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 metronida