 mol . wt form of Clostridium difficile toxin B; Shoshan MC et al.; The potent cytotoxin of Clostridium difficile, toxin B, is internalized by endocytosis and activated intracellularly by an unidentified mechanism . Here it is shown that dithiothreitol treatment of toxin B resulted in (1) a mol . wt of 250,000 which is the smallest species of this toxin shown to be cytotoxic; (2) an increased endpoint titre; and (3) translocation of plasma membrane-bound toxin across the membrane at pH 4.5 . Treatment with dithiothreitol can thus mimic intracellular activation of the toxin . Radiolabelling of highly purified toxin with retained activity, as well as the 32 N-terminal amino acids and the amino acid composition, is also presented.

Appl Microbiol Biotechnol, 1993 Jul, 39(4-5), 553 - 7
A combined polymerase chain reaction-colour development hybridization assay in a microtitre format for the detection of Clostridium spp; Galindo I et al.; We have developed a rapid and sensitive assay for the detection of clostridial cells or spores in liquid food samples . The method recognizes Clostridium 16S (small ribosomal subunit RNA) rDNA (ribosomal DNA) sequences by a polymerase chain reaction-digoxigenin-labelling protocol, coupled to a capture oligonucleotide immobilized on a microtitre plate . The positive results are revealed by means of a colour reaction . In 6 h of non-intensive labour, we can detect as few as two to five clostridial cells or spores in experimentally contaminated soft drinks.

Curr Microbiol, 1993 Jul, 27(1), 27 - 33
Analyses of the gene and amino acid sequence of the Prevotella (Bacteroides) ruminicola 23 xylanase reveals unexpected homology with endoglucanases from other genera of bacteria; Whitehead TR; The DNA sequence for the xylanase gene from Prevotella (Bacteroides) ruminicola 23 was determined . The xylanase gene encoded for a protein with a molecular weight of 65,740 . An apparent leader sequence of 22 amino acids was observed . The promoter region for expression of the xylanase gene in Bacteroides species was identified with a promoterless chloramphenicol acetyltransferase gene . A region of high amino acid homology was found with the proposed catalytic domain of endoglucanases from several organisms, including Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, and Clostridium thermocellum . The cloned xylanase was found to exhibit endoglucanase activity against carboxymethyl cellulose . Analysis of the codon usage for the xylanase gene found a bias towards G and C in the third position in 16 of 18 amino acids with degenerate codons.

Mater Med Pol, 1993 Jul-Dec, 25(3-4), 127 - 31
Clostridium difficile toxin A stimulates enzyme secretion from isolated rat pancreatic acini; Malecka-Panas E et al.; Although Cl difficile bacteremia and the presence of antibodies to toxin A (TxA) have been reported, little information is available at present on TxA effect on the functional properties of various visceral organs . We have, therefore, examined the in vitro effects of TxA on amylase and trypsin secretion from rat isolated pancreatic acini . Dispersed rat pancreatic acini were exposed for 60 min to different concentrations of highly purified TxA and the rate of amylase, trypsin and LDH release were monitored . Free cytosolic calcium release in pancreatic acini after toxin A (10(-10)M to 10(-8)M) treatment was measured with Fura-2/AM, Ca-indicator dye . TxA (10(-10) to 10(-8)M) increased significantly the rate of both the amylase and trypsin secretion without any membrane damage, with toxin A exerting its action via calcium dependent pathway as suggested by intracellular calcium release measured with Fura-2/AM.

FEBS Lett, 1993 Jun 21, 324(3), 353 - 7
Production of monoclonal antibodies that inhibit ADP-ribosylation of small GTP-binding proteins catalyzed by Clostridium botulinum ADP-ribosyltransferase C3; Toratani S et al.; Four monoclonal antibodies that inhibited ADP-ribosylation of 23 kDa protein(s) of ascidian eggs catalyzed by Clostridium botulinum ADP-ribosyltransferase C3 were produced . They also inhibited C3-catalyzed ADP-ribosylation of the 24 kDa protein of rat liver cytosol . By the immunoprecipitation technique, it was found that they recognized small GTP-binding proteins of ascidian eggs and mammalian brains, but did not interact with the rat brain activator of the ADP-ribosyltransferase reaction . The antibody can also immunoprecipitate recombinant Rho A irrespective as to whether the Rho A is the GDP-bound form or the GTPrS-bound form . Thus the antibodies are novel and useful tools in analyzing the physiological roles of the Rho family of GTP-binding proteins.

Biochemistry, 1993 Jun 15, 32(23), 6058 - 64
Using dysprosium complexes to probe the nitrogenase paramagnetic centers; Oliver ME et al.; EPR progressive power saturation techniques were used to monitor relaxation enhancement of the nitrogenase paramagnetic centers produced by interaction with Dy3+ complexes . Three models are presented for the relationship between the degree of enhancement and the distance of closest approach of Dy3+ to the intrinsic metal cluster . In the first model, the perturbing dysprosium ions are represented as a single average site . The second and third models are variations of the treatment of Innes and Brudvig {(1989) Biochemistry 28, 1116-1125} and assume the unknown protein to be spherical with Dy3+ dispersed either randomly over the surface of the protein or randomly in solution . Using these models, the distance of closest approach of the Dy3+ complex to the {4Fe-4S} cluster in the Fe-protein from Azotobacter vinelandii was determined to be 5.0-6.5 A . Similarly, the distance of closest approach to FeMoco in the MoFe-protein was determined to be 0.0-1.2 A, which, when corrected to the fact that FeMoco exists as an S = 3/2 spin state, indicates that the distance is > or = 6 A . These distances did not change when (1) either protein was in the presence of the other, (2) both proteins were cross-linked to each other, or (3) the Fe-protein from A . vinelandii was mixed with the MoFe-protein from Clostridium pasteurianum . On the other hand, formation of the inactive complex of the Fe-protein from C . pasteurianum with the MoFe-protein from A . vinelandii blocked dysprosium-induced relaxation enhancement, implying that each component protein overlaps the metal cluster in the complementing protein.

Tijdschr Diergeneeskd, 1993 Jun 1, 118(11), 361 - 4
{Findings in goats at necropsy}; Akkermans JP et al.; An overview is given of the results of diagnostic investigations into the cause of death of 463 goats performed by the Animal Health Service of West and Middle Netherlands during the period 1 January 1986 to 31 December 1991 . The results are discussed and analysed with regard to the age of the animals, the anamnesis, and the abnormalities found at post-mortem examination and with regard to the results of further parasitological, bacteriological, and virological investigations . Enterotoxaemia caused by (epsilon-toxin-positive) Clostridium perfringens was present in 9% of the animals and fibrinous pneumonia with pasteurella in 15% of the animals . Animals with Aujeszky's disease did not show the 'itching' symptom . There were few parasitic infections.

J Med Microbiol, 1993 Jun, 38(6), 434 - 41
Serogroup F strains of Clostridium difficile produce toxin B but not toxin A; Depitre C et al.; Most toxigenic strains of Clostridium difficile produce two toxins: an enterotoxin (toxin A) and a cytotoxin (toxin B) . Only one strain (strain 8864) has been reported to produce toxin B but no toxin A . Serogroup F strains (44) of C . difficile, often isolated from asymptomatic infants, have been examined for toxin production . These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in vitro when examined in three distinct immunoassays . Nevertheless, all the strain produced a cytotoxin . Immunological differences between the cytotoxin of the serogroup F strains and that produced by C . difficile strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C . difficile strain VPI 10463 or C . sordellii lethal toxin (LT) . Polymerase chain reaction amplification with primers derived from C . difficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene . When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces . However, cytotoxin was detected . Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C . difficile strain VPI 10463 . The serogroup F strains appeared to be homogeneous and distinct from other C . difficile strains with regard to toxin production.

J Bacteriol, 1993 Jun, 175(12), 3838 - 43
Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444; Kim AY et al.; A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444 . Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E . coli . Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E . coli DH11S transformants containing pCAK1 in the absence of cell lysis . This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E . coli DH11S . The single-stranded form of pCAK1 isolated from E . coli supernatant was transformed into E . coli DH5 alpha' or DH11S by electroporation . Isolation of ampicillin-resistant E . coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E . coli . The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1 . Following phagemid construction in E . coli, pCAK1 was transformed into C . acetobutylicum ATCC 824 and C . perfringens 13 by intact cell electroporation . Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C . acetobutylicum and C . perfringens suggested that it was identical to that present in E . coli transformants.

Am J Gastroenterol, 1993 Jun, 88(6), 891 - 7
Clostridium difficile infection is a treatable cause of diarrhea in patients with advanced human immunodeficiency virus infection: a study of seven consecutive patients admitted from 1986 to 1992 to a university teaching hospital; Cappell MS et al.; Of 427 human immunodeficiency virus-seropositive patients admitted to the Robert Wood Johnson University Hospital from January 1986 through August 1992, seven had Clostridium difficile enteric infection documented by the presence of cytotoxin B in the stool, without other enteric infection . All seven patients had AIDS, and all had recently received antibiotics . These patients had a severe clinical presentation of C . difficile infection . All patients had profound watery diarrhea, with a mean of 20 +/- 14 (SD) bowel movements per day . Four had fever > 38.5 degrees C, and another had hypothermia . Three patients had borderline hypotension, and another was orthostatic . The mean pulse was 119 +/- 26 (SD) beats/min . Five patients had abdominal pain and tenderness . Two had occult blood in the stool . Four had metabolic derangements such as hyponatremia, hypokalemia, or prerenal azotemia . Three of four patients undergoing abdominal roentgenography had radiographic findings consistent with severe colitis of colonic dilation, mural thumbprinting, or mural thickening . Sigmoidoscopic findings ranged from diffuse erythema to prominent pseudomembranes . During a mean interval of 14.3 +/- 6.2 (SD) days before institution of specific antibiotic therapy, the diarrhea spontaneously resolved in only one of the seven patients . In the others, the diarrhea resolved on average 7.3 +/- 4.0 (SD) days after instituting antibiotic therapy . During a mean follow-up of 4.4 +/- 6.3 (SD) months, only two patients redeveloped diarrhea . Both patients had recurrent C . difficile colitis; the symptoms again rapidly resolved after repeat antibiotic therapy . We conclude that in patients with AIDS C . difficile may present as a severe enteric infection with profound diarrhea due to immunosuppression, that the diarrhea may be prolonged and not remit spontaneously, and that the diarrhea usually rapidly resolves with specific antibiotic therapy.

J Bacteriol, 1993 Jun, 175(11), 3452 - 8
Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose; Gelhaye E et al.; The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate . The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h) . Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose . Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h . In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis . The stage of growth influenced adhesion of C . cellulolyticum; maximum adhesion was found during the exponential phase . Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose . An exhaustion of the accessible cellulose could be responsible for this release.

J Bacteriol, 1993 Jun, 175(11), 3394 - 400
Sequence and molecular characterization of a DNA region encoding a small heat shock protein of Clostridium acetobutylicum; Sauer U et al.; A DNA region of Clostridium acetobutylicum containing a gene (hsp 18) with significant homology to a family of small eukaryotic heat shock proteins was cloned and sequenced . It is the second reported sequence of a low-molecular-weight heat shock protein from gram-positive bacteria and is induced not only by heat shock but also at the onset of solventogenesis, as determined by Northern (RNA) blot analysis, thus confirming the results of an earlier study performed at the protein level (A . Pich, F . Narberhaus, and H . Bahl, Appl . Microbiol . Biotechnol . 33:697-704, 1990) . By primer extension analysis, a transcriptional start site was identified 149 bp upstream of hsp18 . This site was preceded by a region that exhibits high homology to the consensus promoter sequences of gram-positive bacteria, as well as sigma 70-dependent Escherichia coli . A direct repeat structure was detected in the -35 region . The promoter is located 196 bp from the start of a potential regulatory tRNA(Thr)ACG gene involved in the shift to solventogenesis which is transcribed in the opposite direction . A putative rho-independent transcription termination structure was identified at the 3' end of hsp18.

J Bacteriol, 1993 Jun, 175(11), 3353 - 60
Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG; Lemaire M et al.; The nucleotide sequence of the celG gene of Clostridium thermocellum, encoding endoglucanase CelG, was determined . The open reading frame extended over 1,698 bp and encoded a 566-amino-acid polypeptide (molecular weight of 63,128) similar to the C . thermocellum endoglucanase CelB (51.5% identical residues) . The N terminus displayed a typical signal peptide, followed by a catalytic domain . The C terminus, which was separated from the catalytic domain by a 25-amino-acid segment rich in Pro, Thr, and Ser, contained two conserved stretches of 22 amino acids closely similar to those previously described in other cellulases from the same organism . Expression of the gene in Escherichia coli was increased by fusing the fragment coding for the catalytic domain in frame with the start of the lacZ' gene present in the vector . A low- and a high-M(r) form of the protein were purified . The two forms displayed identical enzymatic properties . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that both forms consist of a major polypeptide of M(r) 50,000 and two minor polypeptides of M(r)s 49,000 and 48,000, resulting from heterogeneous proteolytic cleavage at the C terminus . An antiserum raised against the forms purified from E . coli reacted with an immunoreactive polypeptide of M(r) 66,000, which was associated with the extracellular cellulolytic complex of C . thermocellum known as the cellulosome.

J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1331 - 6
Microbial transformation of nitroaromatic compounds under anaerobic conditions; Gorontzy T et al.; The transformation of several mono- and dinitroaromatic compounds (tested at 50-200 microM) by methanogenic bacteria, sulphate-reducing bacteria and clostridia was studied . Some of the nitroaromatics tested were transformed chemically by 1.5 mM quantities of culture media reducing agents, like cysteine or sulphide . This abiotic reduction occurred at the o-nitro-groups preferentially . Nitrophenols, p-nitroaniline and p-nitrobenzoic acid were completely transformed biologically into the corresponding amino derivatives . The nitroaromatics were transformed by all of the bacterial strains tested . While growing cells of sulphate-reducing bacteria and Clostridium spp . carried out nitroreduction, methanogen cells lysed in the presence of nitroaromatics . Nevertheless these culture suspensions converted nitroaromatics to the corresponding amino derivatives . This was also confirmed by crude cell extracts of methanogenic bacteria . The rate of nitroreduction by sulphate-reducing bacteria depended on the electron donors supplied and the cell density, with molecular hydrogen being the most effective donor of reducing equivalents . The toxicity of p-nitrophenol to some of the organisms tested depended on the concentration of the nitroaromatic compound and the type of organism.

J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1227 - 34
Cloning, sequencing and biochemical characterization of xylose isomerase from Thermoanaerobacterium saccharolyticum strain B6A-RI; Lee YE et al.; The xylose isomerase gene from Thermoanaerobacterium saccharolyticum strain B6A-RI was cloned by complementation using Escherichia coli xyl-5 mutant strain HB101 . One positive clone was detected and the recombinant plasmid, pZX16, was isolated . The clone contained the vector pUC18 and an insert fragment of 4.5 kb . The cloned xylose isomerase gene (xylA) was expressed constitutively in E . coli . The gene contained one open reading frame (ORF) of 1317 bp, which corresponds to 439 amino acid residues . The molecular mass of the gene product was calculated to be 50474 Da from the deduced amino acid sequence . A putative promoter region (Pribnow box), TATAATATATAAT, which repeated twice at the -10 region in E . coli, was found 25 bp upstream of the ribosomal binding site . The deduced amino acid sequence of T . saccharolyticum strain B6A-RI xylose isomerase exhibited very high homology to those from Thermoanaerobacterium thermosulfurigenes 4B (formerly Clostridium thermosulfurogenes 4B) and Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) . Codon usage in xynA, xynB and xylA showed a clear propensity for AT-containing isocodons . The native molecular mass of the purified recombinant thermostable xylose isomerase was 200 kDa, and the enzyme was a tetramer comprised of identical subunits . The apparent temperature and pH optima for activity of the cloned xylose isomerase were 80 degrees C and 7.0 to 7.5, respectively.

Infect Dis Clin North Am, 1993 Jun, 7(2), 277 - 93
The role of the clinical microbiology laboratory in the management of Clostridium difficile-associated diarrhea; Peterson LR et al.; Nosocomial diarrhea due to infection with C . difficile is a major health care problem, causing 20% to 30% of institutionally acquired diarrhea and affecting up to 8% of hospitalized patients . The clinical microbiology laboratory should be able to provide both diagnostic and epidemiologic services for institutions where this disease occurs . Diagnostic testing includes culture for isolation of toxigenic C . difficile and detection of either toxin A or B from stool specimens . Epidemiologic services include providing appropriate media and specimen preparation for surveillance activities and performance of accurate typing of C . difficile strains when necessary to determine organism relatedness for development of effective infection control practices.

Microbiol Rev, 1993 Jun, 57(2), 347 - 66
Bacterial phospholipases C; Titball RW; A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins . Initial speculation that all phospholipases C would have lethal properties has not been substantiated . Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C . The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain . The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis . However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease . Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C . Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome . Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines . A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.

In Vitro Cell Dev Biol Anim, 1993 Jun, 29A(6), 456 - 60
Botulinum toxin A inhibits acetylcholine release from cultured neurons in vitro; Ray P; Clostridium botulinum type toxin A (BoTx) blocks stimulus-induced acetylcholine (ACh) release from presynaptic nerve terminals at peripheral neuromuscular junctions . However, the detailed mechanism of this effect remains elusive . One obstacle in solving this problem is the lack of a suitable in vitro homogenous cholinergic neuronal model system . We studied the clonal pheochromocytoma PC12 cell line to establish such a model . PC12 cells were differentiated in culture by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release properties . Stimulation of these cells with high K+ (80 mM) in the perfusion medium markedly increased calcium-dependent {3H}ACh release compared to undifferentiated cells . Stimulated {3H}ACh release was totally inhibited by pretreatment of cells with 2 nM BoTx for 2 h . BoTx inhibition of {3H}ACh release was time- and concentration-dependent . A 50% inhibition was obtained after 2 h incubation with a low (0.02 nM) toxin concentration . The time required for 2 nM BoTx to cause a measurable inhibition (18%) of stimulated {3H}ACh release was 30 min . Botulinum toxin inhibition of stimulated ACh release was prevented by toxin antiserum and heat treatment, suggesting the specificity of the toxin effect . Our results show that by differentiation with NGF, PC12 cells can be shifted from an insensitive to a sensitive state with respect to BoTx inhibition of stimulated ACh release . This cell line, therefore, may serve as a valuable in vitro cholinergic model system to study the mechanism of action of BoTx.

Appl Environ Microbiol, 1993 Jun, 59(6), 1876 - 82
Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri; Wolff RA et al.; A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase) . C . kluyveri fermented {1,4-13C}succinate primarily to {1-13C}acetate, {2-13C}acetate, and {1,4-13C}butanoate . Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group . Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis . 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized . Neither activity was found in cells grown on acetate and ethanol (acetate cells) . Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts . In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells . These data support the hypothesis that C . kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.

Appl Environ Microbiol, 1993 Jun, 59(6), 1731 - 4
Comparison of the azoreductase and nitroreductase from Clostridium perfringens; Rafii F et al.; The purified azoreductase and nitroreductase of Clostridium perfringens, which have similar electrophoretic properties, both reacted in a Western blot (immunoblot) with a polyclonal antibody raised against the azoreductase . The activity of both enzymes was enhanced by flavin adenine dinucleotide and was inhibited by menadione, o-iodosobenzoic acid, and the antibody against azoreductase . Reduction of the azo dye Direct Blue 15 by the azoreductase was inhibited by nitroaromatic compounds . The apparent Km of the enzyme for reduction of Direct Blue 15 in the presence of 1-nitropyrene was higher than the Km with the azo dye alone, demonstrating competitive inhibition . The data show that the same protein is involved in the reduction of both azo dyes and nitroaromatic compounds.

Am J Forensic Med Pathol, 1993 Jun, 14(2), 151 - 4
Fatal Clostridium perfringens and Escherichia coli sepsis following urea-instillation abortion; Jasnosz KM et al.; Intraamniotic instillation of urea is a common mode of legal second-trimester pregnancy termination . Associated mortality rarely occurs and is most commonly due to amniotic fluid embolism, pulmonary thromboembolism, infection, hemorrhage, and disseminated intravascular coagulation (DIC) . We present the case of an 18-year-old gravida 2, para 1 white woman at 18 weeks' gestation who underwent intraamniotic instillation of hyperosmolar urea and intracervical insertion of laminaria tents; 19 h later, she became unresponsive, academic, and went into shock . Coagulation studies were diagnostic of DIC . Bacilli were seen on peripheral blood smear . Autopsy showed marked subcutaneous emphysema of the anterior abdominal wall, necrosis and emphysema of the uterus, diffuse pulmonary alveolar damage, and renal cortical necrosis . Antemortem blood cultures grew Clostridium perfringens and Escherichia coli . Postmortem culture of the uterus grew E . coli . The source of infection was most likely the introduction of vaginal organisms via laminaria insertion . This is apparently the first reported case of death caused by Clostridium perfringens and E . coli sepsis following urea instillation.

Can J Surg, 1993 Jun, 36(3), 261 - 5
Value of cultures of tissue samples taken at operation for lower intestinal perforation; Fraulin FO et al.; Tissue cultures from perforations of the lower intestinal tract commonly yield both aerobes (coliform organisms) and anaerobes (Bacteroides sp . and Clostridium sp.) . To determine the consistency of this pattern and the value of intraoperative cultures, the authors reviewed the hospital records of 115 patients with perforation of the appendix (100 patients) or colon (15 patients), treated between 1987 and 1990, in whom organisms were cultured from tissue samples taken intraoperatively . Attention was paid to the organisms cultured, their distribution and antibiotic sensitivity in initial samples and in subsequent samples obtained when there were septic complications . On average, 4.7 bacterial isolates per patient were obtained . The common organisms were as expected: Bacteroides fragilis, Escherichia coli and Clostridium sp . Although the culture results did not affect the management of these patients, the sensitivity of Bacteroides fragilis to cefoxitin was found to be lower than expected, indicating a shift in sensitivity.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S439 - 42
Optimal methods for identifying Clostridium difficile infections; Gerding DN et al.; The major controversy in the diagnosis of symptomatic gastrointestinal infection due to Clostridium difficile is whether laboratory evidence of the C . difficile organism in culture is sufficient or if evidence of one of the C . difficile toxins in stool should be required . Cultures performed properly on selective media currently are the most sensitive method for detection of C . difficile, whereas the cell cytotoxin assay for detection of toxin B is the most specific . Stool specimens from patients with clinical diarrhea are sometimes found to be culture-positive for C . difficile but assay-negative for cytotoxin . Samples from these patients can be viewed as false-positive by culture or false-negative by cytotoxin test . Evidence from endoscopy indicates that some patients whose stool is culture-positive for the organism but assay-negative for toxin do have pseudomembranous colitis, but its incidence among such patients (11%) is lower than that among patients whose stool is culture- and assay-positive (51%) . Response to treatment with vancomycin or metronidazole is similar in the two groups of patients, and withholding treatment from patients whose stool contains C . difficile but not cytotoxin may result in increased morbidity and mortality . Up to one-third of C . difficile organisms from stool specimens that are culture-positive but assay-negative are incapable of producing cytotoxin in vitro, a finding that suggests these organisms may not be the cause of diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S435 - 8
How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay?
Citron DM, Appelbaum PC.
Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive . Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory . Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician . Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified . Group- or genus-level identifications may suffice in other instances . The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents . Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin).

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S427 - 34
The friendly anaerobes; Bokkenheuser V; Anaerobic bacteria include the most pathogenic of microorganisms . Their primary function, however, is hardly to cause illness . They rarely are involved in epidemics or in clinically significant infections . Some organisms, e.g . lactobacilli, control the normal vaginal ecosystem, and the intestinal anaerobes probably are instrumental in restraining the growth of Clostridium difficile in human carriers . The main role of anaerobes appears to be the provision of catabolic enzymes for organic compounds that cannot be digested by enzymes of eukaryotic origin . They are needed for the catabolism of cholesterol, bile acids, and steroid hormones; they hydrolyze a number of flavonoid glycosides to anticarcinogens; and they detoxify certain carcinogens . Anaerobic enzymes are used industrially in the production of cheese; the conversion of starch to sweeteners; and the transformation of sawdust, wood chips, and waste paper to fuel . Indeed, the anaerobes may well be the gene bank on which future generations of eukaryotic organisms will rely to adapt successfully to an ever-changing world.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S387 - 9
Antibiotic susceptibility of anaerobic bacteria in Europe; Tuner K et al.; Twenty-two laboratories in 15 European countries determined the MICs of 12 antibiotics for 1,289 isolates of the B . fragilis group by a microdilution method . There was no resistance to metronidazole (breakpoint, 8 mg/L), and only one isolate was found to be resistant to chloramphenicol (breakpoint, 8 mg/L) . Resistance was uncommon to imipenem (0.3% at concentrations > 4 mg/L), amoxicillin/clavulanate (1% at concentrations > 8 mg/L), cefoxitin (3% at concentrations > 32 mg/L), mezlocillin (6% at concentrations > 64 mg/L), and clindamycin (9% at concentrations > 4 mg/L) . The majority of the isolates were resistant to ampicillin (93% at concentrations > 4 mg/L), ciprofloxacin (56% at concentrations > 4 mg/L), and tetracycline (64% at concentrations > 4 mg/L) . Bacteroides fragilis was the most susceptible species of the group, and the most striking regional differences in susceptibility were found in association with clindamycin and tetracycline; more resistance was noted in isolates from the southern part of Europe (Greece, Italy, Portugal, and Spain) . It has been reported that Fusobacterium species and Clostridium species occasionally produce beta-lactamases . A few metronidazole-resistant Clostridium perfringens strains but no metronidazole-resistant fusobacteria have been isolated.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S377 - 81
Patterns of susceptibility to fluoroquinolones among anaerobic bacterial isolates in the United States; Goldstein EJ; The activity of the fluoroquinolone antibiotics against anaerobic bacteria has generally been moderate to poor . Nalidixic acid and cinoxacin show poor activity against almost all anaerobes . Norfloxacin and enoxacin exhibit poor anaerobic activity (MIC90, 8- > 256 micrograms/mL) except against Clostridium perfringens, Bacteroides ureolyticus, and some eubacteria . Norfloxacin is slightly more active than enoxacin against some Bacteroides species . While ciprofloxacin is more active than earlier fluoroquinolones against anaerobes such as Bacteroides fragilis (MIC90, 4-16 micrograms/mL), fusobacteria, and peptostreptococci, its activity is often variable and its MIC90 is frequently close to the maximal level attainable in serum . Ofloxacin is active against B . fragilis (MIC90, 4-8 micrograms/mL) but not against other species of the B . fragilis group (MIC90, 8-32 micrograms/mL); other anaerobes (e.g., peptostreptococci and fusobacteria) are generally susceptible to < or = 8 micrograms of ofloxacin/mL . Several newer quinolones exhibit improved anaerobic activity (although the studies yielding relevant data have often used diverse methods, small numbers and varieties of isolates, and different breakpoints) . CI-960 and WIN 57273 inhibit 95% of strains tested at < or = 2 micrograms/mL . CI-990, sparfloxacin, and temafloxacin inhibit most anaerobes at < or = 2 micrograms/mL, but clustering around the breakpoint and strain variability have been noted . CI-990 is active against more than 85% of B . fragilis strains (MIC90, 4 micrograms/mL), but other species of the B . fragilis group often require > 4 micrograms/mL for inhibition; Prevotella bivia, Fusobacterium varium, and Fusobacterium ulcerans are usually resistant.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S361 - 6
Antibacterial activity of meropenem and selected comparative agents against anaerobic bacteria at seven North American centers; Sheikh W et al.; The antibacterial activity of meropenem and comparative agents against approximately 1,000 anaerobes was determined using the disk dilution methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS) . The organisms represented 27 species of six genera and included the most common pathogens . Meropenem and imipenem were the most active drugs and were comparable in overall activity, generally exhibiting an MIC90 of < or = 1 micrograms/mL . In contrast, the MICs of cefoxitin, clindamycin, and metronidazole were 32, 16, and 2 micrograms/mL, respectively . Meropenem was two- to fourfold more active than imipenem against selected Bacteroides species, Clostridium species, and Fusobacterium species . At a concentration of 1 microgram/mL, meropenem was more active than imipenem against cefoxitin-resistant Bacteroides fragilis or Bacteroides thetaiotaomicron . At a concentration of < or = 0.5 micrograms/mL, meropenem was more active than imipenem against clindamycin-resistant Bacteroides distasonis . At a concentration of 2 micrograms/mL, meropenem was more active than imipenem against cefoxitin-resistant or clindamycin-resistant Clostridium difficile . Thus, meropenem's high potency and broad-spectrum activity against common, rare, and drug-resistant anaerobes confirms its utility in the treatment of mixed anaerobic and aerobic infections.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S239 - 44
Elevated levels of serum immunoglobulins in asymptomatic carriers of Clostridium difficile; Mulligan ME et al.; Serum levels of IgA, IgM, and polyvalent immunoglobulins reactive with Clostridium difficile were determined by ELISA for asymptomatic carriers (n = 5), symptomatic individuals (n = 21), and a pool of 30 "normal" individuals . Mean IgA concentrations expressed as optical density (OD +/- SD) were significantly higher (P < .001) for asymptomatic carriers (1.252 +/- 0.516) than for symptomatic patients (0.374 +/- 0.145) . Mean serum IgM levels also were significantly higher (P < .001) for carriers (1.456 +/- 0.582) than for symptomatic patients (0.727 +/- 0.331), as were mean values for polyvalent immunoglobulins (2.25 +/- 0.718 for carriers vs . 1.457 +/- 0.574 for symptomatic patients; P < .05) . Although the patient populations were small, the levels of immunoglobulins reactive with C . difficile antigens differed significantly . This difference might reflect the ability of the host to mount an immune response and might be a factor influencing whether a patient develops disease due to this organism . The ability to detect differences in immunoglobulin levels might also help differentiate infection from colonization.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S228 - 33
Role of the laboratory in investigations of Clostridium difficile diarrhea; Brazier JS; The laboratory diagnosis of acute diarrhea due to Clostridium difficile can be based solely on the demonstration of either cytotoxin or enterotoxin in the stool . This and other noncultural methods, however, fail to provide isolates from which useful data can be obtained . Outbreaks can be recognized only by comparison of isolated strains . A dual approach of toxin detection and isolation is therefore recommended.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S219 - 27
Purification of a functional receptor for Clostridium difficile toxin A from intestinal brush border membranes of infant hamsters; Rolfe RD et al.; A receptor for Clostridium difficile toxin A was purified from brush border membranes (BBMs) from the small intestine of infant hamsters . The BBMs were solubilized with Triton X-114, and the solubilized receptor was purified with use of a toxin A immobilized affinity-chromatography column and differential temperature elution . SDS-PAGE and silver staining of the purified receptor revealed numerous high-molecular-weight bands . However, ligand blotting analysis with 125I-toxin A used as the probe identified a 163-kD protein as the predominate toxin A-binding molecule . Toxin A bound to the purified receptor at physiological temperature, but the amount of toxin bound increased at lower temperatures . Bovine thyroglobulin bound to toxin A and inhibited its binding to the purified receptor . Preincubation of the receptor with lectins produced by Bandeirea simplicifolia or Datura stramonium reduced specific binding by 125I-toxin A . Our data indicate that the purified toxin A receptor from small intestine BBMs of infant hamsters is a galactose- and N-acetylglucosamine-containing glycoprotein.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S214 - 8
The microecology of Clostridium difficile; Wilson KH; An understanding of the microecology of Clostridium difficile provides for a better understanding of the disease that this organism causes . C . difficile is not a significant component of the microflora in the colon of healthy adult humans or animals; however, it can establish large populations in antibiotic-treated or gnotobiotic animals and in infants before they acquire a complete flora . Major factors that determine whether or not disease develops are: (1) the size of the C . difficile populations; (2) the toxigenicity of the colonizing strain; (3) the presence of other organisms that affect toxin expression or activity; (4) susceptibility of the host; and possibly (5) a strain's adhesion to colonic epithelium . The rest of the colonic flora determines the size of the C . difficile population, at least in part by limiting available nutrients . In outbreaks, most C . difficile disease is caused by nosocomial strains . Environmental contamination with spores and spread via the hands of health care workers have been implicated in transmission . Information with regard to this organism's microecology suggests alternative approaches to the control of disease.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S195 - 9
Role of theta toxin, a sulfhydryl-activated cytolysin, in the pathogenesis of clostridial gas gangrene; Stevens DL et al.; Clostridial infections cause a wide variety of dramatic infections and intoxications . In each case the major virulence factors are extracellular toxins . Clostridium perfringens produces potent exotoxins, which are its major virulence factors . theta Toxin, a thiol-activated cytolysin, causes the clear zone of hemolysis around colonies on blood-agar plates, suppresses myocardial contractility ex vivo, and induces shock within 1 to 2 hours in vivo . Low concentrations of theta toxin induce priming and degranulation of polymorphonuclear leukocytes (PMNs) and functional up-regulation of PMN-dependent adherence molecules such as the integrin CD11/CD18, whereas higher concentrations are cytotoxic . Similarly, theta toxin causes concentration- and time-dependent induction of endothelial cell synthesis of platelet-activating factor, a potent proinflammatory lipid autocoid that mediates endothelial cell-dependent adherence of PMNs . These data suggest that theta toxin in high concentrations is a potent cytolysin and promotes direct vascular injury at the site of infection . At lower concentrations theta toxin activates PMNs and endothelial cells, and in so doing promotes vascular injury distally by activating adherence mechanisms . The rapid tissue necrosis associated with C . perfringens infection may be related to progressive vascular compromise orchestrated by dysregulated host-cell responses induced by theta toxin.

FEMS Microbiol Lett, 1993 Jun 1, 110(1), 77 - 83
Anaerobic breakdown of uroporphyrins I and III to bile pigments by extracts of Clostridium tetanomorphum; Chen CH et al.; Two blue bile pigments were formed under anaerobic conditions from the tetrapyrrole precursor delta-aminolevulinate by cells and cell extracts of Clostridium tetanomorphum . These compounds were also formed by cell extracts from the octacarboxylic tetrapyrrole, uroporphyrin III . Bactobilin, the first bacterial bile pigment to be discovered, is related to uroporphyrin I . The present results hence increase the number of bile pigments related to bactobilin . Bactobilin and its isomers differ markedly from the eukaryotic bile pigments which are all related to the dicarboxylic compound, protoporphyrin IX . The enzyme participating in the formation of the bacterial bile pigments was obligatorily anaerobic, in decided contrast to the only other known bile pigment-forming enzyme, the eukaryotic oxygen-requiring heme oxygenase.

FEMS Microbiol Lett, 1993 Jun 1, 110(1), 45 - 50
Biochemical and immunological properties of the C-terminal domain of the alpha-toxin of Clostridium perfringens; Titball RW et al.; The C-terminal domain of the alpha-toxin (cpa247-370) of Clostridium perfringens has been expressed in Escherichia coli and purified . Antiserum raised against cpa247-370 reacted in an identical manner to anti-alpha-toxin serum when used to map epitopes in the C-terminal domain, suggesting that cpa247-370 was immunologically and structurally identical to this region in the alpha-toxin . The isolated cpa247-370 was devoid of sphingomyelinase activity or haemolytic activity and was not cytotoxic for mouse lymphocytes . Haemolytic activity was detected when cpa247-370 was tested with the N-terminal domain of the alpha-toxin (cpa1-249), confirming that cpa247-370 confers haemolytic properties on the phospholipase C activity of the alpha-toxin . Haemolytic activity was not detected if cpa247-370 was tested with the Bacillus cereus phosphatidylcholine phospholipase C, nor if cpa1-249 and cpa247-370 were incubated sequentially with erythrocytes.

Monatsschr Kinderheilkd, 1993 Jun, 141(6), 474 - 7
{Clostridium difficile in early childhood ulcerative pancolitis}; Hoppen T et al.; Ulcerative colitis is a rare disease in young infants . Less than one per cent of cases occur during the first two years of life . We describe a male child who developed frequent bloody diarrhea at the age of 20 months . More common causes like infections or gastrointestinal food allergy were excluded . Endoscopy and histopathological evaluations revealed ulcerative colitis of the entire colon . Treatment with sulfasalazine and prednisone resulted in a clinical remission after seven weeks . The follow-up of 15 months was complicated by Rotavirus infection . Two relapses were caused by Clostridium difficile infections . The latter were successfully treated with oral vancomycine, but in the last relapse an increased dosage of prednisone was required, too . In relapses of inflammatory bowel disease gastrointestinal infections, especially caused by Clostridium difficile should be considered and treated adequately.

Scand J Immunol, 1993 Jun, 37(6), 634 - 6
In vitro and in vivo measurement of cell-mediated immunity in patients with HIV-1 infection; Borleffs JC et al.; We studied the usefulness of the in vitro lymphoproliferation assay and the in vivo skin test in HIV-1-infected patients by using Clostridium tetani and tuberculin as testing antigens . Moreover, the relationship between data obtained from both assays was studied . In 56 HIV-infected patients not receiving antiretroviral therapy CD4+ cell counting was performed . In addition, in vitro (lymphocyte proliferation assay) and in vivo (delayed type hypersensitivity skin test) measuring of the immune status was done using C . tetani and tuberculin as testing antigens . When using C . tetani a significant correlation between the results of both tests and the CD4+ cell count was found . In contrast to earlier reports from African countries, in vivo skin testing using tuberculin did not yield clinically significant information on the degree of immunodeficiency . We explain our findings by the fact that health care policy in The Netherlands encompasses vaccination with C . tetani, which enables the application of C . tetani as testing antigen for measuring immune function both in vitro and in vivo.

Br J Rheumatol, 1993 Jun, 32(6), 467 - 73
Optimizing the assessment of disease activity during treatment with anti-rheumatoid drugs; Astbury C et al.; Clinical trials in RA usually involve the use of several laboratory assessments of disease activity . Their use is not universal and the relative value of many novel assessments has not been determined in relation to existing clinical and laboratory methods . This study attempts to investigate the value of established and novel assessments of disease activity during treatment with accepted DMARDs . Over a 48-week study period, changes in cytidine deaminase (CD), beta 2-microglobulin, alpha 1-acid glycoprotein (alpha 1-AGP), serum antibodies to Clostridium perfringens alpha-toxin, pre-albumin and caeruloplasmin were compared to a group of established clinical and laboratory assessments including plasma viscosity, CRP haemoglobin and platelet count during treatment with the established second-line drugs, D-penicillamine (n = 20), sulphasalazine (n = 17), gold (n = 12) and hydroxychloroquine (n = 18) . Overall, the assessments showing the greatest degree of change were plasma viscosity, articular index, summated change score, platelet count, CD, white cell count, alpha 1-AGP, CRP and pain score . The assessments showing the greatest degree of change were not homologous between the treatment groups and no single assessment was outstanding for a particular drug treatment.

Clin Infect Dis, 1993 Jun, 16 Suppl 4, S234 - 8
Diagnosis and monitoring of Clostridium difficile infections with the polymerase chain reaction; Kuhl SJ et al.; Toxigenic Clostridium difficile is the etiologic agent of pseudomembranous colitis . We have developed an assay system for the rapid direct detection of toxigenic C . difficile in human stool samples . After DNA extraction, polymerase chain reaction (PCR) amplification is undertaken with primers targeting specific sequences in the C . difficile 16S rRNA gene . Next, toxigenic strains of C . difficile are distinguished from nontoxigenic strains by PCR amplification of toxin A and/or B gene sequences . This study included 12 patients with C . difficile colitis, seven of whom had clinical relapses after discontinuation of vancomycin therapy . We detected toxigenic C . difficile in stools from four (57%) of these seven patients before relapse--at a time when no toxin B was detectable in stools and results of anaerobic culture were negative . The PCR assay is 100-fold more sensitive than anaerobic culture methods . The course of the infection in one patient (both during and after therapy) was monitored by the PCR technique . The multigene analysis approach permitted the detection of colonization with a nontoxigenic strain when this patient's relapses had cleared . This clinically applicable assay allows earlier detection of infection with toxigenic C . difficile . The result is more timely therapeutic intervention.

Arch Biochem Biophys, 1993 Jun, 303(2), 394 - 401
Studies on three reductases which have polycyclic aromatic hydrocarbon quinones as substrates; Jarabak J et al.; Unlike rodent tissues, the major quinone reductase in centrifuged homogenates of human liver and placenta is a carbonyl reductase rather than a DT-diaphorase . When reduction of polycyclic aromatic hydrocarbons is compared, there are differences between the human placental carbonyl reductase, rat liver DT-diaphorase, and Clostridium DT-diaphorase . In a buffer containing 1% albumin and 10 microM quinone, 9,10-phenanthrenequinone is reduced most rapidly by the carbonyl reductase, 2-methyl-1,4-naphthoquinone is reduced most rapidly by the rat enzyme, and 3,6-pyrenequinone is reduced most rapidly by the Clostridium enzyme . In the presence of O2, redox cycling occurs with all of the quinones that are enzyme substrates, but the rate of cycling does not necessarily correlate with that of quinone reduction . Since glutathionyl adducts of certain quinones can undergo redox cycling mediated by the human carbonyl reductase or rat DT-diaphorase, it is unlikely that the conjugation of one of these quinones with glutathione is sufficient to protect against quinone-mediated oxidative stress in cells which contain either of these enzymes . The observation that superoxide dismutase and a dismutase "mimic," 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl, inhibit the redox cycling of 9,10-phenanthrenequinone suggests a mechanism whereby cells could be protected against oxidative stress caused by certain quinones.

Res Microbiol, 1993 Jun, 144(5), 405 - 10
Identification of a Clostridium cocleatum strain involved in an anti-Clostridium difficile barrier effect and determination of its mucin-degrading enzymes; Boureau H et al.; We isolated Gram-positive circular bacterium HB1 from intestinal microflora showing resistance to colonization by Clostridium difficile in mice (Su et . al., 1986a,b) . We studied its enzymatic capacity to degrade mucin the first potential barrier to implantation of strains in the intestine . Its biochemical characteristics, terminal metabolites and the electrophoretic profiles of proteins and DNA-DNA homology indicated that it was a strain of Clostridium cocleatum . This strain displayed numerous glucosidase activities which were assumed to play a role in the degradation of mucin oligosaccharide chains in the digestive tract . These enzymes included alpha- and beta-galactosidases, beta-glucosidase, beta-N-acetylglucosaminidase, sialidase and alpha-N-acetylgalactosaminidase.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1123 - 30
Isolation of a cellobiohydrolase of Clostridium thermocellum capable of degrading natural crystalline substrates; Singh RN et al.; A cellobiohydrolase (CBH3) of Clostridium thermocellum was isolated from the recombinant strains of Escherichia coli . The enzyme was shown to have a Mr of 78 kDa and isoelectric point of 4.75 . The hydrophilic nature of the enzyme was confirmed by its unretarded elution on gel filtration column . The enzyme had a binding affinity for the microcrystalline substrate (Avicel) . The pH and temperature optimum were determined as 6.5 and 60 degrees C . The enzyme was found to be active on p-nitrophenyl cellobioside, carboxymethyl cellulose, Avicel, amorphous cellulose, lichenan and xylan . The most distinguishing feature of CBH3 was the degradation of a variety of natural crystalline substrates, with maximum activity on filter paper . The enzyme was tolerant to high levels of cellobiose, susceptible to heavy metals and was protected by DTT and calcium at higher temperatures . This is the first report of a highly active cellobiohydrolase produced in C . thermocellum.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1115 - 22
Cloning and expression in Escherichia coli of the gene encoding the {2Fe-2S} ferredoxin from Clostridium pasteurianum; Fujinaga J et al.; The gene encoding the {2Fe-2S} ferredoxin from Clostridium pasteurianum has been amplified from genomic DNA by the polymerase chain reaction and cloned under the control of a promoter specifically recognized by T7 RNA polymerase . The protein has been overproduced in E . coli and found to be identical to its native counterpart purified from C . pasteurianum, including the molecular weight, the N-terminal sequence and the spectroscopic properties of the {2Fe-2S} chromophore.

Biochemistry, 1993 May 11, 32(18), 4813 - 9
Resonance Raman studies of iron-only hydrogenases; Fu W et al.; The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy . The results indicate the presence of ferredoxin-like {4Fe-4S}2+,+ and {2Fe-2S}2+,+ clusters in both T . maritima hydrogenase and C . pasteurianum hydrogenase I, but only {4Fe-4S}2+,+ clusters in D . vulgaris hydrogenase . This necessitates a reevaluation of the iron-sulfur cluster composition of C . pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center {Macor, K . A., Czernuszewicz, R . S., Adams, M . W . W., & Spiro, T . G . (1987) J . Biol . Chem . 262, 9945-9947} arise from an indigenous {2Fe-2S}2+ cluster . No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed . This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.

Fortschr Med, 1993 May 10, 111(13), 219 - 23
{Toxic megacolon in pseudomembranous colitis . Complicated course of antibiotic-induced Clostridium difficile colitis}; Rexroth G; We report on a patient with antibiotic-induced pseudomembranous colitis aggravated by toxic megacolon . Colonoscopy not only rapidly permits the diagnosis to be established, but the relief of pressure achieved simultaneously also has a therapeutic effect . If treatment comprising parenteral fluid and electrolyte replacement in combination with oral vancomycin fails to effect an improvement, surgery becomes necessary . Current recommendations for treatment are discussed on the basis of a review of the literature . However, a major preventive measure remains the rational use of antibiotics.

Appl Environ Microbiol, 1993 May, 59(5), 1398 - 402
Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1; Freer SN; The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q) . The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme was rich in acidic and hydrophobic amino acids . The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase . alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0 . The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C . When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1 . The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose . The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.

Antimicrob Agents Chemother, 1993 May, 37(5), 957 - 61
In vitro activity of FK037, a new parenteral cephalosporin, against anaerobic bacteria; Kato N et al.; The activity of FK037, a new parenteral cephalosporin, was compared with those of cefpirome, ceftazidime, and flomoxef against 322 recent clinical isolates of anaerobic bacteria . A fastidious facultative anaerobe, Gardnerella vaginalis, was also studied . FK037 inhibited 90% of isolates of Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus, Clostridium perfringens, Mobiluncus spp., G . vaginalis, and Porphyromonas gingivalis at < or = 0.78 micrograms/ml . The MICs of FK037 for 50 and 90% of Bacteroides fragilis isolates were 25 and > 200 micrograms/ml, respectively; the activity of FK037 was comparable to those of cefpirome and ceftazidime but lower than that of flomoxef . The activity of FK037 against Fusobacterium nucleatum, Fusobacterium varium, and Bilophila wadsworthia decreased when inoculum size was increased from 10(6) to 10(8) CFU/ml . Little influence of inoculum size on the activity of FK037 was observed for other isolates tested . Medium pH affected the activity of FK037 against F . varium (MICs at pHs 5 and 7, 3.13 and 100 micrograms/ml, respectively) and Bacteroides gracilis (MICs at pHs 5 and 7, 12.5 and 1.56 micrograms/ml, respectively) but not against other organisms tested . FK037 was less resistant than flomoxef to hydrolysis by beta-lactamase group 2e derived from B . fragilis GAI 0558 and GAI 10150.

J Infect, 1993 May, 26(3), 309 - 13
Clostridial endocarditis: report of a case caused by Clostridium septicum and review of the literature; Ridgway EJ et al.; We describe a case of fatal infective endocarditis due to Clostridium septicum in a patient with underlying colonic carcinoma . This is believed to be the first reported case of C . septicum endocarditis . The literature on the subject is reviewed.

Diagn Microbiol Infect Dis, 1993 May-Jun, 16(4), 303 - 11
Medical technologists using molecular epidemiology as part of the infection control team; Peterson LR et al.; Two medical technologists were appointed as permanent members of a new epidemiology section in the diagnostic microbiology laboratory of a large Veterans Administration Medical Center in the fall of 1989 . These positions accounted for 9% of the total microbiology staff and were created on a temporary basis 2 years earlier from a need to have dedicated technical expertise for use in the culture, isolation, and typing of nosocomial organisms . The technologists have evaluated outbreaks due to Clostridium difficile, methicillin-susceptible Staphylococcus aureus, and Serratia marcescens, and have begun work on a methicillin-resistant Staphylococcus aureus (MRSA)-typing scheme . Their major responsibility has been the development and application of molecular biology techniques for the typing of nosocomial isolates, including restriction enzyme analysis of genomic DNA, plasmid profiling with and without restriction enzyme analysis, ribosomal RNA probing of restricted genomic DNA, and selected DNA sequencing of target organisms . Medical supervision rests jointly between the directors of the infection control program and the microbiology laboratory . During their tenure, infections due to C . difficile have dropped from 95 cases per year to 57 cases annually, treatment of MRSA colonization with systemic agents has been curtailed, and a case control investigation involving S . marcescens was avoided . The inclusion of medical technologists in the infection control practice of large medical care facilities, particularly with the availability of molecular epidemiologic techniques and the emergence of increasing numbers of multiply-drug-resistant pathogens, will become an essential component of these programs.

J Bacteriol, 1993 May, 175(10), 3031 - 42
Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins; Wang G et al.; The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium . Strains carrying mutations located downstream of nifB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations . DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB . The gene products of modB and modC are homologous to ChlJ and ChlD of Escherichia coli and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively . ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein . The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other . Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum . Compared with that for the parental nifHDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants . No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants . The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase . Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake.

Arch Pathol Lab Med, 1993 May, 117(5), 524 - 7
Neutropenic enterocolitis . Two unusual cases with review of the literature; Lev R et al.; Cases of neutropenic enterocolitis associated with Clostridium septicum infection have been reported with increasing frequency in the past decade . We report two such cases involving unusual hosts and briefly discuss possible pathogenetic mechanisms such as ischemia, mucosal damage related to chemotherapy and neutropenia, and immunosuppression . One case involved a young man with chronic Epstein-Barr infection who developed extensive gas gangrene of the right side of his trunk and thigh and who died within 12 hours of presentation to the emergency department . Diagnosis was only made at postmortem examination . The second, middle-aged patient was admitted with an acute abdomen shortly after he completed chemotherapy for pleural mesothelioma . A right hemicolectomy was performed, but the patient developed antibiotic-associated pseudomembranous colitis and died . These cases indicate that neutropenic enterocolitis may arise in a variety of underlying conditions and that prompt diagnosis and therapy will be required to salvage more patients with this disorder.

Arch Pathol Lab Med, 1993 May, 117(5), 507 - 10
The use of a commercially available enzyme immunoassay for the detection of Clostridium difficile toxin A; Gilligan PH et al.; A commercially available enzyme immunoassay (EIA) for the detection of Clostridium difficile toxin A (Premier Toxin A EIA, Meridian Diagnostics, Cincinnati, Ohio) was compared with tissue culture cytotoxicity assay, enterotoxigenic culture, and latex agglutination test for the laboratory diagnosis of C difficile-associated disease . When evaluated for detection of C difficile-associated disease using clinical specimens, EIA was the most sensitive (83.1%) and tissue culture cytotoxicity assay was the most specific test with EIA, tissue culture cytotoxicity assay and enterotoxigenic culture having similar correlation values (96.6, 96.1, 94.0%, respectively) . The latex agglutination test was not as accurate (89.7% correlation) as the other three tests due mainly to its poor sensitivity (47.9%) . The EIA is a rapid, easy-to-use alternative to tissue culture cytotoxicity assay for detection of C difficile-associated disease.

Urology, 1993 May, 41(5), 458 - 60
Clostridium perfringens emphysematous cystitis; Katz DS et al.; Emphysematous cystitis is a rare disease and is usually caused by aerobic bacteria, most commonly Escherichia coli . Only rarely have anaerobic bacteria been associated with this condition . We report a case of emphysematous cystitis due to Clostridium perfringens with bacteremia in an elderly diabetic woman.

J Med Microbiol, 1993 May, 38(5), 384 - 7
Detection of Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction; Boondeekhun HS et al.; A rapid assay was developed for detection of the Clostridium difficile enterotoxin gene in stool specimens by means of the polymerase chain reaction (PCR) . The PCR primers amplified a 63-bp repetitive sequence of the enterotoxin gene, thereby generating a distinctive ladder pattern of DNA bands following electrophoresis . Crude DNA extracts from stools containing C . difficile produced one (63-bp) or more bands of the characteristic ladder . Of 172 stool specimens from 58 patients, 37 gave positive results by culture (15 specimens) or cytotoxin assay (36 specimens) . When 36 available "positive" specimens were tested by the PCR assay, 34 (94%) gave positive results--24 by direct testing, and 10 after extraction of DNA by the Qiagen procedure . Insufficient material of the remaining two specimens was available for DNA extraction . Of 21 stools "negative" for C . difficile by culture or cytotoxin assay, one gave a positive result by PCR and seven produced atypical bands . The rapid PCR detection technique for C . difficile was more sensitive than standard culture, and of a sensitivity similar to cytotoxin testing . The method has the potential for adoption in routine laboratory practice.

Eur J Epidemiol, 1993 May, 9(3), 327 - 34
Improvement of Clostridium difficile isolation by heat-shock and typing of the isolated strains by SDS-PAGE; Lahn M et al.; Clostridium difficile plays an essential role in causing pseudomembranous colitis . We looked for the presence of these bacteria in the stools of 169 hospitalized patients and 38 nurses from wards with cases of diarrhea (207 subjects) . The study was divided into three parts . In the first part, we compared three methods for isolating Clostridium difficile from stool samples: pre-selection with heat-shock, direct plating on Cycloserine-Cefotaxime-Fructose Agar (CCFA) and culturing in a selective broth medium . Final identification of Clostridium difficile was achieved by gas-chromatography and ApiZym . From the 207 consecutively obtained stool specimens, Clostridium difficile was isolated in 108 (52%) when pre-treated by heat-shock compared to only 26 (13%) when plated on modified CCFA and 23 (11%) when cultured in selective broth medium . Pre-selection significantly increases the isolation rate for Clostridium difficile and should be used in further epidemiological research . In the second part of our study, a retrospective review of subjects' records showed that the heat-shock method detected Clostridium difficile in all age groups at a higher rate than the other methods . In the third part of our study, we typed the 157 isolates of Clostridium difficile strains by protein patterns using SDS-PAGE, and 16 distinct groups were identified . In 19 cases different Clostridium difficile strains were found in the same subject by SDS-PAGE . Finally, the isolated strains were compared with strains from Brussels and Freiburg . Matching patterns were noted in only three cases.

Plasmid, 1993 May, 29(3), 233 - 5
Clostridium perfringens-Escherichia coli shuttle vectors that carry single antibiotic resistance determinants; Bannam TL et al.; Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed . Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms . The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin . Each plasmid contained the pUC18-derived multiple cloning site and the lacZ' gene which enabled direct screening for recombinants in E . coli . These plasmids should prove invaluable for the genetic manipulation of C . perfringens.

Can J Microbiol, 1993 May, 39(5), 480 - 5
Reduction of ferric iron by Listeria monocytogenes and other species of Listeria; Deneer HG et al.; One mechanism by which Listeria monocytogenes is thought to obtain iron required for growth is through the extracellular reduction of a ferric iron source to the ferrous form . To better characterize this reductase activity we have developed a simple plate assay that allows detection of colonies of Listeria species able to reduce ferric iron . Cells are plated on an agar base medium containing a ferric iron source and ethylenediamine dihydroxyphenylacetic acid . Colonies are then overlain with soft agarose containing NADH, flavin mononucleotide, and Ferrozine, a chelator of ferrous iron . Colonies able to reduce the ferric iron source form a red-purple color as the ferrous iron is complexed with ferrozine . Using this qualitative assay we have shown that all species of Listeria are able to reduce ferric iron when presented as ferric ammonium citrate whereas most other species of Gram-positive and Gram-negative bacteria are not . Only Clostridium perfringens was able to reduce ferric iron to the same extent as Listeria . Listeria monocytogenes was further shown to be capable of reducing various ferric iron salts as well as iron bound to ferritin, transferrin, and 2,3-dihydroxybenzoic acid in the agar plate assay . The utility of this assay was demonstrated by using it to screen a bank of Tn916-derived mutants of L . monocytogenes for clones unable to reduce ferric iron . Four such mutants were identified and all were shown to have greatly decreased ferric reductase activity.

J Cell Biochem, 1993 May, 52(1), 116 - 24
Activation of cellular phospholipase A2 by Clostridium difficile toxin B; Shoshan MC et al.; C . difficile toxin B is a potent cytotoxin known to disrupt the microfilaments of cultured cells . We have recently shown also increased phospholipase A2 activity in cells treated with toxin B . The activity was detected as a toxin-induced, dose-dependent release of 14C-arachidonic acid from prelabeled fibroblasts . Here is shown that the toxin elicited a 14C-arachidonic acid release in a cell mutant resistant to the toxin B effect on the microfilaments . The toxin-induced release was further characterized using fibroblasts . Within 20 min high doses of toxin B (6 micrograms/ml) elicited a release which increased exponentially with time . Of the major membrane phospholipids the lipase activity affected mainly phosphatidyl ethanolamine . Neither cycloheximide nor pertussis toxin treatment or target cells inhibited the toxin-induced release, while it could be increased with 12-O-tetradecanoylphorbol-13-acetate . Our results also suggest a toxin-mediated increase in phospholipase C activity occurring at a later stage than the phospholipase A2 activation . We conclude that the ability of toxin B to induce phospholipase activation represents a hitherto unrecognized toxin B effect which is neither a cause nor a consequence of toxin-induced microfilament disorganization.

J Cell Biochem, 1993 May, 52(1), 107 - 15
Signal transduction pathways and cellular intoxication with Clostridium difficile toxins; Shoshan MC et al.; In cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar . The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown . As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed . Both toxin-induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4-bromophenacylbromide . Agents affecting certain G-proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin . Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C-dependent pathway, but not pertussis toxin-sensitive G-proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity . In addition, both toxins were shown to activate phospholipase A2.

J Pediatr Gastroenterol Nutr, 1993 May, 16(4), 419 - 25
Saccharomyces boulardii for Clostridium difficile-associated enteropathies in infants; Buts JP et al.; Based on experimental evidence in animals showing that the oral administration of Saccharomyces boulardii is effective in reducing morbidity and mortality due to Clostridium difficile-induced pseudomembranous colitis, we conducted an open trial to examine the effects of the living yeast, given as primary therapy, in a selected group of infants and children with persistent intestinal symptoms related to toxinogenic C . difficile overgrowth . Over a period of 10 consecutive months, we studied 19 eligible patients (median age 8 months) who presented with enteral symptoms lasting for > 15 days and who had solely C . difficile in stools with positive cytotoxin B assay . Serotyping of the strains and determination in vitro of production of toxins A and B were performed subsequently . The patients presented with persistent or protracted diarrhea, malabsorption, and failure to grow (n = 8), or with repeated attacks of colics, emesis, and hypermeteorism without diarrhea (n = 4), or with both entities (n = 7) . Patients with chronic protracted diarrhea (n = 3) had depressed jejunal disaccharidase activities and ultrastructural changes of enterocytes, including sparce and shortened microvilli . None had evidence of colitis . All the strains of C . difficile tested (n = 17) belonged to pathogenic serotypes (A1, A8, C, F, G, H, and K) and produced in vitro high levels of toxins A (n = 16) and B (n = 17) . S . boulardii was given orally in a lyophilized form over 15 days (250 mg two to four times per day according to age).(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1993 May 1, 213(3), 935 - 45
The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination; Basso LA et al.; The binding of NAD+ and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy . The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40% . This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail . The ternary complex can be formed via the GDH-NAD+ or the GDH-L-Glu binary complexes, but the route via the GDH-NAD+ binary complex is the preferred pathway . The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined . These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme . The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ.mol-1 . We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme . Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ.mol-1.

J Bacteriol, 1993 May, 175(10), 2961 - 9
Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences; Tiboni O et al.; The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe . An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp . The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family . The P . woesei GSI was aligned with available homologs from other archaea (S . solfataricus, M . voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria . Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments . In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny . On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles . One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation) . The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P . woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S . coelicolor . The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism . However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.

Enzyme Microb Technol, 1993 May, 15(5), 393 - 400
Purification and properties of the Clostridium thermocellum bglB gene product expressed in Escherichia coli; Romaniec MP et al.; The Clostridium thermocellum beta-glucosidase B was purified to homogeneity in its recombinant form from Escherichia coli . The purification protocol included ion exchange, hydrophobic interaction and hydroxyapatite chromatography . The polypeptide was found to have a molecular mass of 84,000 daltons and a pI of 4.4 . There was a differential effect of temperature on the aryl-beta-glucosidase and cellobiase activities of the purified protein . The cellobiase activity had an optimum of 45 degrees C, and aryl-beta-glucosidase 60 degrees C . Both activities had an optimum pH of 5.6, although the aryl-beta-glucosidase had a secondary peak at 7.0 . Both activities were stimulated by divalent cations and DTT, but inhibited by thiol reagents . The enzyme was found to have a broad substrate specificity . Using cellobiose as substrate and a temperature of 45 degrees C, the Km and Vmax values were 1.6 mM and 5.5 U mg-1 respectively . The aryl-beta-glucosidase when assayed against pNP glucopyranoside and a temperature of 60 degrees C had Km and Vmax of 2.9 mM and 1.1 U mg-1 respectively . The enzyme was very stable at 45 degrees C, but rapidly inactivated at 60 degrees C.

Biochemistry, 1993 Apr 27, 32(16), 4420 - 9
Properties of a high-potential flavin analogue and its use as an active site probe with clostridial flavodoxin; Raibekas AA et al.; The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin . This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5) . 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption . This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring . It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position . The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5) . Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins . The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins . Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP . 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding . 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment . As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN . This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin . This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring . 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1993 Apr 25, 268(12), 8529 - 35
Identification of potential active-site residues in the human poly(ADP-ribose) polymerase; Simonin F et al.; The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases . To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context . Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity . The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid . In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J . (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.

Neurosci Lett, 1993 Apr 16, 153(1), 61 - 4
Sprouting of mammalian motor nerve terminals induced by in vivo injection of botulinum type-D toxin and the functional recovery of paralysed neuromuscular junctions; Comella JX et al.; Paralysis of the mouse levator auris longus muscle by in vivo injection of Clostridium botulinum type-D neurotoxin (BoNT/D) triggered a marked outgrowth of the motor nerve from the original terminal arborization . The increase in total nerve terminal length was due to both increase in the number of terminal branches and in average branch length . Asynchronous quantal transmitter release in response to nerve impulses was a prominent feature in paralysed junctions that started 24 h after poisoning and lasted for about 15 days . The functional recovery of poisoned junctions occurred 25-30 days after poisoning and was characterized by the synchronous quantal transmitter release upon nerve stimulation that triggered synaptically evoked action potentials and muscle fibre contraction.

FEMS Microbiol Lett, 1993 Apr 15, 108(3), 319 - 23
Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824; Lee SY et al.; The copy number and stability of several plasmid vectors in Clostridium acetobutylicum ATCC 824 were determined . The protocols were modified from the traditional ones to overcome the problems associated with unusual behavior of C . acetobutylicum cells on solid medium . The plasmid copy numbers of pSYL2, pFNK1, pFNK3, and pFNK5 in strain ATCC 824 were 14, 8, 6, and 6, respectively . pSYL2 and pFNK1 were segregationally stable, since the fractions of plasmid-carrying cells after 60 generations of growth without antibiotic (erythromycin) were 73% and 77%, respectively . Vector pFNK1 carrying fermentative genes was found to be rather unstable . The observed instability seemed to be due to the complex host-plasmid interactions by amplified expression of enzymes involved in the tightly regulated primary metabolism of C . acetobutylicum.

Biochem J, 1993 Apr 15, 291 ( Pt 2), 409 - 12
ADP-ribosylation of Drosophila indirect-flight-muscle actin and arthrin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin; Just I et al.; Purified Drosophila indirect-flight-muscle actin and arthrin, an actin-ubiquitin conjugate, were ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin . Phalloidin treatment inhibited the ADP-ribosylation of Drosophila actin and arthrin . Like actin, the ADP-ribose-arthrin linkage was sensitive towards hydroxylamine treatment, indicating arginine as the amino acid acceptor . Actin translated in vitro from the indirect-flight-muscle-specific gene Act88F was ADP-ribosylated by C . botulinum C2 toxin and C . perfringens iota toxin . Actin from the R177Q mutant of Act88F translated in vivo was not ADP-ribosylated confirming Arg-177 as the ADP-ribose acceptor . Mutant L176M actin was modified by both toxins, indicating that amino acid 176 of actin does not define the substrate specificity of C . botulinum C2 toxin . Whereas the gene products of various C-terminal mutants of Act88F translated in vitro (E334K, V339I, E364K, G368E, R372H) were substrates for ADP-ribosylation by C . botulinum C2 toxin and by C . perfringens iota toxin, neither toxin modified the N-terminal O-12 deletion mutant.

Biochem Pharmacol, 1993 Apr 6, 45(7), 1409 - 16
Enhancement of Clostridium botulinum C3-catalysed ADP-ribosylation of recombinant rhoA by sodium dodecyl sulfate; Just I et al.; The influence of sodium dodecyl sulfate (SDS) on ADP-ribosylation by Clostridium botulinum C3 exoenzyme (C3) was studied . SDS increased the ADP-ribosylation of recombinant rhoA and human platelet cytosolic proteins maximally at 0.01% whereas higher concentrations of the detergent (> 0.01%) inhibited the ADP-ribosylation . In contrast, ADP-ribosylation of human platelet membranes and of recombinant rhoB was inhibited by the detergent . The Km for NAD of the ADP-ribosylation of rhoA was decreased by SDS from about 10 to 0.6 microM . Whereas in the absence of SDS, the C3-induced ADP-ribosylation of recombinant rhoA is not affected by the amphiphilic wasp venom mastoparan, in the presence of SDS (0.01%) mastoparan (100 microM) inhibited the ADP-ribosylation . C3-associated NAD-glycohydrolase activity was maximally and half-maximally inhibited by 0.1 and 0.013% SDS, respectively . Inhibition of NAD-glycohydrolase activity was reversed by diluting out SDS indicating that C3 was not irreversibly denatured by SDS treatment . SDS (0.01%) completely inhibited the {3H}GTP binding of rhoA whereas the release of previously bound nucleotide was not affected . The data indicate that changes in the lipophilicity of rhoA protein largely affect its ability to serve as a substrate for C3-like ADP-ribosyltransferases.

Protein Sci, 1993 Apr, 2(4), 640 - 9
Modeling the structure of Pyrococcus furiosus rubredoxin by homology to other X-ray structures; Wampler JE et al.; The three-dimensional structure of rubredoxin from the hyperthermophilic archaebacterium, Pyrococcus furiosus, has been modeled from the X-ray crystal structures of three homologous proteins from Clostridium pasteurianum, Desulfovibrio gigas, and Desulfovibrio vulgaris . All three homology models are similar . When comparing the positions of all heavy atoms and essential hydrogen atoms to the recently solved crystal structure (Day, M . W., et al., 1992, Protein Sci . 1, 1494-1507) of the same protein, the homology model differ from the X-ray structure by 2.09 A root mean square (RMS) . The X-ray and the zinc-substituted NMR structures (Blake, P . R., et al., 1992b, Protein Sci . 1, 1508-1521) show a similar level of difference (2.05 A RMS) . On average, the homology models are closer to the X-ray structure than to the NMR structures (2.09 vs . 2.42 A RMS).

New Microbiol, 1993 Apr, 16(2), 171 - 5
Antimicrobial compounds from Lactobacillus casei and Lactobacillus helveticus; Vescovo M et al.; Three strains of Lactobacillus casei and one of Lactobacillus helveticus were examined for antagonistic activity toward twenty five indicator strains of different species . Under conditions eliminating the effects of organic acid and hydrogen peroxide, culture supernatants of all the Lactobacillus strains exhibited a wide spectrum of inhibitory activity toward microorganisms of different genera . The inhibitory compound secreted by one strain of L . casei was active against Clostridium tyrobutyricum . The active components were insensitive to proteolytic enzyme and heat treatment.

J Clin Gastroenterol, 1993 Apr, 16(3), 192 - 4
Clostridium difficile diarrhea in patients with AIDS versus non-AIDS controls . Methods of treatment and clinical response to treatment; Cozart JC et al.; We reviewed the hospital charts of 17 patients with AIDS and Clostridium difficile diarrhea to determine antibiotic use before C . difficile infection, methods of treatment for C . difficile diarrhea, and response of diarrhea to treatment . Left shift and total white blood cell count before and after treatment for C . difficile were also determined . Non-HIV-infected patients with C . difficile diarrhea served as controls . In the patients with AIDS, resolution of diarrhea was noted in 15 (88%) patients . In 25 (76%) control patients, diarrhea resolved with treatment . The patients with AIDS also had a significant decrease (p < 0.05) in left shift in white blood cell count with treatment; the controls did not . Our study therefore suggests that C . difficile diarrhea is at least as likely to resolve with antibiotic therapy in patients with AIDS as it is in those with the non-AIDS-related disorder . We also found that patients with AIDS and C . difficile diarrhea are more likely than patients without AIDS to have a decreased left shift in white blood cell count after antibiotic therapy.

Scand J Gastroenterol, 1993 Apr, 28(4), 323 - 30
Tumor necrosis factor-alpha potentiates phospholipase A2-stimulated release and metabolism of arachidonic acid in cultured intestinal epithelial cells (INT 407); Gustafson-Svard C et al.; Tumor necrosis factor-alpha (TNF-alpha), a known pro-inflammatory cytokine, has been suggested to play a role in the pathogenesis of inflammatory bowel disease (IBD) by mediating damage to the intestinal epithelial cells . The present study demonstrates that TNF-alpha potentiates release and metabolism of 14C-labeled arachidonic acid (14C-AA) in cultured intestinal epithelial cells (INT 407) . Although TNF-alpha on its own was but a weak stimulator of cellular 14C-AA turnover, it significantly potentiated the release of 14C-AA and 14C-labeled prostaglandin E2(14C-PGE2) after stimulation with three known phospholipase A2 activators: phospholipase . C from Clostridium perfringens, the calcium ionophore A23187, and the phorbol ester 4-beta-phorbol-12-myristate-13-acetate (PMA) . The phospholipase A2 inhibitor quinacrine significantly reduced both AA and PGE2 release after combined stimulation with phospholipase C and TNF-alpha . In contrast to its effect on the AA turnover, TNF-alpha did not affect the phospholipase C-stimulated production of platelet-activating factor (PAF-acether) . Taken together, these findings indicate that a) TNF-alpha potentiates phospholipase A2-stimulated AA release from cultured intestinal epithelial cells; b) TNF-alpha may stimulate phospholipase A2-dependent AA release without affecting the formation of PAF-acether and c) pretreatment with TNF-alpha potentiates the formation of PGE2 after stimulation with phospholipase A2 activators . In summary, the present investigation points to the possibility that TNF-alpha may stimulate intestinal epithelial cells to produce biologically active AA metabolites and that this stimulation may be modulated by components of the intestinal luminal content, like bacterial toxins.

J Dairy Sci, 1993 Apr, 76(4), 972 - 7
Growth of Listeria monocytogenes and Clostridium sporogenes in cottage cheese in modified atmosphere packaging; Chen JH et al.; Low fat cottage cheese (pH 5.14) was inoculated with three strains of Listeria monocytogenes, serotypes 1a and 4b, an isolate from a dairy processing plant, and Clostridium sporogenes ATCC 3584 . The cheese was packaged with or without added dissolved CO2 in polystyrene tubs overwrapped with or without high barrier heat shrink film and stored at 4, 7, and 21 degrees C for up to 63 d . The concentration of CO2 in the container headspace was 35% (vol/vol) . The CO2 concentration in that headspace declined by one-third over the 63 d of storage at 4 degrees C . Clostridium sporogenes failed to grow under any condition applied in this study . In the conventionally packaged cottage cheese, L . monocytogenes increased from 10(4) to 10(7) cfu/g after lag phases of 28 and 7 d at 4 and 7 degrees C, respectively . In contrast, L . monocytogenes failed to grow in cottage cheese packaged with CO2 and stored at 4 degrees C up to 63 d and increased from 10(4) to 10(5) cfu/g in products packaged with CO2 at 7 degrees C . These data suggest that the addition of CO2 to cottage cheese to extend shelf-life does not represent an increased Listeria or botulism hazard but that cottage cheese could be a vehicle for listeriosis.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 175 - 82
Nucleotide sequence of the gene coding for Clostridium barati type F neurotoxin: comparison with other clostridial neurotoxins; Thompson DE et al.; The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published . The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141,049 Da . The sequence identity between the C . barati ATCC43756 and non-proteolytic C . botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain . This is much lower than reported identities for the type E neurotoxins from C . botulinum and C . butyricum (96% identity between light chains and 98.8% between the heavy chains) . Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C . barati . An ORF upstream of the toxin coding region was revealed . This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C . botulinum type C neurotoxin.

Unfallchirurg, 1993 Apr, 96(4), 181 - 91
{Necrotizing soft tissue infections}; Kach K et al.; Necrotizing soft tissue infections are a group of life- and limb-threatening infections . They are caused by aerobic and anaerobic bacteria occasionally in a synergistic polymicrobial combination . The literature describing necrotizing soft tissue infections is controversial and often contradictory . Depending on their clinical appearance, tissue level and microbiological findings, necrotizing soft tissue infections are classified into two major groups, infections within the subcutaneous/fascia niveau and within the muscle level . Necrotizing infections of the subcutaneous level are further differentiated in hemolytic streptococcal gangrene, necrotizing fasciitis, clostridium fasciitis, and anaerobic nonclostridium fasciitis . In particular, necrotizing fasciitis is a rapidly progressing process, which is characterized by suppurative fasciitis, following by vascular thrombosis and cutaneous gangrene and is often accompanied by severe systemic toxicity, seen as septic-toxic shock and progressive (multi-) organ failure . Nineteen cases of necrotizing soft tissue infections were treated at the Department of Surgery, University Hospital of Zurich, between 1989 and 1992 . These infections originated from "neglected trauma" in 9 (9/19), drug injections in 4 (4/19), acute infections in 3 (3/19), operative wounds in 2 (2/19) and perforation of the intestine in 1 case (1/19) . Most of the patients (13/19) suffered from chronic debilitating diseases and were compromised by a suppressed immune system . We treated two groups of patients, one with septic-toxic clinical course and the other without . Eleven patients (11/19) belonged to group one and four of them, showing necrotizing fasciitis of the trunk, died as a result of multiorgan failure (MOF) . Furthermore, three patients in this group had a limb amputated . In the other group without septic-toxic signs, no one died or lost a limb . The two groups differed also in length of hospital stay, an average of 60 days in group one (23 days intensive care) and 25 days in group two . Our results suggest that prompt recognition and treatment of necrotizing soft tissue infections are essential for the patient's survival . Often the full extent of the infections is underestimated initially, resulting in delayed surgical therapy . To control the rapidly spreading necrosis, early diagnosis and radical debridement of the affected tissue are essential and should be done without compromise, even if the affected limb must be amputated.

Mol Gen Genet, 1993 Apr, 238(1-2), 145 - 54
Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted endo-beta-1,3-1,4-glucanases and ExoH resembles membrane proteins; Becker A et al.; Nucleotide sequencing of a 4.15 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 revealed the location of the genes exoH, exoK and exoL . The putative proteins encoded by these genes have molecular weights of 41, 30, and 44 kDa, respectively . The hydrophobicity profile of the ExoH amino acid sequence resembles that of transmembrane proteins . The predicted exoL gene product does not contain hydrophobic regions, indicating a cytoplasmic localization . The exoK gene product is characterized by a putative signal peptide and exhibits significant homology to endo-beta-1,3-1,4-glucanases of bacilli and Clostridium thermocellum . R . meliloti exoK mutants induced pink nodules and synthesized a reduced amount of exopolysaccharide (EPS) . Colonies of this mutant showed a delay in the appearance of the Calcofluor white fluorescence . In addition, the formation of the characteristic halo was strongly delayed . R . meliloti exoL and exoH mutants induced pseudonodules . The exoH, but not the exoL mutant, synthesized an EPS that could be precipitated by cetyl pyridinium chloride (CPC) and also by ethanol . Plasmid integration mutagenesis revealed promoter regions preceding exoH, exoK and exoL.

Cornell Vet, 1993 Apr, 83(2), 143 - 51
Clostridium septicum septicemia in a neonatal foal with hemorrhagic enteritis; Jones SL et al.; Clostridium septicum was isolated by anaerobic culture of blood collected from a 3-day-old foal with hemorrhagic enteritis and signs suggestive of septicemia . The foal responded well to treatment with intravenous fluids, antibiotics, plasma, and oral gastrointestinal protectants . One month after apparent complete recovery from the septicemia and hemorrhagic enteritis, the foal was euthanized during an acute episode of colic that was caused by severe, strangulating intestinal adhesions, thought to have formed as a result of peritonitis secondary to the hemorrhagic enteritis . The value of anaerobic culture of blood in foals with signs suggestive of septicemia is emphasized by the case presented here, as is the importance of evaluating the presence and extent of peritoneal inflammation in foals with hemorrhagic enteritis . To our knowledge, Clostridium septicum has not previously been reported to cause septicemia in neonatal foals.

Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2636 - 40
Function of yeast cytoplasmic C1-tetrahydrofolate synthase; Song JM et al.; The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cytoplasmic trifunctional C1-tetrahydrofolate (THF) synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) activities . However, it has been suggested that the ADE3-encoded C1-THF synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosynthesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis {Barlowe, C . K . & Appling, D . A . (1990) Mol . Cell . Biol . 10, 5679-5687} . This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF synthase, whether catalytically active or not, is correlated with the Ade+ phenotype . In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF synthase is enough to complement the adenine requirement in ade3 strains . These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains . Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine . These results suggest that the catalytic activity of the C1-THF synthase is involved in purine biosynthesis.

J Clin Microbiol, 1993 Apr, 31(4), 963 - 7
Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea; Barbut F et al.; Enzyme immunoassays (EIAs) based on monoclonal antibodies for the detection of Clostridium difficile toxins have recently been developed for clinical use . The aim of this study was to compare three commercially available EIAs, two for toxin A (Premier C . difficile Toxin A; Meridian, Osi, Elancourt, France; and Vidas C . difficile Toxin A; bioMerieux, Marcy l'Etoile, France) and one for toxins A and B (Cytoclone A + B EIA; Cambridge Biotech Corp., Codiapharm, Evian, France), with a cytotoxicity assay and toxigenic culture for the diagnosis of C . difficile-associated diarrhea (CDAD) . The study was performed with 285 fresh stools from 285 patients with suspected CDAD . In case of disagreement, the tests were repeated on a frozen aliquot of the same stool sample, and the patient's chart was reviewed . CDAD diagnosis was established in 55 cases (incidence, 19.3%) . The sensitivities and specificities of the methods were, respectively, 92.7 and 100% for the cytotoxicity assay, 96.4 and 99.1% for toxigenic culture, 75.5 and 97.8% for Cytoclone, 65.4 and 99.6% for Premier, and 65.4 and 100% for Vidas . The results were uninterpretable in 3.2% of cases with Cytoclone, 0.3% with Premier, and 2.5% with Vidas . We conclude that the cytotoxicity assay and toxigenic culture remain the best methods for the diagnosis of CDAD even though they lack standardization and require 48 to 96 h to obtain the result . Despite their rapidity and simplicity, EIAs are not sensitive enough to be relied on as the sole laboratory test.

Gastroenterology, 1993 Apr, 104(4), 1108 - 15
Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum; Pothoulakis C et al.; BACKGROUND: Saccharomyces boulardii is a nonpathogenic yeast used for the prevention and treatment of Clostridium difficile-associated diarrhea and colitis . However, the mechanism by which S . boulardii exerts its protective effects remains unclear . METHODS: The binding of {3H}toxin A to its brush border receptor preincubated with S . boulardii-cultured suspension or filtered conditioned medium was measured in vitro . The effect of toxin A on secretion, epithelial permeability, and morphology in rat ileal loops in vivo was also examined in rats pretreated with S . boulardii . RESULTS: S . boulardii reduced {3H}toxin A-receptor binding in a dose-dependent fashion . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of ileal brush border exposed to S . boulardii-conditioned medium revealed a diminution of all brush border proteins . Treatment of rats with S . boulardii suspension reduced fluid secretion and mannitol permeability caused by toxin A . CONCLUSIONS: S . boulardii may reduce some of the enterotoxic effects of toxin A by inhibiting toxin A-receptor binding . This effect appears to be manifested by a secreted product of the yeast, possibly a protease.

J Bacteriol, 1993 Apr, 175(7), 1891 - 9
Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface; Fujino T et al.; The nucleotide sequence was determined for a 9.4-kb region of Clostridium thermocellum DNA extending from the 3' end of the gene (now termed cipA), encoding the S1/SL component of the cellulosome . Three open reading frames (ORFs) belonging to two operons were detected . They encoded polypeptides of 1,664, 688, and 447 residues, termed ORF1p, ORF2p, and ORF3p, respectively . The COOH-terminal regions of the three polypeptides were highly similar and contained three reiterated segments of 60 to 70 residues each . Similar segments have been found at the NH2 terminus of the S-layer proteins of Bacillus brevis and Acetogenium kivui, suggesting that ORF1p, ORF2p, and ORF3p might also be located on the cell surface . Otherwise, the sequence of ORF1p and ORF2p gave little clue concerning their potential function . However, the NH2-terminal region of ORF3p was similar to the reiterated domains previously identified in CipA as receptors involved in binding the duplicated segment of 22 amino acids present in catalytic subunits of the cellulosome . Indeed, it was found previously that ORF3p binds 125I-labeled endoglucanase CelD containing the duplicated segment (T . Fujino, P . Beguin, and J.-P . Aubert, FEMS Microbiol . Lett . 94:165-170, 1992) . These findings suggest that ORF3p might serve as an anchoring factor for the cellulosome on the cell surface by binding the duplicated segment that is present at the COOH end of CipA.

CMAJ, 1993 Apr 1, 148(7), 1161 - 9
Comparison of cefoxitin and ceftizoxime in a hospital therapeutic interchange program; Martinusen S et al.; OBJECTIVE: To determine whether (a) ceftizoxime can replace cefoxitin in the prevention and treatment of various infections in a major teaching hospital, (b) a previously applied two-stage intervention program is an effective method of instituting a therapeutic interchange of ceftizoxime for cefoxitin and (c) the replacement of cefoxitin with ceftizoxime results in a more cost-effective therapy . DESIGN: Two-phase, open, sequential study . SETTING: Tertiary care teaching hospital . PATIENTS: One hundred patients who received cefoxitin during the 6 months immediately before the start of the interchange program (phase 1) and 100 who received ceftizoxime during the 6 months immediately after the start of the program (phase 2) were randomly selected . RESULTS: The demographic characteristics of the two patient groups were similar except for sex (p < 0.05) . The cefoxitin doses were usually given every 6 hours (in 33% of the cases) or every 8 hours (in 61%), whereas the ceftizoxime doses were usually given every 12 hours (in 98%) . Prescriber distribution was stable throughout the study period, the Department of General Surgery being responsible for about 70% of the orders . Prophylactic indications accounted for over 60% of the treatment courses . The proportion of prophylactic treatment courses that resulted in a successful clinical outcome did not differ between the two groups (cefoxitin 92% and ceftizoxime 91%) . Of the empiric or directed treatment courses clinical success or improvement was observed in 89% of the cefoxitin and 91% of the ceftizoxime recipients . Microbiologic eradication was seen in 65% of the cefoxitin and 90% of the ceftizoxime directed treatment courses . Pathogens isolated during therapy were similar in the two treatment groups . Diarrhea was the most common adverse effect, occurring in 8% of the cefoxitin and 10% of the ceftizoxime recipients; no Clostridium difficile or C.-difficile-producing toxin was identified in these patients . The ceftizoxime therapy was 36% less expensive than the cefoxitin therapy on average, and the annual savings was estimated to be $83,123 . An estimated 5615 drug doses were avoided annually, for an additional savings of $24,875 in drug administration . Therefore, the total estimated annual cost savings resulting from this two-stage interchange program was $107,998 . Given the cost of $4856 to implement and maintain the program, the estimated net savings for the first year was $103,142 . CONCLUSION: Ceftizoxime can replace cefoxitin in the prevention and treatment of various infections . The form of evaluation described herein is valuable when any formulary modification is being considered in a hospital.

Antimicrob Agents Chemother, 1993 Apr, 37(4), 868 - 74
In vitro and in vivo antibacterial activities of E-4868, a new fluoroquinolone with a 7-azetidin ring substituent; Guinea J et al.; E-4868, (-)-7-{3-(R)-amino-2-(S)-methyl-1-azetidinyl}-1-(2,4- difluorophenyl)-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid, is a new fluoroquinolone with an azetidine moiety at the 7 position . The in vitro activity of E-4868 has been compared with those of ciprofloxacin, ofloxacin, and fleroxacin, while the activity of ciprofloxacin was used as reference for in vivo studies . The MICs of E-4868 for 90% of the isolates tested (MIC90s) were 0.06 to 0.5 microgram/ml against gram-positive organisms, including Staphylococcus, Streptococcus, and Enterococcus spp . In general, the in vitro potency of E-4868 against gram-positive bacteria was higher than those of all of the other fluoroquinolones tested . MIC90s against members of the family Enterobacteriaceae between 0.03 and 1 microgram/ml were observed, with the exception of those against Serratia marcescens and Providencia spp., and a MIC90 of 2 micrograms/ml against Pseudomonas aeruginosa was obtained . E-4868 inhibited 90% of the Clostridium spp . and Bacteroides spp . at 2 micrograms/ml and was twofold more active than ciprofloxacin . An increase in the Mg2+ concentration from 1 to 10 mM increased the MIC between two and three times . Human urine caused a significant decrease in activity of E-4868, which was more pronounced at pH 5.5 than at pH 7.2 . The presence of serum also decreased the activity of E-4868 . Fifty percent effective dose (ED50) values against experimental Escherichia coli HM-42 infections in mice were 3.9 mg/kg of body weight with E-4868 and 3.5 mg/kg of body weight with ciprofloxacin . Corresponding ED50 values against P . aeruginosa HS-116 were 93.2 and 107.8 mg/kg, respectively, and those against Staphylococcus aureus HS-93 were 6.5 and 44.6 mg/kg, respectively . In experimental infections with Streptococcus pneumoniae 84551, the ED50 value of E-4868 was 154.4 mg/kg, while ciprofloxacin proved totally inactive at a dose of 400 mg/kg . When E-4868 was administered orally at a dose of 50 mg/kg in mice, the area under the concentration-time curve (0 to 4 h) value was 28.4 microgram . h/ml, while an area under the concentration-time curve value of 2.3 microgram . h/ml was observed for ciprofloxacin at the same dose . In these studies, levels of the two agents in blood 1 h postadministration were 7.6 and 1.2 microgram/ml, respectively.

Appl Environ Microbiol, 1993 Apr, 59(4), 1077 - 81
In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824; Mermelstein LD et al.; The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L . D . Mermelstein, N . E . Welker, G . N . Bennett, and E . T . Papoutsakis, Bio/Technology 10:190-195, 1992) . Methylation by the phi 3T I methyltransferase encoded by Bacillus subtilis phage phi 3T was shown to protect plasmid DNA from restriction by Cac824I . Expression in Escherichia coli of the phi 3tI gene (which encodes the phi 3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E . coli-C . acetobutylicum shuttle vectors with ColE1 origins of replication . Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824 . These vectors could not be introduced into strain ATCC 824 when unmethylated because the E . coli portions of the plasmids contain a large number of Cac824I sites . This method obviates the need to use B . subtilis-C . acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C . acetobutylicum ATCC 824.

Mol Microbiol, 1993 Apr, 8(2), 325 - 34
Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL-protein reveals an unusual degree of internal homology; Gerngross UT et al.; It is known that two proteins of the cellulosomal complex of Clostridium thermocellum (SL and SS) together degrade crystalline cellulose . SL is a glycoprotein of 210,000 Da which enhances the binding to cellulose and the activity of SS, an endoglucanase of 83,000 Da . We have previously reported the cloning of a DNA fragment encoding the N-terminal end of the SL protein using antibodies raised against the native protein . A chromosomal walking approach using an EcoRI and a Bam HI-Sau3A gene library allowed us to isolate the C-terminal end of the gene . Sequencing of both fragments revealed the existence of a leader peptide as has been found in cellulases of the same organism . This leader sequence is followed by a stretch of 14 amino acids that is identical to the N-terminal amino acid sequence of the native secreted protein . The open reading frame (ORF) of this gene encodes a protein of 196,800 Da and is followed by a hairpin loop that could be involved in transcription termination . Within the open reading frame (ORF), we found nine internal repeated elements (IREs) of about 500 nucleotides each . Seven of these sequences displayed 98-100% homology and were located adjacent to each other within the structural gene without intervening regions . The remaining two, located on the N-terminal end of the gene, showed a significantly lower homology . Bearing in mind the inherent instability of reiterated regions, we confirmed the authenticity of our clones by Southern blot analysis using chromosomal C . thermocellum DNA and ruled out the possibility of rearrangements during the cloning and sequencing process . The sequenced gene is designated cipA and the encoded SL protein CipA.

Pathol Biol (Paris), 1993 Apr, 41(4), 421 - 7
{Survey with three epidemiological markers after 22 cases of diarrhea caused by Clostridium difficile in a geriatric hospital}; Vautrin AC et al.; In a geriatric hospital of Saint-Etienne (Charite), among 153 patients having presented a nosocomial diarrhea from September 1990 to August 1991 Clostridium difficile (C.d.) has been isolated in 22 cases . Two of the nine units of the hospital had the highest incidence rates: 4.6 and 3.7% . In the faeces of 16 patients, C.d . was toxinogenic . In all cases, except one, antibiotic preceded diarrhea . Amoxicillin + clavulanic acid treatment was the most frequently responsible (65%) . For detecting an eventual outbreak, several epidemiologic markers were evaluated: Clindamycin MIC, protein profiles, serotyping . Clindamycin susceptibility differentiated two Cd . types, but has no epidemiologic value . Protein profiles, performed by SDS-Page, individualized 6 different profiles, but 10 strains gave no classifiable profiles . Serotyping, applied by M . Delmee, appeared as the most interesting marker . Inquiry allowed to eliminate an outbreak but revealed two episodes of cross contaminations in the 3 units, 2 of them having the highest incidence rates . Markers proved persistence of the same C.d . strain in some patients who were correctly treated by metronidazole.

Toxicon, 1993 Apr, 31(4), 427 - 35
Effect of drugs acting on the central nervous system on the lethality in mice of Clostridium perfringens epsilon toxin; Nagahama M et al.; Lethal activity of Clostridium perfringens epsilon toxin was significantly reduced by the prior administration of barbiturates . Phenothiazine derivatives such as chlorpromazine and trifluoperazine and butyrophenone derivatives such as haloperidol and spiperone delayed the lethal effects in mice . Reserpine completely protected mice against the toxin when 10 mg/kg of the drug was administered 24 hr before the injection of the toxin, but did not protect when the same dose of the drug was given within 60 min before the injection of the toxin . Diazepam, apomorphine and gamma-butyrolactone also resulted in a significant increase in the time to death after the toxin . On the other hand, atropine, diphenhydramine, chlorpheniramine and verapamil provided no protection against the toxin . The administration of the toxin resulted in a significant decrease of dopamine levels in the brain, but no effect on levels of epinephrine and norepinephrine . The data suggest that drugs which directly or indirectly inhibit release and receptors of dopamine may lessen the lethal effects of epsilon toxin in mice.

Kitasato Arch Exp Med, 1993 Apr, 65 Suppl, 117 - 26
Space microbiology--lethality, mutagenicity and cytological effects of terrestrial microorganisms by irradiation of cosmic proton under simulated space condition; Koike J et al.; We have been discussing in connection with a space quarantine . The subject is not merely an academic problem, but it contains a fundamental problem which avoid the contamination of other planets by terrestrial microflora . The space environments in the solar system were simulated by using an apparatus of cryostat (low temperature of 110-310K, high vacuum of 1 x 10(-8) torr) and proton irradiation from the Van de Graaff generator . After exposure to a barrage of protons corresponding to about 250 years in solar space, Tobacco mosaic virus, Bacillus subtilis spore, Staphylococcus aureus . Micrococcusflavus, Clostridium mangenoti spore and Aspergillus niger spore showed considerably high survival rates . Furthermore, it was found firstly that an irradiation of proton induced considerable mutation frequency compared to that of spontaneous and caused also the cytological effects based on a damage of chromosome.

Int J Syst Bacteriol, 1993 Apr, 43(2), 314 - 8
Assignment of the agent of Tyzzer's disease to Clostridium piliforme comb . nov . on the basis of 16S rRNA sequence analysis; Duncan AJ et al.; The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing . By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G . J . Olsen, R . Overbeek, N . Larsen, T . L . Marsh, M . J . McCaughey, M . A . Maciukenas, W.-M . Kuan, T . J . Macke, Y . Xing, and C . R . Woese, Nucleic Acids Res . 20:2199-2200, 1992) . Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp . The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii . Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble . We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.

Int J Syst Bacteriol, 1993 Apr, 43(2), 232 - 6
Clostridium ljungdahlii sp . nov., an acetogenic species in clostridial rRNA homology group I; Tanner RS et al.; Clostridium ljungdahlii sp . nov . strain ATCC 49587T (T = type strain) was isolated from chicken yard waste for its ability to produce ethanol from synthesis gas . This gram-positive, motile, sporeforming rod's metabolism was primarily acetogenic . C . ljungdahlii grew with carbon monoxide, hydrogen and carbon dioxide, ethanol, pyruvate, arabinose, xylose, fructose, or glucose . Methanol, ferulic acid, lactate, galactose, and mannose did not support growth . The G+C content was 22 to 23 mol% . C . ljungdahlii is the first acetogen in clostridial 23S rRNA homology group I.

FEBS Lett, 1993 Mar 15, 319(1-2), 84 - 9
Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum; Brecht M et al.; The nucleotide sequence of the large subunit E of glutamate mutase of Clostridium tetanomorphum was determined . The protein consists of 483 amino acids and is not made in a precursor form, thus excluding the possibility of subunit E being a pyruvoyl enzyme . It shows no homology to any other protein in the database, and while binding coenzyme B12, a conspicuous B12 binding motif, shared amongst other proteins, is not detectable at the sequence level.

FEBS Lett, 1993 Mar 15, 319(1-2), 125 - 7
The projection structure of perfringolysin O (Clostridium perfringens theta-toxin); Olofsson A et al.; The cytolysin Perfringolysin O was applied to lipid layers and the obtained ring-shaped oligomers analyzed by electron microscopy and image processing . The final result shows the periodic repeat of 2.4 nm along the outer rim of the ring . The asymmetric protein unit, corresponding to one monomer, spans the ring from the convex to the concave surface . It shows a clear protein peak close to the outer radius and less density in the middle of the oligomer . The number of monomers in the average ring is 50, and the inner radius of the aggregate is approximately 15 nm.

J Biol Chem, 1993 Mar 15, 268(8), 5605 - 14
Sequence and expression of the gene encoding the corrinoid/iron-sulfur protein from Clostridium thermoaceticum and reconstitution of the recombinant protein to full activity; Lu WP et al.; The corrinoid/iron-sulfur protein (C/Fe-SP) from Clostridium thermoaceticum acts as a methyl group carrier in the anaerobic acetyl-CoA pathway of CO and CO2 fixation . Consisting of a small (approximately 33 kDa) and a large (approximately 55 kDa) subunit, the C/Fe-SP contains 1 mol of cobalt in a corrinoid cofactor and 1 mol of {4Fe-4S}2+/1+ cluster/mol of alpha beta dimer . Cobalt is the site of methylation, and the {4Fe-4S} center appears to serve an electron transfer function . The genes encoding both subunits have been cloned previously and are located within a gene cluster that includes other genes required for CO2 fixation by anaerobic bacteria . When the genes encoding the C/Fe-SP were expressed in Escherichia coli, the protein was found to be inactive . We report the amino acid sequences of the large and small subunits of the C/Fe-SP based on the DNA sequences of the cloned genes . The {4Fe-4S} cluster was found to be located in the large subunit . Although the primary structural lattice for cobamide binding resides in the small subunit, both subunits are required for formation of a stable cobamide-binding protein . Based on sequence comparisons with other {4Fe-4S}-containing proteins, 3 of the 4 cysteine residues that serve as ligands to the iron sites in the cluster have been located . The two subunits were independently overexpressed in E . coli to a level of 30-50% of cell protein; however, the resulting protein was inactive, lacked stoichiometric amounts of Fe-S cluster, and lacked cobamide . By combining the recombinant subunits, unfolding them with urea, and refolding in the presence of cobamide, iron, and inorganic sulfide, the resulting C/Fe-SP was found to contain stoichiometric amounts of cobamide and {4Fe-4S} cluster and had spectroscopic and enzymatic properties similar to those of the native protein . We expect that the methods developed here may be used for heterologous overexpression and reconstitution of other complex metalloenzymes . The C/Fe-SP was found to utilize with equal efficiency either vitamin B12 or the natural cofactor 5-methoxybenzimidazolylcobamide as a methyl carrier.

Gene, 1993 Mar 15, 125(1), 85 - 9
Secretion of a prokaryotic cellulase in bacterial and mammalian cells; Soole KL et al.; The catalytic domain of mature Clostridium thermocellum endoglucanase E (EGE') and derivatives of the enzyme fused to prokaryote and eukaryote signal peptides (SP), were produced in Chinese hamster ovary (CHO) cells and Escherichia coli . All three forms of the endoglucanase were secreted into the periplasm of Escherichia coli, but only derivatives of the enzyme containing an N-terminal SP were exported from CHO cells . Extracellular EGE', purified from E . coli and CHO cultures, displayed similar properties suggesting that glycosylation of the enzyme in the eukaryote did not significantly alter the protein's properties . Data presented in this report indicate that mature EGE' contains secretion signals which are recognised only by the E . coli protein export apparatus, suggesting that there are differences in the recognition of certain secretion signals in eukaryotes and prokaryotes . As mature EGE' does not contain secretion signals recognised by the mammalian cell, membrane translocation of the bacterial cellulase in a higher eukaryote is directed by an N-terminal prokaryotic SP.

FEMS Microbiol Lett, 1993 Mar 15, 108(1), 103 - 10
Genetic interrelationships of saccharolytic Clostridium botulinum types B, E and F and related clostridia as revealed by small-subunit rRNA gene sequences; Hutson RA et al.; The phylogenetic interrelationships of saccharolytic C . botulinum types B, E and F together with eleven other saccharolytic clostridia were examined by 16S rRNA gene sequencing . Comparative analysis of the sequence data revealed that the saccharolytic C . botulinum types B, E and F were highly related and represent a single genetic group . Strains of C . barati and C . butyricum that produce botulinal neurotoxin revealed almost 100% 16S rRNA sequence identity with their respective non-toxigenic counterparts and were phylogenetically distinct from saccharolytic C . botulinum (types B, E and F) . Proteolytic C . botulinum type F was shown to be phylogenetically remote from the saccharolytic C . botulinum group . The implications of the sequence data for the taxonomy of the C . botulinum complex are discussed.

Vet Rec, 1993 Mar 13, 132(11), 273 - 5
A paralytic-like disease of the ostrich (Struthio camelus masaicus) associated with Clostridium chauvoei infection; Lublin A et al.; Two adult Masai ostriches in a zoological collection became recumbent . The birds could not raise the neck or the head, had difficulties in breathing, and died eight and 13 days, respectively, after the first clinical signs appeared . On post mortem examination the heart had a globous appearance accompanied by gelatinous atrophy . The lungs were hyperaemic and oedematic, while the intestines had prominent haemorrhages in their mucosa . The liver and kidney were enlarged, and the former had also necrotic foci . Smears taken from the hyperaemic regions of the intestines and the necrotic foci of the liver were positive to Clostridium chauvoei after staining with specific fluorescein-labelled antiserum . No other pathogenic agents were identified or isolated from these birds . This report appears to be the first of a pathogenic condition associated with C chauvoei in an avian species.

Mol Gen Mikrobiol Virusol, 1993 Mar-Apr, (2), 16 - 21
{Extrachromosomal genetic elements of Clostridium botulinum . I . Plasmids of toxigenic and atoxigenic strains of Clostridium botulinum type A}; Evstigneev VI et al.; Plasmid profiles in fifteen toxigenic and six nontoxigenic strains of Clostridium botulinum type A were investigated . The electrophoretic patterns of nucleic acids from the bacterial lysates were shown to vary in relation to the mode of bacterial cells lysis and the stage of microbial population growth . Two toxigenic strains harbour a unique 3.9 MD plasmid, five strains have a plasmid of about 6 Md and seven other strains carry an 11.5 Md plasmid . The nontoxigenic derivatives of two toxigenic strains harbouring a 6 Md plasmid have lost this extrachromosomal element . The analysis of the derivatives has not indicated the 6 Md plasmid to be connected with bacteriocin production or antibiotic resistance in Clostridium botulinum . Nontoxigenic variants of three other strains possess an 11.5 Md plasmid similar to their toxigenic parents' one . One of the studied toxigenic strains harboured no plasmids, as well as its nontoxigenic derivative . The functions of all studied plasmids remain unknown . Physical maps of the 11.5 and 6 Md plasmids were constructed on the basis of restriction analysis . The obtained results can be used for identification and differentiation of Clostridium botulinum strains and in further molecular biological experiments with this organism.

J Laryngol Otol, 1993 Mar, 107(3), 208 - 10
The use of clostridium botulinum toxin in palatal myoclonus . A preliminary report; Saeed SR et al.; Palatal myoclonus is a rare syndrome characterized by involuntary rhythmical movements of the soft palate giving rise to clicking objective tinnitus . The intrusive nature of the tinnitus prompts patients to seek medical advice but to date no single treatment modality has been shown to be consistently effective . We present three cases in whom various management regimens were unsuccessful and in whom botulinum toxin injection to the palatal muscles was undertaken . All three cases were rendered free of their tinnitus with complete abolition of the myoclonus . The questions of optimum dosage as well as frequency of injection will be answered as greater numbers are treated by this method.

Appl Environ Microbiol, 1993 Mar, 59(3), 828 - 36
Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity; Van Rijssel M et al.; An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren . The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain . The protein consisted of 230- and 25-kDa subunits . The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose) . Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used . Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures . The smallest substrate utilized by the hydrolase was a tetragalacturonate . Vmax with oligogalacturonates increased with increasing chain length . The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively . The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively . The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively . Both enzyme activities were stable for more than 1 h at 70 degrees C . The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified.

J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 617 - 22
Similarity in the EDTA-soluble antigens of Clostridium chauvoei and C . septicum; Hamaoka T et al.; The EDTA-soluble antigens were prepared from whole cells of six strains of Clostridium chauvoei and five strains of C . septicum and were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis . SDS-PAGE profiles of the 11 strains were nearly identical, although there were slight variations in molecular mass in adjacent bands . In immunoblot analysis with two antisera against C . chauvoei and three against C . septicum, the antigens of all strains tested reacted with all five antisera and there were no differences in reactivities to the same antiserum between homologous and heterologous antigens . In an immunoblot reacted with a single antiserum, band patterns of 10 of the 11 strains were quite similar . After cross-absorption, antisera to both species lost most of their reactivities not only to heterologous antigens but also to homologous antigens . These results indicate that the two species share many common antigens and that there is a marked similarity in the antigenic properties of EDTA-soluble material.

Acta Chir Belg, 1993 Mar-Apr, 93(2), 49 - 53
Atraumatic splenic rupture in the course of a pneumonia with Streptococcus pneumoniae . Case report and literature review; Salame J et al.; Atraumatic splenic ruptures in the course of infectious diseases are rare but have been reported . Various germs of viruses can be at the origin of such rupture . The more often quoted viral disease is infectious mononucleosis . The more frequently involved bacteria are Streptococcus non pneumoniae, Pseudomonas, staphylococci and Clostridium . Rupture mechanism is not clearly elucidated; it can be connected with sepsis diffusion at spleen level via haematogenic way and consequently splenomegaly . Splenic rupture following septicaemia does not always entail major splenomegaly nor abscess formation but the attack of the splenic tissue itself is sometimes sufficient to bring about the rupture . The present case of atraumatic splenic rupture on spleen sepsis, no abscess, starting from a pulmonar infection with Streptococcus pneumoniae is, to our knowledge, the first case reported in literature.

Antimicrob Agents Chemother, 1993 Mar, 37(3), 474 - 82
Oral enoxacin for infection prevention in adults with acute nonlymphocytic leukemia . The Enoxacin Prophylaxis Study Group; Talbot GH et al.; A randomized, double-blind, placebo-controlled trial was conducted in eight hematologic units to determine the efficacy and safety of oral enoxacin for infection prevention in adult patients with acute nonlymphocytic leukemia . One hundred nineteen patients undergoing remission induction or consolidation chemotherapy were enrolled; 62 of them received enoxacin (400 mg orally every 12 h) . Patients received antifungal prophylaxis with oral mycostatin (1,000,000 U four times daily) or clotrimazole (1 troche five times daily) . Analysis was performed on an intent-to-treat basis . There was no significant difference between groups in race, age, or type and stage of leukemia, but there were more males in the placebo group (P = 0.073 {Fisher's exact test}) . Fewer enoxacin patients had gram-negative bacteremia (1 versus 14 {P < 0.001}), gram-negative infection at any site (2 versus 19 {P < 0.001}), or bacterial and/or fungal infection (17 versus 26 {P = 0.056}) . There was no significant difference in the number of patients with gram-positive infection at any site (12 versus 16), gram-positive bacteremia (9 versus 10), deep fungal infection (6 versus 2), death (2 versus 3), other antimicrobial therapy required (48 versus 48), therapy with amphotericin B (15 versus 7 {P = 0.105}), any adverse event (45 versus 36), or any study drug-associated adverse events (13 versus 6) . Logistic regression confirmed (odds ratios and 95% confidence intervals are given in parentheses) that enoxacin reduced the risk of gram-negative infection (0.07; 0.01 to 0.30), especially gram-negative bacillary bacteremia (0.05; 0.01 to 0.37), without altering the risk of gram-positive bacterial (0.63; 0.26 to 1.5), deep fungal (2.57; 0.47 to 13.9), or Clostridium difficile (1.16; 0.3 to 4.56) infection . The median time to the onset of fever of more than or equal 102.8 F (39.3 degree C) was 32 days for the enoxacin group versus 15 days for patients receiving placebo (P=0.0007 {Wilcoxon test}) . In patients with acute nonlymphocytic leukemia, oral enoxacin prevents gram-negative infections, delays the onset of fever, does not alter the incidence of gram-positive or proven deep fungal infections, and is well tolerated.

J Clin Microbiol, 1993 Mar, 31(3), 740 - 2
Comparison of enterotoxin production, cytotoxin production, serogrouping, and antimicrobial susceptibilities of Clostridium difficile strains isolated from AIDS and human immunodeficiency virus-negative patients; Barbut F et al.; We analyzed and compared Clostridium difficile strains isolated from diarrheic stools of 49 human immunodeficiency virus (HIV)-negative and 50 AIDS patients . Our results suggest that distribution patterns of serogroups are different in these two populations . Serogroup C (which has been previously reported to be very resistant to antimicrobial agents) represents 66.0 and 18.4% of the isolates from AIDS and HIV-negative patients, respectively (P < 0.001); the selection of serogroup C could be explained by multiple antibiotic pressure to which AIDS patients have been subjected.

EMBO J, 1993 Mar, 12(3), 921 - 31
A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly; Aullo P et al.; We have developed a new tool for studying the role of rho in actin stress fibre formation . Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT) . The resulting chimeric toxins were tested on Vero cells . Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT . Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification . In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin . The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol . DC3B inhibited the bacterially induced microfilament assembly indicating that L . monocytogenes utilizes a cellular rho dependent mechanism in this process.

J Bacteriol, 1993 Mar, 175(6), 1637 - 44
Cloning, nucleotide sequence, and regulatory analysis of the Lactococcus lactis dnaJ gene; van Asseldonk M et al.; The dnaJ gene of Lactococcus lactis was isolated from a genomic library of L . lactis NIZO R5 and cloned into pUC19 . Nucleotide sequencing revealed an open reading frame of 1,137 bp in length, encoding a protein of 379 amino acids . The deduced amino acid sequence showed homology to the DnaJ proteins of Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis, and Clostridium acetobutylicum . The level of the dnaJ monocistronic mRNA increased approximately threefold after heat shock . The transcription initiation site of the dnaJ gene was determined and appeared to be preceded by a typical gram-positive vegetative promoter sequence (TTGCCA-17 bp-TAAAAT) . Upstream of the promoter region, an inverted repeat is located that is identical to those detected upstream of heat shock genes of other gram-positive organisms . A transcriptional fusion between the dnaJ expression signals and a usp45-amyS secretion cassette caused a significant increase in alpha-amylase activity after heat shock induction . Deletion mutagenesis showed that the inverted repeat is involved in heat shock regulation of the dnaJ gene . The conservation of this palindromic sequence in gram-positive heat shock genes suggests a common regulatory pathway distinct from the system used in gram-negative bacteria.

J Bacteriol, 1993 Mar, 175(5), 1293 - 302
Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component; Wang WK et al.; Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose . The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies . However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J . H . D . Wu, W . H . Orme-Johnson, and A . L . Demain, Biochemistry 27:1703-1709, 1988) . To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein . The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues . It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein . A putative ribosome binding site was identified at the 5' end of the gene . A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein . The predicted molecular weight of the secreted protein was 80,670 . The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes . A palindromic structure was found downstream from the open reading frame . The celS gene is unique among the known cel genes of C . thermocellum . However, it is highly homologous to the partial open reading frame found in C . cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.

J Bacteriol, 1993 Mar, 175(5), 1250 - 6
Complete structure of the tyrosine-linked saccharide moiety from the surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70; Christian R et al.; In this study, we have extended and completed a previous investigation (P . Messner, R . Christian, J . Kolbe, G . Schulz, and U . B . Sleytr, J . Bacteriol . 174:2236-2240, 1992) in which we demonstrated for the first time in prokaryotic organisms the presence of a novel O-glycosidic linkage via tyrosine . The surface layer glycoprotein of the eubacterium Clostridium thermohydrosulfuricum S102-70 is arranged in a hexagonal lattice, with center-to-center spacings of approximately 16.3 nm . Molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both glycosylated and chemically deglycosylated surface layer glycoprotein showed values for the monomeric subunits of 94,000 and 87,500, respectively . Glycopeptide fractions obtained after exhaustive pronase digestion of purified, intact glycoprotein were isolated by reversed-phase liquid chromatography . One- and two-dimensional nuclear magnetic resonance studies, together with chemical analyses and plasma desorption time-of-flight mass spectrometry, were used to elucidate the structure of the hexasaccharide moiety linked by the novel O-glycosidic linkage to tyrosine . The combined evidence suggests the following structure: beta-D-Galf-(1-->3)-alpha-D-Galp- (1-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Manp-(1--3)-alpha-L- Rhap-(1-->3)-beta- D-Glcp-(1-->4)-L-Tyr.

Infect Immun, 1993 Mar, 61(3), 1082 - 90
Effects of Clostridium difficile toxin B on human monocytes and macrophages: possible relationship with cytoskeletal rearrangement; Siffert JC et al.; Toxin B from Clostridium difficile is cytopathic in vitro for various types of cells, including polymorphonuclear cells, lymphocytes, and monocytes . Since intestine lamina propria is rich in macrophages, we studied the effect of toxin B on human monocytes and on human macrophages generated in vitro by long-term culture of purified circulating blood monocytes . Upon addition of toxin B, human monocytes exhibited few modifications whereas macrophages adopted a stellate morphology, with rounding up of the perikaryon . Toxin B made microfilaments of actin disappear and induced an important reorganization of vimentin and a redistribution of tubulin . Membrane area increased by approximately 16% . Toxin B did not affect the viability of human mononuclear phagocytes and did not exert any significant lytic effect . It profoundly altered the phagocytic function of macrophages . When activated by gamma interferon in the presence of toxin B, monocytes were more cytotoxic for U-937 target cells than control monocytes activated in absence of toxin . Finally, the combined treatment of monocytes with gamma interferon and toxin B increased significantly the secretion of tumor necrosis factor alpha, whereas toxin B alone was unable to induce tumor necrosis factor production . These results suggest that morphological and functional alterations induced in human mononuclear phagocytes by toxin B from C . difficile are due to the disorganization of the cytoskeleton and the resulting impairment of the membrane traffic equilibrium.

Acta Med Port, 1993 Mar-Apr, 6(3-4), 135 - 40
{Nosocomial infections . Some aspects}; Monteiro JA; Nosocomial infection is not a recent problem . However, it presents nowadays as a serious issue, not only due to the associated morbidity and mortality but also due to the economic burden on hospitals . The increased susceptibility of patients and the increased resistance to antibiotics by bacterial agents are important factors in the present situation . Among the different nosocomial pathogens, methicillin-resistant Staphylococcus aureus and Clostridium difficile are two of the most distressing for hospitals worldwide . Knowledge of their epidemiology, pathological and clinical particularities and treatment are fundamental in the observation and control of these infections . As a result of my recent experience in two teaching hospitals in the United States of America, I have decided to write the present paper.

Nippon Saikingaku Zasshi, 1993 Mar, 48(2), 417 - 27
{Studies on the locality and the function of spirosome in bacterial cells: relationship between the production of spirosome and anaerobic glycolysis}; Matayoshi S; The locality and the function of spirosome were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy in Peptostreptococcus productus, Clostridium sp . HD-17, Lactobacillus fermentum, Eubacterium aerofaciens and Escherichia cali B . When the bacterial cells in phosphate buffered saline were disrupted by sonication and fractionated by differential centrifugation, the spirosome protein was found in the cytoplasmic fraction and not in the fraction of cell wall and cell membrane (the envelope fraction) . The spirosome protein was found, however, in the envelope fraction when the bacterial cells were treated with a proper concentration of SDS . The spirosome protein in the envelope fraction disappeared after treatment with 2% Triton X-100 for 2 h . These results suggest that the spirosome protein anchored to the cell membrane upon SDS treatment . So it is presumed that the locality of spirosome is close to the cell membrane . Spirosome production increased in parallel to the concentration of glucose in the medium in obligate and aerotolerant anaerobes as well as in facultative anaerobes . This result indicates that the spirosome production was induced during the process of anaerobic glycolysis . Fructose as the sole carbon source in the minimal medium induced the spirosome production by E . coli as did glucose, but sodium pyruvate did not induce it either under aerobic or anaerobic condition.

Wiad Lek, 1993 Mar, 46(5-6), 211 - 5
{Study of obligate anaerobic bacterial sensitivity to tinidazole and metronidazole (determination of minimal inhibiting concentration--MIC)}; Kalowski M et al.; Comparative susceptibility testing of 428 strains of obligate anaerobic bacteria belonging to genera Propionibacterium, Arachnia, Actinomyces, Bacteroides, Prevotella, Porphyromonas, Anaerorhabdus, Fibrobacter, Fusobacterium, Peptostreptococcus and Clostridium to metronidazole and tinidazole was performed . The study of the susceptibility of anaerobic bacteria was carried out by the method of serial dilution in Brucella agar according to Finegold and Sutter (1972) . Strains of B . fragilis species, B . fragilis group, other Bacteroides spp. . Fusobacterium spp . and Clostridium spp . were susceptible to both chemotherapeutics in clinically attainable concentrations . Of particular significance is the greater susceptibility of these bacteria to tinidazole . Taking into account this activity, tinidazole is a superior agent in the treatment of anaerobic infections . Both chemotherapeutics were not active against, rarely isolated from severe infections . Gram-positive anaerobic rods from genera Propionibacterium . Actinomyces and Arachnia and were partially active against peptostreptococci.

Res Microbiol, 1993 Mar-Apr, 144(3), 171 - 80
Studies on the large subunit ribosomal RNA genes and intergenic spacer regions of non-proteolytic Clostridium botulinum types B, E and F; Campbell KD et al.; The large-subunit ribosomal ribonucleic acid (23S rRNA) genes of non-proteolytic (group II) strains of Clostridium botulinum toxin types B, E and F were amplified using the polymerase chain reaction (PCR), and cloned in Escherichia coli . Sequence determination showed that the 23S rRNA genes were 2910 nucleotides in length, and comparative analysis revealed approximately 99.5% sequence similarity . The 23S rRNA gene sequence of a strain phenotypically resembling non-proteolytic C . botulinum, except in not producing botulinal neurotoxin, was also determined and displayed 99.5% sequence similarity with those from toxigenic strains . A diagnostic sequence within the 23S rRNA characteristic for non-proteolytic C . botulinum was identified and used for the design of an oligonucleotide probe . Molecular hybridizations with PCR-amplified rDNA targets provided a precise and reliable method of identifying non-proteolytic (or Group II) C . botulinum and closely related non-toxigenic strains.

Appl Microbiol Biotechnol, 1993 Mar, 38(6), 750 - 4
Purification and properties of two truncated endoglucanases produced in Escherichia coli harbouring Clostridium cellulolyticum endoglucanase gene celCCD; Shima S et al.; The endoglucanase gene, celCCD, of Clostridium cellulolyticum has been expressed in Escherichia coli . Multiple active polypeptides were detected in the E . coli cells . The relative molecular mass (M(r)) of two major active polypeptides were 56,000 (D56) and 38,000 (D38), which were smaller than the deduced M(r) of the mature protein (63,401) . D56 and D38 were purified from the periplasmic fraction . The N-terminal sequences of the two purified polypeptides were identical to that of the mature endoglucanase (Ala-Ile-Asn-Ser-Gln-Asp-Met-Val---) deduced from the nucleotide sequence . These data indicated that these polypeptides were produced by processing the original mature protein in the C-terminal region . The enzymatic properties of these two polypeptides were very similar, except that the specific activity of D38 was 2-3.5-fold higher than that of D56, and D38 was more heat stable than D56.

Biotechnology (N Y), 1993 Mar, 11(3), 376 - 9
Manipulation of the repertoire of digestive enzymes secreted into the gastrointestinal tract of transgenic mice; Hall J et al.; In non-ruminant livestock the energy which can be derived from dietary cellulose and xylan is limited by the inefficient microbial fermentation of these polymers in the hind-gut . Furthermore, in poultry, cereal-derived plant structural polysaccharides impair normal digestive function through the formation of gel-like structures, which trap nutrients rendering them unavailable to the animal . The nutrition of non-ruminant livestock could be significantly improved by the depolymerization of plant structural polysaccharides, through the introduction of cellulase activity into the small intestines of these animals . Here we describe the expression of Clostridium thermocellum endoglucanase E in the exocrine pancreas of transgenic mice . A non-glycosylated active enzyme is secreted into the small intestines, and is resistant to proteolytic inactivation, demonstrating the feasibility of generating non-ruminant animals with the endogenous capacity to depolymerize plant structural polysaccharides in the small intestines.

Mol Microbiol, 1993 Mar, 7(5), 755 - 63
Structural analysis and demonstration of the 29 kDa antigen of pathogenic Entamoeba histolytica as the major accessible free thiol-containing surface protein; Flores BM et al.; The 29 kDa protein of pathogenic Entamoeba histolytica is a cysteine-rich surface antigen which we recently characterized by cDNA sequencing and by using monoclonal antibodies which differentiated between pathogenic and non-pathogenic clinical isolates . To determine the structure and biochemical attributes of this protein, a repertoire of immunological techniques using monoclonal antibodies, and radiolabelling were employed . We demonstrated that the 29 kDa protein forms a 60 kDa dimer and a high-molecular-mass oligomer(s) on the surface of the organism through disulphide bonds, and is the major accessible free thiol-containing surface protein of E . histolytica . The deduced amino acid sequence encoding the 29 kDa protein was found to share a common amino acid domain with sequences reported for Helicobacter pylori, Salmonella typhimurium, MER5 gene expressed in murine erythroleukemia cells, Clostridium pasteurianum, and a Bacillus spp.

Biochemistry, 1993 Feb 23, 32(7), 1803 - 9
Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis; Thrall SH et al.; The intermediacy of a pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) in the Clostridium symbiosum pyruvate phosphate dikinase catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) was examined using transient kinetic techniques . Single-turnover experiments with {gamma-32P}ATP or {14C}ATP and PPDK were carried out in the presence and absence of Pi to test for pyrophosphorylenzyme and AMP formation, respectively . Formation of the E-PP.AMP complex was found to be followed by Pi binding and the formation of the E-P.AMP.PPi complex . The level of pyrophosphorylenzyme accumulated during a single turnover was found to be dependent on the divalent metal cofactor used (Mn2+ > Co2+ > Mg2+) . Single-turnover experiments with {32P}PEP and PPDK were carried out in the presence and absence of PPi and pyruvate to test for phosphorylenzyme formation in the reverse, ATP-forming direction of the reaction . Phosphorylenzyme formed from the reaction of the E.PEP complex was converted in the presence of AMP and PPi to free enzyme at a rate exceeding the steady-state turnover rate . The reaction sequence for pyruvate phosphate dikinase was determined to be {formula see text} 31P NMR analysis of the phosphorylenzyme in the native (-4.0 ppm) and denatured form (-3.9 ppm) revealed a 3-N-phosphohistidine residue . Complexation of Mg2+ resulted in a 0.3 ppm upfield shift of the phosphorus resonance from native phosphorylenzyme while Mn2+ complexation lead to extensive line broadening, indicative of metal cofactor binding in close vicinity to the phosphoryl group.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1993 Feb 20, 229(4), 1153 - 6
Crystallization and preliminary crystallographic data for formyltetrahydrofolate synthetase from Clostridium thermoaceticum; Lewinski K et al.; Formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been crystallized using the hanging-drop method from ammonium sulphate and PEG solutions . Crystals are trigonal, the space group is R32, a = 163 A, c = 259 A . A 4.2 A resolution data set has been collected . Analysis of the data using the self-rotation function shows that tetramers have approximate 222 symmetry and are positioned on a crystallographic 2-fold axis.

Eur J Biochem, 1993 Feb 15, 212(1), 121 - 7
Purification and characterization of a coenzyme-A-dependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri; Sohling B et al.; Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinyl-CoA:CoA transferase (0.28 U/mg), a coenzyme-A-dependent succinate-semialdehyde dehydrogenase (0.73 U/mg) and a NAD(+)-dependent 4-hydroxybutyrate dehydrogenase (0.25 U/mg) . The semialdehyde dehydrogenase, which catalyzed the NADPH-dependent reduction of succinyl-CoA to succinate semialdehyde, was purified 59-fold to homogeneity . A molecular mass of 115000 Da was determined for the native enzyme; SDS/PAGE revealed one protein band at 55,000, indicating that the active form is a dimer . The enzyme was highly specific for succinyl-CoA and succinate semialdehyde . The pH optimum was 7.0 for the reduction of succinyl-CoA, and 8.5 for the reverse reaction . Km values were determined for both the forward and reverse directions . The kinetic data suggest a ping-pong mechanism.

FEBS Lett, 1993 Feb 15, 317(3), 259 - 62
Analysis of the catalytic center of cyclomaltodextrinase from Thermoanaerobacter ethanolicus 39E; Podkovyrov SM et al.; The amino acid sequences of cyclomaltodextrinase (CDase) from Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) and other amylolytic enzymes were compared by using linear alignment and hydrophobic cluster analysis . Two Asp and one Glu residue, which were considered to be the catalytic residues of the compared enzymes according to crystallographic or protein engineering experiments, were also conserved in CDase . Asp325, Asp421 and Glu354 of the CDase were individually replaced by means of site-directed mutagenesis . The mutant enzymes completely lost activity, suggesting that these residues play an important role in catalysis.

Biochim Biophys Acta, 1993 Feb 13, 1156(2), 213 - 8
Purification and characterization of the ganglioside-binding fragment of Clostridium botulinum type E neurotoxin; Kamata Y et al.; A way of fragmentation of Clostridium botulinum neurotoxin was carried out to elucidate the structure-function relationship of neurotoxin . The hitherto only plausible fragment was isolated from the trypsin-treated heavy chain of botulinum type E neurotoxin . In the presence of 4 M urea, one protein peak emerged from QAE-Sephadex column loaded with the heavy chain mildly treated with trypsin by elution with 0.1 M sodium chloride . Although many protein bands were detected in SDS-PAGE of the treated heavy chain, the eluted protein migrated in a single band to the position of 41,000 Da . The recovery of the 41,000-Da fragment was 28.6%, but with a 2 M urea-containing buffer as eluant, the recovery was less than 12% . The 41,000-Da fragment bound to gangliosides GD1a, GT1b, and GQ1b, to which neurotoxin and the heavy chain bound . The 41,000-Da fragment partially interfered with the binding of 125I-labeled neurotoxin to mouse brain synaptosomes . We have proposed a three-fragment structure (L.H-1.H-2) for botulinum type E neurotoxin . The characters of the 41,000-Da fragment described in this paper seem to substantiated our proposal that type E neurotoxin consists of three fragments, L.H-1.H-2, and that the ganglioside-binding fragment is H-2.

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 317 - 22
Low spin quantitation of NiFeC EPR signal from carbon monoxide dehydrogenase is not due to damage incurred during protein purification; Shin W et al.; Evidence is presented that the O2-sensitive, nickel- and iron-containing enzyme carbon monoxide dehydrogenase from Clostridium thermoaceticum was purified without significantly inactivating either its CO oxidation or CO/acetyl-CoA exchange activities . All CO oxidation activity from the crude extract was recovered in the purified enzyme (and side fractions) . The exchange activity could not be quantified similarly, because the crude extract and early purification step fractions exhibited little or no exchange activity . Later purification fractions exhibited much more exchange activity, suggesting that an inhibitor was present in the impure fractions . The NiFeC EPR signal intensity was used as an indicator of the enzyme's capacity to catalyze exchange . This signal was extremely sensitive to oxygen; exposure to as little as 0.5 equiv/mol enzyme dimer resulted in substantial loss of intensity . The NiFeC intensities at each step in the purification were virtually invariant, indicating that the enzyme had not been exposed to oxygen and had not been inactivated towards catalyzing exchange . The ability to purify carbon monoxide dehydrogenase (CODH) without inactivating nearly any of the molecules suggests that it is quite stable under anaerobic conditions . The purified enzyme, which could not have lost functional metal ions during purification, contained 1.9 Ni and 11.3 Fe, similar to previous reports . The NiFeC EPR signal intensity from each purification fraction (0.2 spins/mol enzyme dimer) was as low as from previous preparations, indicating that its low spin quantitation is not the result of damage incurred during purification . If the low intensity arises from heterogeneity as proposed earlier, the heterogeneity must originate prior to purification.

FEBS Lett, 1993 Feb 8, 317(1-2), 44 - 8
Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum . Organization of the mut genes; Holloway DE et al.; The gene encoding component E, the large subunit, of adenosylcobalamin (coenzyme B12)-dependent glutamate mutase from Clostridium tetanomorphum has been cloned and sequenced . The mutE gene encodes a protein of 485 amino acid residues, with M(r) 53,708 . The mutE gene is situated some 1,400 bp downstream of the mutS gene, which encodes the small subunit of glutamate mutase . Between the two is an open reading frame encoding a protein of 462 amino acids, with M(r) 50,171, and of unknown function . All three genes appear to be transcribed as an operon and lie immediately upstream of the gene encoding beta-methylaspartase, the next enzyme in the pathway of glutamate fermentation . Local homology exists between mutE and a region of beta-methylaspartase which contains an active-site serine residue.

FEBS Lett, 1993 Feb 8, 317(1-2), 17 - 21
F0 and F1 parts of ATP synthases from Clostridium thermoautotrophicum and Escherichia coli are not functionally compatible; Das A et al.; F1-stripped membrane vesicles from Clostridium thermoautotrophicum and Escherichia coli were reconstituted with F1-ATPases from both bacteria . Reconstituted F1F0-ATPase complexes were catalytically active, i.e . capable of hydrolyzing ATP . Homologous-type ATPase complexes having F0 and F1 parts of ATP synthases from the same origin were DCCD sensitive and supported ATP-driven enhancement of anilinonaphthalene sulfonate (ANS) fluorescence . Hybrid-type ATPase complexes having F0 and F1 parts of ATP synthases from different origins were neither DCCD sensitive nor did they support ATP-driven enhancement of ANS fluorescence . Analyzing these results it has been demonstrated that the F0 and F1 parts of ATP synthases of these two bacteria are not functionally compatible.

FEBS Lett, 1993 Feb 8, 317(1-2), 101 - 4
Evidence for an {Fe}-type hydrogenase in the parasitic protozoan Trichomonas vaginalis; Payne MJ et al.; The hydrogenase of the pathogenic protozoan Trichomonas vaginalis was extracted and partially purified . The catalytic and spectroscopic properties of the enzyme indicate that it belongs to the class of {Fe}-hydrogenases, rather than the {NiFe}-hydrogenases . The hydrogenase activity was highly sensitive to carbon monoxide, 50% inhibition being attained by 1 microM CO . The EPR spectrum of the most active fractions from chromatography, after reduction by hydrogen and partial reoxidation under argon, showed an EPR spectrum at g = 2.10, 2.04, 2.00 . This unusual spectrum is characteristic of the 'H-cluster', as seen in {Fe}-hydrogenases of anaerobic bacteria such as Clostridium spp.

Genetika, 1993 Feb, 29(2), 217 - 24
{Specific proteolysis of Clostridium thermocellum endoglucanase upon heterologous expression in Escherichia coli cells}; Aminov RI et al.; The cel(1) gene of Clostridium thermocellum encoding endoglucanase cloned earlier in Escherichia coli was deleted at specific sites and fused with the E . coli gene lacZ' . Analysis of heterologous expression of the produced variants was performed . It was established that the post-translational modification of the clostridial endoglucanase in E . coli involves a membrane-linked N-terminal cleavage of 6 kDa polypeptide during secretion through the cytoplasmic membrane and a C-terminal cleavage of 4 kDa protein not related to the secretion . A comparative study of the authors' and literature data supports the suggestions that: (i) the cel(1) cloned in this laboratory is a deleted variant of the cloned cel(1) gene of C . thermocellum described previously by other authors, and (ii) the C-terminal proteolysis of the endoglucanase precursor takes place in a linker region connecting the catalytic and cellulose binding domains . An account of similar kind of proteolysis responsible for multiple forms of cellulolytic enzymes is given.

Int J Food Microbiol, 1993 Feb, 17(4), 343 - 8
A 48 kilodalton enterotoxin-related protein from Clostridium perfringens vegetative and sporulating cells; Ryu S et al.; Sporulating and vegetative cell extracts of enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens were examined by Western blotting using enterotoxin antiserum . A 48-kDa enterotoxin-related protein was found in vegetative and sporulating cell extracts of both toxin types . The results question previous reports about the ability of vegetative cells and presumptive enterotoxin-negative strains of this organism to produce enterotoxin, a protein with a molecular weight of 34 kDa.

Mol Microbiol, 1993 Feb, 7(3), 371 - 81
Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture; Eveillard M et al.; Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells . Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells . The binding of C . difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C . difficile-binding sites are differentiation-related brush border protein(s) . To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C . difficile with adherence assays . It was shown that heating of C . difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells . A labile surface-associated component was involved in C . difficile adhesion since washes of C . difficile grown in the presence of blood without heat shock decreased adhesion . After heating, washes of C . difficile grown in the presence of blood did not modify adhesion . Analysis of surface-associated proteins of C . difficile subjected to different culture conditions was conducted . After growth of C . difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared . Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhesion of C . difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins . Indeed, adhesion to Caco-2 cell monolayers of C . difficile strains grown in the presence of blood, without or with heat-shock, was blocked . Taken together, our results suggest that C . difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.

J Clin Pathol, 1993 Feb, 46(2), 180 - 3
Neutropenic enterocolitis associated with Clostridium tertium; Coleman N et al.; A 15 year old boy being treated for relapsed acute lymphoblastic leukaemia developed severe diarrhoea and abdominal pain which worsened despite empirical antibiotic treatment . A right hemicolectomy was performed . The caecum and ascending colon showed changes typical of neutropenic enterocolitis . Clostridium tertium was isolated from faeces, blood cultures, and from the resected gut wall, with no evidence of other organisms capable of causing such a condition . As far as is known, this is the first reported case in which neutropenic enterocolitis has been associated with well documented C tertium infection, an organism previously described as a cause of bacteraemia in neutropenic patientsPublication Types:

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