N Engl J Med, 1998 Sep 24, 339(13), 868 - 74 High-level chloramphenicol resistance in Neisseria meningitidis; Galimand M et al.; BACKGROUND: Neisseria meningitidis is nearly always susceptible to the penicillins, the cephalosporins, and chloramphenicol . Between 1987 and 1996, however, chloramphenicol-resistant strains were isolated from 11 patients in Vietnam and 1 in France . METHODS: The minimal inhibitory concentration of chloramphenicol was determined for the 12 isolates . The isolates were analyzed by monoclonal-antibody-based serotyping and subtyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis . Bacterial DNA was analyzed by hybridization, the polymerase chain reaction, and sequencing to identify the resistance gene and determine the origin of the resistance . RESULTS: The isolates were resistant to chloramphenicol (minimal inhibitory concentration, > or =64 mg per liter) and produced an active chloramphenicol acetyltransferase . All 12 strains belonged to serogroup B but had a high degree of diversity, and 10 could not be typed with the use of monoclonal antibodies . The nucleotide sequence of the resistance gene and the flanking regions was identical to that of an internal portion of transposon Tn4451 that carries the catP gene in Clostridium perfringens . Moreover, this gene was located in the same genomic site in the chloramphenicol-resistant isolates . CONCLUSIONS: The high-level chloramphenicol resistance that we describe in N . meningitidis isolates is of great concern, since in developing countries, chloramphenicol given intramuscularly is the standard therapy for meningococcal meningitis . The resistance to chloramphenicol is due to the presence of the catP gene on a truncated transposon that has lost mobility because of internal deletions, and the transformation of genetic material between strains of N . meningitidis probably played an important part in the dissemination of the gene. J Biolumin Chemilumin, 1998 Jul-Aug, 13(4), 189 - 93 Prospects for chemiluminescent and bioluminescent immunoassay and nucleic acid assays in food testing and the pharmaceutical industry; Kricka LJ; The sensitivity, speed and convenience of chemiluminescent (CL) and bioluminescent (BL) immunoassays and probe assays have led to a diverse range of applications for these technologies, mainly in the clinical laboratory . These methods are now being explored by the food and pharmaceutical industries . Demanding detection limits and the complexity of sample preparation for food and pharmaceutical analyses present daunting challenges for the analyst . Immunoassay and nucleic acid amplification technologies have been applied to food testing, but these have mostly favoured non-luminescent endpoints . Food assays with CL or BL endpoints are now emerging, e.g., Clostridium botulinum type A detection using a CL immunosorbent assay; Salmonella and Zygosaccharomyces detection using a combination of PCR and CL detection . The analytical challenges posed by the pharmaceutical industry include testing for contaminants in raw materials and drug products, and drug discovery . The sensitivity and rapid signal acquisition characteristics of CL and BL are advantageous for the high throughput, massively parallel testing of micro-sized samples demanded in drug discovery . Current progress and the prospects for CL and BL immunoassay and nucleic acid technologies in this and other pharmaceutical and food applications is reviewed. Protein Eng, 1998 Jul, 11(7), 569 - 75 Use of protein engineering to explore subunit interactions in an allosteric enzyme: construction of inter-subunit hybrids in Clostridium symbiosum glutamate dehydrogenase; Aghajanian S et al.; Hybrids of different forms of clostridial glutamate dehydrogenase (GDH) have been constructed in order to probe the basis of allosteric interaction in this hexameric enzyme . It was shown that the C320S mutant, which is fully active and shows allosteric behaviour similar to that of the wild-type enzyme, can also be renatured after unfolding in urea . Mixtures of unfolded wild-type and C320S subunits gave rise to hybrids upon refolding . A purely random reassembly would lead to a simple binomial distribution . However there was a slightly better overall recovery of wild-type subunits and there appears to be a tendency for rapidly formed structured wild-type subunits in a mixture to nucleate further refolding in a way that biases the final distribution against the formation of C320S hexamers . Only the wild-type subunits in such hybrid mixtures are able to react with Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) . Accordingly, after modification of hybrid hexamers with DTNB only the mutant subunits can bind NAD+ . This permits fractionation on an NAD+-agarose affinity column . The elution pattern in itself indicates cooperativity since DTNB modification of just one subunit in a 1:5 wild-type/C320S hybrid largely abolished binding to the column . Kinetic studies were carried out on a fractionated preparation in which hexamers containing only one C320S subunit and five wild-type subunits were the predominant active species . Measurements of activity were made both before and after treatment with an excess of beta-mercaptoethanol to remove the blocking thionitrobenzoate moieties . Before beta-mercaptoethanol treatment this sample, with only one active subunit per hexamer, gave strictly hyperbolic (Michaelis-Menten) kinetics with NAD+ at pH 7.0, whereas after beta-mercaptoethanol (all six subunits now active) the markedly kinked Eadie-Hofstee plot characteristic of wild-type enzyme was obtained . On the other hand the sigmoid response to glutamate at high pH persisted (Hill coefficient=3.6) even without beta-mercaptoethanol, reflecting the fact that the inactive subunits can still bind glutamate . Beta-mercaptoethanol treatment restored full positive cooperativity (Hill coefficient=5.2) . These results prove beyond doubt that the non-classical kinetic behaviour of clostridial GDH is a direct result of interaction between NAD+ binding sites on the six (normally) identical subunits of a hexamer. Structure, 1998 Aug 15, 6(8), 1021 - 33 How a protein prepares for B12 binding: structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum; Tollinger M et al.; BACKGROUND: Glutamate mutase is an adenosylcobamide (coenzyme B12) dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate . The enzyme from Clostridium tetanomorphum comprises two subunits (of 53.7 and 14.8 kDa) and in its active form appears to be an alpha 2 beta 2 tetramer . The smaller subunit, termed MutS, has been characterized as the B12-binding component . Knowledge on the structure of a B12-binding apoenzyme does not exist . RESULTS: The solution structure and important dynamical aspects of MutS have been determined from a heteronuclear NMR study . The global fold of MutS in solution resembles that determined by X-ray crystallography for the B12-binding domains of Escherichia coli methionine synthase and Propionibacterium shermanii methylmalonyl CoA mutase . In these two proteins a histidine residue displaces the endogenous cobalt-coordinating ligand of the B12 cofactor . In MutS, however, the segment of the protein containing the conserved histidine residue forms part of an unstructured and mobile extended loop . CONCLUSIONS: A comparison of the crystal structures of two B12-binding domains, with bound B12 cofactor, and the solution structure of the apoprotein MutS has helped to clarify the mechanism of B12 binding . The major part of MutS is preorganized for B12 binding, but the B12-binding site itself is only partially formed . Upon binding B12, important elements of the binding site appear to become structured, including an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the cofactor. J Antimicrob Chemother, 1998 Aug, 42(2), 245 - 8 In-vitro antibiotic susceptibility and molecular analysis of anaerobic bacteria isolated in Cape Town, South Africa; Koch CL et al.; One hundred and twenty-three anaerobic isolates from Cape Town, South Africa, were tested in vitro against cefoxitin, chloramphenicol, metronidazole, co-amoxiclav, benzylpenicillin and clindamycin . Forty-five Gram-positive organisms were tested against RP59500 (a streptogramin) . Resistance in the Gram-positive isolates was as follows: of the Clostridium perfringens isolates, 4% were resistant to benzylpenicillin and 4% to clindamycin; of the Peptostreptococcus anaerobius isolates, 10% were resistant to benzylpenicillin, 10% to cefoxitin and 10% to metronidazole; of the isolates of Peptostreptococcus spp., 12% were resistant to benzylpenicillin, 6% to metronidazole, 6% to chloramphenicol and 12% to RP59500 . Of the Gram-negative organisms, those in the Bacteroides fragilis group were resistant to benzylpenicillin (83%), cefoxitin (5%), clindamycin (5%) and co-amoxiclav (2%) . One clindamycin-resistant B . fragilis isolate carried a plasmid homologous to the ermF erythromycin resistance determinant. J Clin Microbiol, 1998 Oct, 36(10), 2957 - 63 Comparison of restriction enzyme analysis, arbitrarily primed PCR, and protein profile analysis typing for epidemiologic investigation of an ongoing Clostridium difficile outbreak; Rafferty ME et al.; During an outbreak of diarrhea in a general hospital in 1992, 166 Clostridium difficile isolates from 102 patients were typed by restriction enzyme analysis (REA), arbitrarily primed PCR (AP-PCR), and protein profile analysis (PP) techniques . A total of 18 types and 5 subtypes were identified by REA, 32 types were identified by AP-PCR, and 9 types were identified by PP . Analysis of the data indicated the presence of a predominant strain among 76, 75, and 84% of the isolates by REA, AP-PCR, and PP, respectively . Subsequently, 45 C . difficile isolates which had been collected in 1990 from 33 patients in the same hospital following a significant increase in the number of cases of diarrhea caused by C . difficile were studied by REA, AP-PCR, and PP typing techniques . Thirteen types and one subtype were identified by REA, 12 types were identified by AP-PCR, and 5 types were identified by PP . As with the isolates from 1992, a dominant strain was identified . This strain was represented by 53, 64, and 70% of the total number of isolates when the strains were typed by REA, AP-PCR, and PP, respectively . Every isolate (210 of 211) from both 1990 and 1992 that was available for typing was typeable by all three methods . Furthermore, the same dominant strain was identified in both 1990 and 1992 by each method . This study demonstrates that each of the three typing methods can be useful in epidemiologic investigations of C . difficile outbreaks and that one strain can be dominant in an institution over a number of years. Rapid Commun Mass Spectrom, 1998, 12(16), 1045 - 50 Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Srinivasan JR et al.; The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS) . The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis . An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis . By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved . Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E. J Vasc Surg, 1998 Sep, 28(3), 404 - 11; discussion 411-2 Gastrointestinal complications after aortic surgery; Valentine RJ et al.; BACKGROUND AND PURPOSE: A major gastrointestinal complication (GIC) after aortic surgery may be disastrous, but these complications have received scant attention . This study was performed to determine the risk factors, associated events, and outcomes for patients with GIC . METHODS: We performed a secondary analysis of a prospective study that examined 120 consecutive patients who underwent transperitoneal aortic revascularization for aneurysmal or occlusive disease . RESULTS: The following 29 GICs developed in 25 patients (21%) within 30 days of aortic surgery: paralytic ileus that required replacement of nasogastric tubes (n = 12), upper gastrointestinal bleeding (n = 5), Clostridium difficile enterocolitis (n = 5), acute cholecystitis (n = 2), mechanical obstruction (n = 2), ascites (n = 2), and colon ischemia (n = 1) . Seven patients required operations for GICs after aortic revascularization . A comparison of patients with and without GICs showed no differences in the prevalence of risk factors, presence of mesenteric artery stenoses, coexisting medical illnesses, antecedent gastrointestinal history, operative indication, preoperative fluid administration, or duration of operation . However, patients with GICs had more intraoperative complications (P = .004), greater intraoperative blood loss (P = .02), and more fluids during the postoperative period (P = .008) . The mean duration of mechanical ventilation was 71 +/- 23 hours for patients with GICs versus 7 +/- 2 hours for patients without GICs (P = .006) . A higher prevalence of pulmonary (P = .004) and renal (P = .001) complications was seen in the patients with GICs . The mean stay in the intensive care unit was 16 +/- 2 days for patients with GICs as compared with 5 +/- 0.4 days for patients without GICs (P < .001) . Four deaths occurred, all caused by multisystem organ failure: 3 patients had GICs, and 1 did not have a GIC (P = .007) . CONCLUSIONS: These results show that GICs are prevalent in transperitoneal aortic surgery and are associated with severe morbidity rates, increased hospital costs because of prolonged stay, and increased mortality rates . Some GICs appear to be associated with intraoperative events that lead to visceral hypoperfusion, and others can be attributed to mechanical causes . However, none of the variables examined in this study were predictive of GICs . In all, GICs should be considered serious adverse sequela after aortic revascularization . Because no risk factors for GICs have been identified, these complications currently cannot be prevented. J Med Microbiol, 1998 Sep, 47(9), 767 - 71 Inhibition of enhanced toxin production by Clostridium difficile in biotin-limited conditions; Yamakawa K et al.; Production of toxins A and B by Clostridium difficile is enhanced in a defined medium with biotin-limited conditions . In the present study compounds inhibitory to enhanced toxin production by a C . difficile strain were examined . Increases in biotin concentration from 0.05 nM to 50 nM accelerated growth and inhibited enhanced toxin production . Asparagine, glutamic acid and glutamine (10 mM) showed an effect on growth and toxin production similar to that of biotin . Lysine (10 mM) suppressed growth and inhibited toxin production . Addition of these toxin-inhibitory compounds within an incubation period of 2 days inhibited the enhanced toxin production, but later addition showed only slight inhibition of toxin production . Amino acids contained in the defined medium under the biotin-limited conditions were actively utilised in the presence of the three toxin-inhibitory amino acids, but in the presence of lysine, amino-acid utilisation was suppressed . Different mechanisms of action of these toxin-inhibitory molecules, which may be divided into excess biotin, asparagine-glutamic acid-glutamine group, and lysine, are discussed. Biochim Biophys Acta, 1998 Aug 14, 1373(1), 47 - 58 A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms; Bennik MH et al.; A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin . To our knowledge, this is the first report on bacteriocin production by this organism . The elucidation of the full primary amino acid sequence of mundticin (KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATGGAAGWSK) revealed that this antimicrobial peptide belongs to the class IIa bacteriocins of lactic acid bacteria which share a highly conserved N-terminal 'YGNGV' motif . Data obtained by computer modelling indicated an oblique orientation of the alpha-helical regions of mundticin and homologous class IIa bacteriocins at a hydrophobic-hydrophilic interface, which may play a role in the destabilization of phospholipid bilayers . The average mass of mundticin, as determined by electron spray mass spectrometry, was found to be 4287.21+/-0.59 Da . With respect to its biological activity, mundticin was shown to inhibit the growth of Listeria monocytogenes, Clostridium botulinum and a variety of lactic acid bacteria . Moreover, it was demonstrated to have a bactericidal effect on L . monocytogenes as a result of the dissipation of the membrane potential, and a loss of intracellular ATP in absence of ATP leakage . Its good solubility in water, and its stability over a wide pH and temperature range indicate the potential of this broad spectrum bacteriocin as a natural preservation agent for foods. J Biol Chem, 1998 Sep 18, 273(38), 24433 - 8 UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of Clostridium perfringens phospholipase C; Flores-Diaz M et al.; A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated . We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell . To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C . perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA . Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector . Exposure of these two types of transfectant clones to C . perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase . Further experiments with genetically engineered C . perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells. Curr Microbiol, 1998 Oct, 37(4), 262 - 8 Analysis of the botulinum neurotoxin type F gene clusters in proteolytic and nonproteolytic Clostridium botulinum and Clostridium barati; East AK et al.; Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx . 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT) . Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx . 69-88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx . 96% identity) and the NTNHs (approx . 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto . Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Biochem J, 1998 Sep 15, 334 ( Pt 3), 625 - 31 Tyrosine-phosphorylation-dependent and rho-protein-mediated control of cellular phosphatidylinositol 4,5-bisphosphate levels; Rumenapp U et al.; The polyphosphoinositide PtdIns(4,5)P2, best known as a substrate for phospholipase C isozymes, has recently been recognized to be involved in a variety of other cellular processes . The aim of this study was to examine whether the cellular levels of this versatile phospholipid are controlled by tyrosine phosphorylation . The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M3 muscarinic acetylcholine receptor . Inhibition of tyrosine phosphatases by pervanadate induced an up-to-approx.-2 . 5-fold increase in the total cellular level of PtdIns(4,5)P2, which was both time- and concentration-dependent . In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, caused a rapid and specific fall in the cellular PtdIns(4,5)P2 level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P2 formation . Inactivation of Rho proteins by Clostridium difficile toxin B caused a similar fall in the HEK-293 cell PtdIns(4,5)P2 level, which was not altered by additional genistein treatment . Furthermore, toxin B treatment abolished the pervanadate-induced increase in PtdIns(4,5)P2 levels . As PtdIns(4,5)P2 is an essential stimulatory cofactor for phospholipase D (PLD) enzymes, we finally examined the effects of the agents regulating PtdIns(4,5)P2 levels on PLD activity in HEK-293 cells . Inhibition of tyrosine phosphatases by pervanadate caused an increase in PLD activity, which was susceptible to genistein and tyrphostin 23, and which was abolished by prior treatment with toxin B . In conclusion, the data presented indicate that the cellular level of the multifunctional phospholipid, PtdIns(4,5)P2, in HEK-293 cells is controlled by a tyrosine-kinase-dependent mechanism and that this process apparently involves Rho proteins, as found similarly for tyrosine-phosphorylation-induced PLD activation. Int J Food Microbiol, 1998 Jul 21, 42(3), 195 - 200 Amount of enterotoxigenic Clostridium perfringens in meat detected by nested PCR; Miwa N et al.; The incidence and quantity of enterotoxigenic Clostridium perfringens in beef, pork, and chicken meat were determined and compared with that of the total enterotoxigenic and nonenterotoxigenic C . perfringens . The method for the detection and quantification of enterotoxigenic C . perfringens consisted of a combination of the most probable number (MPN) method and a nested polymerase chain reaction after culturing of the sample . The results obtained by this method for inoculated meat samples were significantly correlated with those obtained by the plate count method . When the method was applied to the detection and quantification of enterotoxigenic C . perfringens found in randomly selected meat samples, the organism was found in 2% of the beef pieces (< 10(2) MPN/100 g) and 12% of the chicken pieces (< 10(2)-4.3 x 10(2) MPN/100 g) out of the 50 pieces of each meat tested . No enterotoxigenic C . perfringens was found in pork . Total C . perfringens was found in 16% of the beef (< 10(2)-4.3 x 10(2) MPN/100 g), 10% of the pork (< 10(2) MPN/100 g), and 84% of the chicken (< 10(2)-9.3 x 10(3) MPN/100 g) when 50 pieces of each meat was tested by the conventional MPN method . As shown in the above methods, the majority of cells were not enterotoxigenic cells in the population of C . perfringens . A small number of enterotoxigenic cells of C . perfringens co-existed with a large number of nonenterotoxigenic cells in the same meat sample. J Biol Chem, 1998 Sep 11, 273(37), 24266 - 71 Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase; Laufs U et al.; The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown . To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml) . Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively . Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects . Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression . Immunoblot analyses and {35S}GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP . Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression . In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression . These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity. Appl Environ Microbiol, 1998 Sep, 64(9), 3359 - 67 Characterization of a defined 2,3,5, 6-tetrachlorobiphenyl-ortho-dechlorinating microbial community by comparative sequence analysis of genes coding for 16S rRNA; Holoman TR et al.; Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3, 5,6-tetrachlorobiphenyl . In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp . was predominant in the absence of added PCB, but undescribed species in the delta subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB . Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures . Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community . Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected . Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener . Deletion of Clostridium spp . from the community profile by addition of vancomycin only slightly reduced dechlorination . However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria . With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the delta, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species. Appl Environ Microbiol, 1998 Sep, 64(9), 3195 - 201 Characterization of the bactericidal effects of sodium nitroprusside and other pentacyanonitrosyl complexes on the food spoilage bacterium Clostridium sporogenes; Joannou CL et al.; The potent bactericidal activity of sodium nitroprusside (SNP; Na2{Fe(CN)5(NO)}) towards Clostridium sporogenes has been investigated . SNP inhibited cell growth in the concentration range of 10 to 40 microM . Concentrations above 80 microM caused irreversible loss of cell viability and cell lysis . Inhibition of cell growth was similar in complex and in defined media . SNP was found to be unreactive towards individual components of the defined medium, with the exception of cysteine . The chemical characteristics responsible for the potency of SNP were investigated by synthesizing analogs of SNP in which the Fe was replaced by different metals . The inhibitory potency of the pentacyanonitrosyl complexes decreased in the order Fe > Cr > V, which correlates with N-O stretching frequency (vNO) . In contrast, the Ru complex which had a vNO comparable to that of Fe was a poor inhibitor . Electron paramagnetic resonance spectroscopy showed that SNP was rapidly reduced to the paramagnetic Fe(I) compound {Fe(CN)4(NO)}(2-) on contact with cells . Analysis of fractions from SNP-treated cells showed 90% oxidation of thiols in the cell walls compared with those in control cells . The toxicity of SNP involves S-nitrosation and reduction, the lack of toxicity of the Ru analog being consistent with the fact that it has poor reactivity towards thiols . When C . sporogenes cells were exposed to sublethal concentrations of SNP and viewed under the electron microscope, they showed blisters on the surface . These results point to the cell wall surface as a primary point of attack of the nitrosyl complex. Mol Biol Cell, 1998 Sep, 9(9), 2639 - 53 Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function; Vouret-Craviari V et al.; Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces . In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells . Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding . Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response . In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction . The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase . Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity . Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding . Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding . The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants . Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect . These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells. Am J Physiol, 1998 Sep, 275(3 Pt 1), G402 - 9 In vitro evidence that rabbit distal colonic muscularis mucosae has a Clostridium difficile toxin A receptor; Percy WH et al.; In the rabbit ileum Clostridium difficile toxin A causes inflammation and mucosal damage via a specific glycoprotein receptor that contains alpha-D-galactose . In rabbit colon toxin A also causes inflammation, and this is associated with increased myoelectric activity and eicosanoid production . The present in vitro study was undertaken to determine if a toxin A receptor on one or more layers of colonic smooth muscle could mediate the motor effects of this agent . Toxin A (20-100 microg/ml) was without effect on longitudinal and circular muscle but had two different effects on the muscularis mucosae . Initial exposure to the toxin caused increased numbers of spontaneous contractions and a small, atropine-, tetrodotoxin-, and indomethacin-resistant increase in resting tone . More importantly, however, 30-min exposure to toxin A resulted in attenuated muscularis mucosae responses to acetylcholine and K+ . Both the small excitatory and the larger inhibitory effects of toxin A were abolished by pretreatment with the lectin BS-1, which binds to toxin A receptors, but not by the nonreceptor-binding lectin DBA . These data strongly suggest that toxin A causes significant motor effects on the distal colonic muscularis mucosae via a receptor-mediated mechanism . These mechanical data were supported by the presence of histologically demonstrable toxin A and BS-1 binding sites on the muscularis mucosae but not on either the longitudinal or circular muscle layers, both of which were unresponsive to the toxin . By depressing muscularis mucosae function and, ultimately, mucosal movement as a result of toxin A production, C . difficile may promote its own proliferation, thus further contributing to the development of antibiotic-associated colitis. Endocrinology, 1998 Sep, 139(9), 3752 - 62 Prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by apoptosis; Li G et al.; Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs . The prolonged effect of such agents on insulin-secreting beta-cells (HIT-T15 and INS-1) was investigated . Both MPA and mizoribine inhibited mitogenesis, as reflected by {3H}thymidine incorporation . Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g . 1 microg/ml) of MPA for 1, 2, and 4 days, respectively . Mizoribine (48 h) similarly induced the death of HIT cells . INS-1 cells also were damaged by prolonged MPA treatment . MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp . DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 microg/ml MPA, respectively) . Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli . MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion . An inhibitor of protein isoprenylation (lovastatin, 10-100 microM for 2-3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins . Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin . These findings indicate that prolonged depletion of GTP induces beta-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins . Treatment of intact adult rat islets (the beta-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods . Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts. Nucleic Acids Res, 1998 Sep 15, 26(18), 4196 - 204 The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7; Burland V et al.; The complete DNA sequence of pO157, the large virulence plasmid of EHEC strain O157:H7 EDL 933, is presented . The 92 kb F-like plasmid is composed of segments of putative virulence genes in a framework of replication and maintenance regions, with seven insertion sequence elements, located mostly at the boundaries of the virulence segments . One hundred open reading frames (ORFs) were identified, of which 19 were previously sequenced potential virulence genes . Forty-two ORFs were sufficiently similar to known proteins for suggested functions to be assigned, and 22 had no convincing similarity with any known proteins . Of the newly identified genes, an unusually large ORF of 3169 amino acids has a putative cytotoxin active site shared with the large clostridial toxin (LCT) family and proteins such as ToxA and B of Clostridium difficile . A conserved motif was detected that links the large ORF and the LCT proteins with the OCH1 family of glycosyltransferases . In the complete sequence, the mosaic form can be observed at the levels of base composition, codon usage and gene organization . Insights were obtained from patterns of DNA composition as well as the pathogenic and 'housekeeping' gene segments . Evolutionary trees built from shared plasmid maintenance genes show that even these genes have heterogeneous origins. J Appl Microbiol, 1998 Mar, 84(3), 357 - 67 Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertium KT-5A, from lotus soil; Kataoka N et al.; Of 10 strains of mannanase-producing anaerobic bacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A, which was isolated from lotus soil, produced high amounts of extracellular beta-1,4-mannanase . The isolate was an aerotolerant anaerobe without quinon systems; the cell growth cultivated with no addition of reducing agents was also stable . High yields of mannanase were obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively . Fermentation end products on galactomannan fermentation were formate, acetate, lactate, butyrate, carbon dioxide and hydrogen . The extracellular mannanase displayed high activity on galactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spino gum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan . As far as is known, this is the first report on the isolation of an active mannanase-producing anaerobic bacterium from natural environments. J Chemother, 1998 Aug, 10(4), 280 - 4 In vitro activity of HMR 3647 against anaerobic bacteria; Edlund C et al.; The aim of the present investigation was to determine the in vitro activity of HMR 3647 compared with other antimicrobial agents against anaerobic bacteria . The activity of HMR 3647 was determined against 342 clinical isolates of anaerobic bacteria by the agar dilution method and was compared with azithromycin, clarithromycin, roxithromycin, erythromycin, cefoxitin, imipenem, clindamycin and metronidazole . Among the macrolides HMR 3647 and among the beta-lactams imipenem were the most active agents tested . Anaerobic cocci (50 strains) had the following minimum inhibitory concentrations (MICs): HMR 3647, range 0.016-0.125 mg/l; imipenem, range 0.016-0.064 mg/l . Propionibacterium acnes (30 strains): HMR 3647, 0.016-1.0 mg/l; imipenem, 0.032-0.064 mg/l . Clostridium perfringens (30 strains): HMR 3647, 0.125 mg/l; imipenem, 0.016-0.5 mg/l . Clostridium difficile (50 strains): HMR 3647, 0.125-256 mg/l; imipenem, 4.0-8.0 mg/l . Bacteroides fragilis (102 strains): HMR 3647, 0.032-16 mg/l; imipenem, 0.064-0.25 mg/l . Bacteroides and Prevotella species (50 strains): HMR 3647, 0.016-4.0 mg/l; imipenem, 0.016-0.25 mg/l . Fusobacterium nucleatum (30 strains): HMR 3647, 0.016-8.0 mg/l; imipenem, 0.008-0.064 mg/l . HMR 3647 may be useful as treatment and prophylaxis for infections due to anaerobic bacteria. J Med Chem, 1998 Aug 27, 41(18), 3450 - 60 Beta-amino-thiols inhibit the zinc metallopeptidase activity of tetanus toxin light chain; Martin L et al.; Tetanus neurotoxin is a 150-kDa protein produced by Clostridium tetani, which causes the lethal spastic paralytic syndromes of tetanus by blocking inhibitory neurotransmitter release at central synapses . The toxin light chain (50 kDa) has a zinc endopeptidase activity specific for synaptobrevin, an essential component of the neuroexocytosis apparatus . Previous unsuccessful attempts to block the proteolytic activity of this neurotoxin with well-known inhibitors of other zinc proteases led us to study the design of specific inhibitors as a possible drug therapy to prevent the progressive evolution of tetanus following infection . Starting from the synaptobrevin sequence at the level of the cleavage site by tetanus neurotoxin (Gln76-Phe77), a thiol analogue of glutamine demonstrated inhibitory activities in the millimolar range . A structure-activity relationship performed with this compound led us to determine the requirement for the correct positioning of the thiol group, the primary amino group, and a carboxamide or sulfonamide group on the side chain . This resulted in the design of a beta-amino-(4-sulfamoylphenyl)glycine-thiol, the first significantly efficient inhibitor of tetanus neurotoxin with a Ki value of 35 +/- 5 microM. J Protein Chem, 1998 Jul, 17(5), 417 - 28 Covalent structure of botulinum neurotoxin type B; location of sulfhydryl groups and disulfide bridge and identification of C-termini of light and heavy chains; Antharavally BS et al.; Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a approximately 150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues {Whelan et al . (1992), Appl . Environ . Microbiol . 58, 2345-2354} The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulfide bridge . The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a approximately 50-kDa light chain (Pro 1 through Lys 440) and a approximately 100-kDa heavy chain (Ala 441 through Glu 1290) . The limited in vivo nicking of the single-chain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440-Ala 441 bond appear to be due to the adjacent Ala 441-Pro 442 imide bond's probable cis configuration in a mixed population of molecules with cis and trans configurations . The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445 . Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form . In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT . A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysis; thus 29.7% of the protein's entire amino acid sequence predicted from the nucleotide sequence was confirmed . The 6 amino acids, residues 945-950, did not match with the sequence predicted in 1992, but did match with a later report of 1995 . The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp-Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without 14C label), separation and isolation of the fragments by SDS-PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions. J Lipid Res, 1998 Aug, 39(8), 1641 - 6 Synthesis and intestinal metabolism of ursodeoxycholic acid conjugate with an antiinflammatory agent, 5-aminosalicylic acid; Batta AK et al.; 5-Aminosalicylic acid conjugate of ursodeoxycholic acid was synthesized in above 90% yield by adding a basic solution of 5-aminosalicylic acid into the mixed anhydride formed with ursodeoxycholic acid and ethyl chloroformate . The 5-aminosalicylic acid conjugate of ursodeoxycholic acid was poorly secreted into the bile and was deconjugated with cholylglycine hydrolase and Clostridium perfringens, that deconjugate naturally occurring glycine and taurine conjugates of bile acids . However, ursodeoxycholic acid 5-aminosalicylic acid conjugate was not absorbed from the duodenum but was concentrated in the colon where it was partially hydrolyzed by the intestinal bacteria to ursodeoxycholic acid and 5-aminosalicylic acid . We believe that this unique conjugation of ursodeoxycholic acid with 5-aminosalicylic acid may facilitate the transport of both 5-aminosalicylic acid and ursodeoxycholic acid to the colon and may be useful for the treatment of colonic inflammatory bowel diseases, ulcerative colitis and Crohn's disease. Lett Appl Microbiol, 1998 Jun, 26(6), 442 - 6 The detection of a deletion in the type B neurotoxin gene of Clostridium botulinum A(B) strains by a two-step PCR; Franciosa G et al.; Differences between the type B neurotoxin gene sequence of Clostridium botulinum type A(B) and Cl . botulinum type B, including a six nucleotide deletion, were recently proposed as a cause of the lack of expression of this gene in the type A toxigenic strains . A polymerase chain reaction (PCR) based on two sets of primers was designed to investigate the absence of the 6-nucleotide sequence in the apparently unexpressed type B toxin gene of 42 strains of Cl . botulinum type A(B) . Thirty-five strains were shown to exhibit a deletion in their type B toxin gene; two strains did not have the deletion and actually produced small amounts of type B toxin when tested by the mouse bioassay . This two-step PCR might be useful for the rapid determination of the presence of the six nucleotide deletion and consequently, whether the type B toxin is likely to be produced. J Appl Microbiol, 1998 Jun, 84(6), 1156 - 62 Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes; LaMontagne MG et al.; The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments . Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated . Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium . The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture . Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration . Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs . Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species. Eur J Biochem, 1998 Jul 15, 255(2), 336 - 46 Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family; Yip KS et al.; The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion-pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme . In the light of this, we have carried out a homology-based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures . We have attempted to analyse the extent of the formation of ion-pair networks in these different enzymes and tried to correlate this with the observed thermal stability . The results of this analysis indicate that the ion-pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures. J Food Prot, 1998 Aug, 61(8), 1052 - 6 Microbiological quality of hot meals served by airlines; Hatakka M; The microbiological quality of 1,012 hot meals served on aircraft was studied in 1991 to 1994 . The material included dishes prepared in 33 countries all over the world . The geometric means of aerobic colony counts and Escherichia coli were significantly lower in breakfasts (P < 0.05) than in main dishes . Pathogenic bacteria were found in 30 samples (3.0%), Bacillus cereus being the most common pathogen . The frequencies of B . cereus and Staphylococcus aureus did not differ between breakfasts and main dishes . Many of the samples exceeded the minimum acceptable microbiological standards recommended by the Association of European Airlines (AEA) for E . coli, S . aureus, B . cereus, Clostridium perfringens, and Salmonella; i.e., 8.2%, 0.6%, 0.7%, 0.7% and 0.3%, respectively . There were considerable differences in aerobic colony counts and E . coli counts between countries where the food was prepared. J Food Prot, 1998 Aug, 61(8), 988 - 93 Investigation of the ability of proteolytic Clostridium botulinum to multiply and produce toxin in fresh Italian pasta; Del Torre M et al.; The ability of proteolytic Clostridium botulinum (types A, B, and F) to produce toxin in filled fresh Italian pasta (tortelli) packed under a modified atmosphere was investigated . Four types of tortelli (filled with artichoke, meat, ricotta-spinach, or salmon) were inoculated with a suspension of heat-shocked spores to give an initial concentration of approximately 10(3) spores per piece . Samples were incubated at both 12 and 20 degrees C for up to 50 days and examined at selected time intervals for the presence of toxin by an ELISA and the mouse test . Toxin was not detected in any tortelli stored at 12 degrees C . When storage was at 20 degrees C, toxin was detected in the salmon-filled tortelli at day 30, in the meat and ricotta-spinach tortelli at day 50, but not in the artichoke-filled tortelli at day 50. J Biol Chem, 1998 Aug 28, 273(35), 22554 - 62 cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha; Dong JM et al.; Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels . Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor . The protective effect of thrombin was shown to be Rho-dependent . Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels . A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP . RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA) . We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation . This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo . ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes . Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha . These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism. Infect Immun, 1998 Sep, 66(9), 4531 - 6 Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences; Billington SJ et al.; Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR . These genotype E isolates were demonstrated to express alpha and iota toxins, but, despite carrying sequences for the gene (cpe) encoding C . perfringens enterotoxin (CPE), were unable to express CPE . These silent cpe sequences were shown to be highly conserved among type E isolates . However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site . The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the iota toxin genes, on episomal DNA . These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries iota toxin genes, to several different type A C . perfringens isolates. Microb Pathog, 1998 Aug, 25(2), 91 - 9 Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins; Kozaki S et al.; Clostridium botulinum type B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site . Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor . BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant . The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane . A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes . In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons . These results suggest that GT1b functions as a component of the receptor complex. FEMS Microbiol Lett, 1998 Aug 1, 165(1), 193 - 200 Molecular analysis of the malR gene of Clostridium butyricum NCIMB 7423, a member of the LacI-GalR family of repressor proteins; Goda SK et al.; Deletion of a region of DNA 5' to a previously characterised malQ gene of Clostridium butyricum resulted in increased production of the enzyme activity encoded by malQ, 4-alpha-glucanotransferase . Nucleotide sequence analysis revealed the presence of an open reading frame capable of encoding a protein of 335 amino acids . This protein was found to share 33% amino acid sequence identity with the Bacillus subtilis CcpA (catabolite control protein) repressor, 28% identity with the Streptomyces coelicolor MalR repressor, and 30%, 25%, and 21% amino acid identity with the Escherichia coli repressors GalR, LacI and MalI, respectively . The amino-terminal domain was predicted to be able to form a helix-turn-helix structure, and shared highest similarity with the equivalent functional domain from the E . coli LacI repressor . Interruption of malR by the generation of a frameshift mutation led to a 10-fold increase in MalQ activity . These data suggest that the identified open reading frame encodes a repressor of the C . butyricum malQ gene, and of the adjacent malP gene . The gene has, therefore, been designated malR, and its encoded gene product MalR. Clin Infect Dis, 1998 Aug, 27 Suppl 1, S75 - 83 Enterococci and vancomycin resistance; French GL; The frequency of infections with multiply antibiotic-resistant gram-positive bacteria is increasing, and in some cases these organisms remain susceptible only to the glycopeptides vancomycin and teicoplanin . The appearance of transferable high-level glycopeptide resistance in enterococci--producing some strains that are now resistant to all available antibiotics--is thus a cause for concern . The enterococci readily colonize the bowel, spread rapidly among hospital patients, and transfer their antibiotic resistances widely among themselves and other gram-positive species . Glycopeptide resistance has not yet transferred in vivo to other significant pathogens, but experimental transfer to Staphylococcus aureus has been achieved in vitro . The emergence of glycopeptide-resistant enterococci has been encouraged by the increasing use of aminoglycosides, cephalosporins, and quinolones for the treatment of infections due to gram-negative bacteria and glycopeptides for infections due to staphylococci and clostridium difficile . In Europe this antibiotic pressure has been aggravated by the use of the glycopeptide avoparcin in animal feeds . The enterococci may now be poised to disseminate glycopeptide resistance among other more pathogenic gram-positive bacteria. J Food Prot, 1998 Jun, 61(6), 693 - 9 Evaluation of microbial hazards during processing of Spanish prepared Flamenquín; Cordoba MG et al.; Flamenquin is a traditional, prepared, frozen meat product from the south of Spain made with minced pork, chicken, and cooked ham . Since it is a prepared raw meat product some microbial hazards could be associated with the process of making it . Microbiological analyses have been performed throughout the various steps of processing over a 1-year period to evaluate microbial hazards in the commercial process . High levels of microorganisms were observed all through the processing of this product, the mincing and mixing steps being where major microbial contamination was observed . Pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Escherichia coli and Pseudomonas aeruginosa were detected during processing . Raw materials and food handlers were the principal sources of microbial contamination . A modification of processing to include a heating step after mincing and mixing and an improvement in hygiene practices could eliminate the microbial hazards . Both modifications should be noted for the implementation of a hazard analysis of critical control points (HACCP) program in commercial flamenquin processing. J Food Prot, 1998 Apr, 61(4), 450 - 7 Duplex (thermotroph-psychrotroph) quadrant plates: convenient, error-avoiding tools for monitoring of HACCP-contained food lines and for epidemiological investigations under conditions of military or other constraints; Mossel DA et al.; A set of two "two-tier" (thermotroph-psychrotroph) single quadrant plates (QPs) was developed previously to allow convenient enumeration of numbers of colony-forming units of most pertinent pathogenic bacteria or marker bacteria in foods . These include Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, thermotrophic and psychrotrophic Enterobacteriaceae, Clostridium spp., and Enterococcus spp . As the QPs had given excellent results in monitoring samples of marketed food products potentially involved in food-transmitted illnesses, the approach was tested for practicability under military deployment and other constraints . Three approaches were envisaged: (i) validating lapse-free adherence to meticulously codified good military catering practices; (ii) acceptance/rejection testing of locally procured foods or meals; and (iii) employing rapid culture in support of evidence obtained by microscopy in attempts to identify foods involved in infectious or toxic disease outbreaks occurring in the field . The method was found to be elegant, avoiding confusion when larger number of specimens were to be screened, as well as easy to teach to staff with little or no training in microbiology, and it provided entirely reliable results . For use outside the laboratory, preparation of food macerates by use of shake flasks containing glass or plastic beads and peptone saline as a substitute for stomaching was found acceptable, though the shake flask technique led to slightly diminished colony counts . Results obtained with incubation times shortened to ca . 12 h could be relied on only when the results were alarmingly positive, but not when the colony counts at the 12-h point did not yet indicate a reason for concern. J Food Prot, 1998 Mar, 61(3), 324 - 8 Growth and toxin production by Clostridium botulinum on inoculated fresh-cut packaged vegetables; Austin JW et al.; To determine the safety of fresh-cut vegetables packaged in modified atmosphere, challenge studies using both nonproteolytic and proteolytic strains of Clostridium botulinum were performed with a variety of fresh-cut packaged salads and vegetables stored at different temperatures . When vegetables were inoculated with spores of C . botulinum and incubated in low-O2 atmospheres, spore germination and growth and toxin production were observed . Botulinum toxin was produced by proteolytic types A and B on onion, butternut squash, rutabaga, salad, and stir-fry vegetables . Nonproteolytic C . botulinum produced toxin on butternut squash and salad . Nonproteolytic C . botulinum was capable of producing neurotoxin at temperatures as low as 5 degree C whereas proteolytic strains produced neurotoxin at 15 degrees C and higher . Although most samples were visibly spoiled before detection of botulinum toxin, samples of butternut squash and onion remained acceptable after detection of toxin . The strict maintenance of low temperatures (< 5 degrees C) is recommended in order to control the potential growth of C . botulinum on fresh-cut vegetables packaged in a modified atmosphere. J Food Prot, 1998 Mar, 61(3), 318 - 23 The incidence of Listeria spp., Salmonella spp., and Clostridium botulinum in smoked fish and shellfish; Heinitz ML et al.; The frequency of occurrence of Listeria spp., Salmonella spp., and Clostridium botulinum is samples of smoked finfish and smoked shellfish was analyzed over a 5-year period . Listeria monocytogenes were isolated from 14% of 1,080 samples . For those samples where the smoke process was known, the incidence of L . monocytogenes was higher in cold-smoked than hot-smoked products (51 of 240 cold-smoked compared to 19 of 215 hot-smoked products) . Listeria species other than L . monocytogenes were also detected (in 7.2% of cold-smoked and 3.8% of hot-smoked products) . The time and temperature smoke processing guidelines are reviewed for a few state authorities . L . monocytogenes was isolated from 15.2% of the 559 samples of foreign origin . There were four countries for which more than 70 samples were analyzed: Canada, Norway, the Philippines, and the United Kingdom . The occurrence of L . monocytogenes in samples from these four countries was 14.3%, 23.7%, 0%, and 16.1%, respectively . The 521 samples originating in the United States were processed by 194 plants . Thirty-seven plants in 13 states produced contaminated product . Salmonella species were isolated from 5 (3.2%) of 156 samples tested for this organism . All positive samples were of foreign origin (4 from the Philippines and 1 from the United Kingdom) . No C . botulinum spores were detected in any of the 201 vacuum-packed samples tested for this organism. J Food Prot, 1998 Feb, 61(2), 240 - 3 Prevalence of the enterotoxin gene and clonality of Clostridium perfringens strains associated with food-poisoning outbreaks; Ridell J et al.; The prevalence of the enterotoxin gene in a well-characterized collection of 71 Clostridium perfringens strains from 36 separate food-poisoning cases or outbreaks was analyzed with the polymerase chain reaction (PCR) . The clonality of 39 strains originating from 14 outbreaks where at least two isolates were available was studied with pulsed-field gel electrophoresis (PFGE) using SmaI and ApaI restriction endonucleases . The cpe gene PCR assay was found to correlate well with Clostridium perfringens enterotoxin (CPE) production in vitro with reverse passive latex agglutination . Of the C . perfringens food and clinical food-poisoning isolates 24 (86%) and 38 (88%) were cpe-positive, respectively . Different PFGE patterns indicated that multiple cpe-positive clones are frequently present within one outbreak . The existence of cpe-positive and negative isolates with identical or nearly identical PFGE patterns in a single outbreak suggests that the cpe gene may be in a movable genetic element. J Food Prot, 1998 Feb, 61(2), 201 - 4 Detection of enterotoxigenic Clostridium perfringens in spices used in Mexico by dot blotting using a DNA probe; Rodriguez-Romo LA et al.; Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease . In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C . perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin degoxigenin-labeled DNA probe . C . perfringens counts varied from <100 to 433 CFU/g in garlic powder, from <100 to 200 CFU/g in black pepper, from <100 to 433 CFU/g in cumin seed, from <100 to 340 CFU/g in oregano, and from < 100 to 450 CFU/g in bay leaves . The dot-blot technique detected the enterotoxin gene in 8 (4.25%) of 188 confirmed isolates of C . perfringens . dot-blot. J Food Prot, 1998 Jan, 61(1), 126 - 8 Growth and toxin production by Clostridium botulinum on sliced raw potatoes in a modified atmosphere with and without sulfite; Solomon HM et al.; The ability of Clostridium botulinum type A or B spores to grow and produce toxin on fresh raw potatoes in a modified atmosphere with or without sulfite was investigated at 22 degrees C . Fresh, peeled, sliced potatoes, untreated or dipped for 2 min into 0.7% sulfite solution and drained, were surface-inoculated at several concentration levels with a mixture of C . botulinum spores, either type A or B . They were placed in a modified atmosphere (30% N/70% CO2) within oxygen-impermeable bags (200 g/bag) and incubated at room temperature (22 degrees C) . Toxicity was tested on days 0, 3, 4, 5, 6, and 7 . After incubation, the potatoes were blended and centrifuged, and the Millipore-filtered supernatant fluid was injected intraperitoneally into mice . Sensory evaluation, except taste, was also performed . Potatoes inoculated with C . botulinum type A spores but untreated with NaHSO3 became toxic in 4 to 5 days, which coincided with the sensory evaluation "unfit for human consumption" . Potatoes treated with NaHSO3 regardless of inoculum size or residual SO2 levels appeared acceptable for human consumption through day 7, even though they were toxic after 4 days of incubation . Although toxicity from type B spores occurred later and in fewer test samples than toxicity from type A, some potatoes again appeared acceptable but were toxic . Thus, although NaHSO3 markedly extended the consumer acceptability of peeled, sliced, raw potatoes at the abuse temperature, it did not inhibit outgrowth and toxin production by C . botulinum under these conditions. Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 10218 - 23 Propagation by sporulation in the guinea pig symbiont Metabacterium polyspora; Angert ER et al.; The Gram-positive bacterium Metabacterium polyspora is an uncultivated symbiont of the guinea pig gastrointestinal tract . Here we present evidence that in M . polyspora vegetative cell division has taken on a minor, and apparently dispensable, role in propagation . Instead, this unusual bacterium has evolved the capacity to produce progeny in the form of multiple endospores . Endospore formation is coordinated with transit of the bacterium through the gastrointestinal tract of the guinea pig . For the majority of cells, sporulation is initiated in the ileum, whereas later stages of development take place in the cecum . We show that multiple endospores are generated both by asymmetric division at both poles of the cell and by symmetric division of the endospores at an early stage of their development . Our findings suggest that M . polyspora represents an intermediate step in the evolution of a novel mode of cellular propagation that originates with endospore-forming Bacillus and Clostridium spp., which reproduce by binary fission, and extends to Epulopiscium spp., which create multiple viviparous offspring by a process of internal reproduction. Acta Trop, 1998 Jun 15, 70(1), 87 - 99 Trypanosoma rangeli sialidase: kinetics of release and antigenic characterization; Saldana A et al.; The epimastigote stage of Trypanosoma rangeli release a sialidase with a high sialic acid hydrolysis capacity . We demonstrate that sialidase secretion is an active process that is reduced at low temperatures and in the presence of sodium azide . The enzyme is continuously released until certain maximally active concentrations are attained in the BHI culture medium when the parasite density reaches 2-3 x 10(6) cells . When introduced into culture medium already containing such enzyme levels, freshly harvested parasites do not secrete additional sialidase . These findings suggest a self-regulating mechanism and a biological role for the secreted T . rangeli sialidase . The secreted enzyme was purified to homogeneity by fractionation with ammonium sulphate and affinity chromatography . Antibodies raised against the purified molecule recognized antigens of similar molecular weights (73 kDa) in western immunoblotting analyses of T . rangeli and T . cruzi whole cell lysates . No antigenic recognition was recorded against T . cruzi active sialidase/trans-sialidase polypeptides or Clostridium perfringens and Vibrio cholerae commercial sialidases . These observations may indicate the expression of different antigenic domains in T . rangeli, T . cruzi and bacterial sialidases. Int J Food Microbiol, 1998 Jun 16, 41(3), 239 - 41 A survey of human serum samples for antibody against Clostridium perfringens type A enterotoxin in humans in Korea; Kim Y et al.; Serum samples from humans in Chongju City, Korea, were tested for the presence of Clostridium perfringens enterotoxin antibody by enzyme linked immunosorbent assay (ELISA) . About half of the 1054 samples tested had high ELISA readings, which could be the result of exposure to enterotoxin produced by C . perfringens . The high prevalence of the antibody among the Korean population suggests that foodborne illnesses caused by C . perfringens may be common in Korea even though the occurrence has not been monitored. J Clin Microbiol, 1998 Sep, 36(9), 2571 - 4 Prevalence of astroviruses in a children's hospital; Shastri S et al.; An enzyme immunoassay for astrovirus was used to screen 357 stool samples from 267 symptomatic inpatients at a tertiary-care children's hospital . Thirty stool samples from 26 patients contained astrovirus antigen, while rotavirus was found in 34 samples and Clostridium difficile toxin was found in 40 . Half of the astrovirus infections were nosocomial . Additional pathogens were identified in six of the astrovirus antigen-positive stool samples . Most (80%) of the astroviruses recovered were of serotype 1 . Astrovirus infections were significantly more common than rotavirus or C . difficile infections in very young infants and in those with surgical short-bowel syndrome. J Biol Chem, 1998 Aug 21, 273(34), 21950 - 7 Binding and transcytosis of botulinum neurotoxin by polarized human colon carcinoma cells; Maksymowych AB et al.; T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin . Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells . Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction . Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxic in vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations . Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8-10% of serotype A) . MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin . Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 microM brefeldin A, decreased 42% when microtubules were disrupted by 10 microM nocodazole, and decreased 74% at 18 degreesC . Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%) . These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not. J Biol Chem, 1998 Aug 21, 273(34), 21867 - 74 Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells; Essler M et al.; The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated . The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction . Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction . These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells . In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation . C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction . These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells. Plant Physiol, 1998 Aug, 117(4), 1185 - 94 Bacterial cellulose-binding domain modulates in vitro elongation of different plant cells Shpigel E, Roiz L, Goren R, Shoseyov O. Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro . In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 &mgr;g mL-1 CBD . Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes . At low concentrations CBD enhanced elongation of Arabidopsis roots . At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner . Maximum effect on root hair elongation was at 100 &mgr;g mL-1, whereas root elongation was inhibited at that concentration . CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan . When Acetobacter xylinum L . was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control . Electron microscopy examination of the cellulose ribbons produced by A . xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. Infect Control Hosp Epidemiol, 1998 Jul, 19(7), 494 - 9 A randomized crossover study of disposable thermometers for prevention of Clostridium difficile and other nosocomial infections; Jernigan JA et al.; OBJECTIVE: To test the hypothesis that use of disposable thermometers would result in lower rates of nosocomial Clostridium difficile diarrhea and of total nosocomial infections, compared with electronic thermometers . DESIGN: Prospective randomized crossover trial . SETTING: A 700-bed university hospital providing primary and tertiary care . PATIENTS: All patients admitted to a group of 20 inpatient nursing units . INTERVENTIONS: 20 nursing units were randomized into two groups . One group randomly was assigned exclusive use of single-use disposable thermometers for patient temperature measurement, and the other group was assigned exclusive use of electronic thermometers . After 6 months, the assignments were reversed . MAIN OUTCOME MEASURES: Rates of C difficile infections, total nosocomial diarrheal episodes, and total nosocomial infections were prospectively followed in each study unit over 11 months . RESULTS: 26,350 patients were admitted to the study units and hospitalized for 120,529 patient days . There were 947 nosocomial infections (7.86 per 1,000 patient days) . Nosocomial C difficile-associated diarrhea defined by positivity to both toxin B (titer > or = 1:10) and toxin A was detected in 32 patients (3.4% of all nosocomial infections) . A significantly lower rate of nosocomial C difficile-associated diarrhea was observed with disposable thermometer use (0.16 per 1,000 patient days) compared with electronic thermometer use (0.37 per 1,000 patient days, relative risk {RR} = 0.44; 95% confidence interval {CI95}, 0.21-0.93, P = .026) . There was no difference in overall rates of nosocomial infection between the disposable and electronic groups (8.03 and 7.68 infections per 1,000 patient days, respectively; RR, 1.04; CI95, 0.92-1.19; P = .52) or in the overall rate of nosocomial diarrhea (3.34 and 3.40 per 1,000 patient days, respectively; RR, .98; CI95, 0.81-1.19; P = .87) . CONCLUSIONS: The incidence of nosocomial C difficile diarrhea was reduced significantly by using single-use, disposable thermometers as compared with electronic thermometers, but there was no effect on either the overall rate of nosocomial diarrhea or the rate of total nosocomial infections. Nat Struct Biol, 1998 Aug, 5(8), 738 - 46 Structure of the key toxin in gas gangrene; Naylor CE et al.; Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals . The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium . The crystal structure of the enzyme reveals a two-domain protein . The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC) . The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains . We believe this is the first example of such a domain in prokaryotes . This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin . Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C. Nat Struct Biol, 1998 Aug, 5(8), 659 - 62 Structure of the gangrene alpha-toxin: the beauty in the beast; Derewenda ZS et al.; The crystal and molecular structure of the Clostridium perfringens alpha-toxin crowns over a century-long research into the mechanisms of pathogenesis of gas gangrene . The structure reveals a two-domain enzyme, with a catalytic all-helical N-terminal domain, and a C-terminal domain similar in its jelly-roll topology to those found in pancreatic lipase and lipoxygenases. J Hosp Infect, 1998 Jul, 39(3), 189 - 93 Antimicrobial associations of an outbreak of diarrhoea due to Clostridium difficile; Zadik PM et al.; An increased incidence of diarrhoea due to Clostridium difficile (CDD) at the Northern General Hospital, Sheffield, prompted an investigation into antibiotic use on the renal, medical and geriatric wards, those mostly affected . For the first half of 1997 affected patients on these wards were identified and data collected as to which antimicrobials they had taken between admission and diagnosis . Rates were then calculated of the number of affected patients on a drug over the quantity prescribed (N/1000 x defined daily doses) . These were expressed as rate ratios (RR) over the rate of ampicillin/amoxycillin . A corrective procedure was also applied to the RR in an attempt to compensate for bias from the effect of drug combinations . Additionally, quarterly data were collected of numbers of patients with CDD and the amount of cefotaxime, ceftriaxone and cefuroxime issued between January 1996 and September 1997 . Those drugs with the greatest association with CDD were cefotaxime (RR = 27.5, P < 0.000001), ceftriaxone (15.1, 0.00004), cefuroxime (8.6, < 0.000001) and ceftazidime (6.4, 0.00008) . Several other drugs had increased uncorrected RR (P < 0.05) . However, after correction for drug combinations, only the above four drugs remained with significantly increased RRs of 16.9, 8.6, 5.3 and 4.8, respectively . The time course of the outbreak showed a correlation with the use of cefotaxime and ceftriaxone . The incidence peaked in the first months of 1997, following greatest use of cefotaxime in the last quarter of 1996 and of ceftriaxone in the first quarter of 1997 . This study confirms the association of cephalosporin and particularly cefotaxime usage with CDD incidence and shows the need to review the use of these drugs, especially in the treatment of respiratory infection . The method is useful for looking at local effects of prescribing with a view to controlling outbreaks of antibiotic resistant bacteria. J Hosp Infect, 1998 Jul, 39(3), 181 - 7 Incidence and impact of Clostridium difficile infection in the UK, 1993-1996; Wilcox MH et al.; Questionnaires were sent to 360 UK medical microbiologists to determine the incidence of Clostridium difficile infection in the UK between 1993-1996, and to establish the current laboratory testing protocols . Replies were received from 104 laboratories (29% response rate), 86, 7, 4 and 3% of which are in England, Scotland, Wales and Northern Ireland, respectively . The laboratories serve a total of approximately 90,000 hospital beds (median 750) . C . difficile testing was performed by 83% of the laboratories, and 52, 45 and 31% used toxin A +/- B kits, cell cytotoxicity, and culture +/- isolate toxigenicity testing methods, respectively . Forty-seven percent of laboratories only performed testing when specifically requested, 19% if antibiotic use was stated, 15% tested all diarrhoeal specimens, and 14% examined all specimens except those from infants and community patients . The annual totals of positive C . difficile reports and cases increased from 3132 to 12,775, and from 1576 to 8211, respectively, between 1993-1996 . In 1993 C . difficile infection caused ward closures in 5% of hospitals but 16% in 1996 . Antibiotic policy changes, due to C . difficile infection, occurred in 21% of hospitals in 1996 compared with only 4% in 1993 . C . difficile infection appears to be increasing markedly in the UK with major implications on hospital inpatient activity . Wide variations in laboratory selection and testing methods are likely to be masking the true epidemiology of C . difficile infection, and consensus is required on optimal protocols. J Bacteriol, 1998 Aug, 180(16), 4303 - 8 Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome; Kakiuchi M et al.; The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced . CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains . The molecular architecture of C . josui CipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticum CipC, but C . josui CipA is considerably smaller than the other scaffolding proteins . CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain . N-terminal amino acid sequence analysis of the C . josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome. Ann Intern Med, 1998 Aug 1, 129(3), 221 - 8 Botulism in the United States: a clinical and epidemiologic review; Shapiro RL et al.; Botulism is caused by a neurotoxin produced from the anaerobic, spore-forming bacterium Clostridium botulinum . Botulism in humans is usually caused by toxin types A, B, and E . Since 1973, a median of 24 cases of foodborne botulism, 3 cases of wound botulism, and 71 cases of infant botulism have been reported annually to the Centers for Disease Control and Prevention (CDC) . New vehicles for transmission have emerged in recent decades, and wound botulism associated with black tar heroin has increased dramatically since 1994 . Recently, the potential terrorist use of botulinum toxin has become an important concern . Botulism is characterized by symmetric, descending, flaccid paralysis of motor and autonomic nerves, usually beginning with the cranial nerves . Blurred vision, dysphagia, and dysarthria are common initial complaints . The diagnosis of botulism is based on compatible clinical findings; history of exposure to suspect foods; and supportive ancillary testing to rule out other causes of neurologic dysfunction that mimic botulism, such as stroke, the Guillain-Barre syndrome, and myasthenia gravis . Laboratory confirmation of suspected cases is performed at the CDC and some state laboratories . Treatment includes supportive care and trivalent equine antitoxin, which reduces mortality if administered early . The CDC releases botulism antitoxin through an emergency distribution system . Although rare, botulism outbreaks are a public health emergency that require rapid recognition to prevent additional cases and to effectively treat patients . Because clinicians are the first to treat patients in any type of botulism outbreak, they must know how to recognize, diagnose, and treat this rare but potentially lethal disease. Nurse Pract, 1998 Jul, 23(7), 25 - 6, 29-30, 39-43; quiz 44-5 Clostridium difficile and older adults: what primary care providers should know; Melillo KD; Clostridium difficile poses particular risk for older adults, who are subject to more serious symptoms than younger patients . Antibiotic exposure and other risk factors are associated with the pathogenesis of C . difficile-associated disease . Treatment goals include prescribing anti-C . difficile activity agents (when indicated), attending to volume status and prescribing oral rehydration therapy as needed, avoiding the use of antiperistaltic drugs, discontinuing any offending antibiotics, avoiding the indiscriminate use of broad-spectrum antibiotics, providing supportive therapy, following infection control protocols, and eliminating environmental contaminants. Epidemiol Infect, 1998 Jun, 120(3), 245 - 50 Prevalence of Clostridium botulinum type E in Finnish fish and fishery products; Hyytia E et al.; The prevalence of Clostridium botulinum type E gene in fish and fishery products of commercial importance in Finland was determined using a quantitative PCR analysis . The contamination level in 438 raw fish samples from intestines, surface and whole fish and 208 fish roe samples varied from 10-40% and from 4-14% respectively, depending on the fish species studied . The presence of C . botulinum in European wild freshwater fish and roe was demonstrated for the first time by isolation of the organism from PCR-positive samples . Five percent of 214 vacuum-packed and 3% of 123 air-packed fishery product samples examined at retail level were positive for the botulinum neurotoxin type E gene . A contamination level of 10% in vacuum-packed hot-smoked whitefish was detected . The results demonstrate that C . botulinum type E poses a serious health risk for those consuming fishery products from the Baltic Sea area. Vet Res, 1998 May-Aug, 29(3-4), 219 - 32 Clostridial diseases of small ruminants; Songer JG; Members of the genus Clostridium are extraordinarily diverse in their natural habitats, and, when introduced to animal hosts, a few produce acute and often fatal disease . In sheep and goats, as in many other species of domestic animals, pathogenesis is often mediated by one or more of the many toxic proteins produced by these organisms . Prevention and control strategies are frequently based upon amelioration, by immunoprophylaxis, of the effects of these molecules . In spite of their recognition for many years, clostridial diseases still present challenges to veterinary practitioners, diagnosticians and animal producers worldwide. Curr Microbiol, 1998 Sep, 37(3), 166 - 71 Effect of yeast extract on growth and metabolism of H2-utilizing acetogenic bacteria from the human colon; Leclerc M et al.; The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon . Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1-2 g . L-1 in the medium did not enhance utilization of H2/CO2 . Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain . Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain . In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate . The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria . Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species. Hereditas, 1998, 128(2), 173 - 8 Nitrogen-fixing aerobic bacteria have higher genomic GC content than non-fixing species within the same genus; McEwan CE et al.; The genomic GC contents of both nitrogen-fixing and non-fixing members of eight genera of bacteria are investigated . Analysis by t-tests showed that in the two aerobic general investigated (Aquaspirillum and Vibrio) there is a significantly higher GC content in the nitrogen-fixing members of the genus than in those unable to fix nitrogen, whilst in aerobic genera there is either no GC bias, or in the case of two genera (Rhodospirillum and Clostridium) there is a significantly higher GC content in the non-fixing organisms . This suggests that, in many genera, directional mutational pressures are different in nitrogen-fixing and non-fixing organisms . These results are discussed in the light of known mechanisms of mutation pressure and their relation to environmental variables. Jpn J Cancer Res, 1998 May, 89(5), 571 - 7 Intracellular Signal-transducing elements involved in transendothelial migration of lymphoma cells; Tsuzuki S et al.; To investigate the molecular mechanisms underlying transendothelial migration of tumor cells, an essential process for their hematogenous dissemination, we developed an in vitro model system that allows the separate monitoring of cell adhesion and transmigration processes . This system uses a human pre-B lymphoma cell line, Nalm-6, and a cultured mouse endothelial cell line, KOP2.16 . Nalm-6 cells rapidly adhered to KOP2.16 and subsequently transmigrated underneath them . Using this model, we examined the effects on transendothelial migration, of various reagents which specifically interfere with the function of intracellular signal transduction molecules . Treatment of Nalm-6 cells with wortmannin (WMN), herbimycin A, pertussis toxin, or C3 exoenzyme of Clostridium botulinum, which specifically inhibit P13 kinase and/or myosin light chain kinase, herbimycin-sensitive tyrosine kinases, heterotrimeric G proteins, and the small G proteins, and the small G proteins rho/rac, respectively, reduced transmigration in a dose-dependent manner, Pretreatment of KOP2.16 endothelial cells with WMN also reduced transmigration in a dose-dependent manner . Binding of Nalm-6 binding to KOp2.16 was not affected, even when Nalm-6 or KOP2.16 cells were pretreated with these inhibitors, indicating that the reduction of transmigration was not due to a reduction of Nalm-6 to KOP2.16 . These results also indicate that the signal transduction pathway(s) involved in transmigration can be dissociated from that of adhesion . Our results support the notion that endothelial cells are not a passive barrier in lymphoma extravasation, but that they assist lymphoma cell extravasation. Appl Microbiol Biotechnol, 1998 Jun, 49(6), 669 - 76 Development of markers for product formation and cell cycle in batch cultivation of Clostridium acetobutylicum ATCC 824 Schuster KC, van den Heuvel R, Gutierrez NA, Maddox IS. Experiments were performed to identify relationships between morphological and physiological events during batch fermentation of Clostridium acetobutylicum ATCC 824 . Differing fermentation conditions were obtained by manipulation of the culture pH value during the process . The bacterium showed marked changes in morphology during its cultivation, similar to those previously observed for other strains . However, although the acidogenic phase was characterized by the presence of rod-shaped cells, and the solventogenic phase by clostridial forms, there was no simple relationship between the proportion of clostridial forms present and the ratio of solvents to acids . Nevertheless, the shift from acidogenesis to solventogenesis was always coincident with the presence of some clostridial forms and with the accumulation of granulose within the cells . In addition, the "solvent shift" was associated with major changes in the cellular protein pattern, as analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis . Hence, the potential solventogenic ability of any particular culture may be recognised by its morphological appearance and/or its cellular protein pattern. Appl Microbiol Biotechnol, 1998 Jun, 49(6), 639 - 48 New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation Durre P. Clostridial acetone/butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known . Its decline since about 1950 has been caused by increasing substrate costs and the availability of much cheaper feedstocks for chemical solvent synthesis by the petrochemical industry . The so-called oil crisis in 1973 led to renewed interest in novel fermentation and product recovery technologies as well as in the metabolism and genetics of the bacterial species involved . As a consequence, almost all of the enzymes leading to solvent formation are known, their genes have been sequenced (in fact, Clostridium acetobutylicum has been recently included in the microbial genome sequencing project), the regulatory mechanisms controlling solventogenesis have begun to emerge and recombinant DNA techniques have been developed for these clostridia to construct specific production strains . In parallel, cheap agricultural-waste-based feedstocks have been exploited for their potential as novel substrates, continuous culture methods have been successfully established and new on-line product recovery technologies are now available, such as gas tripping, liquid/liquid extraction, and membrane-based methods . In combination with these achievements, a reintroduction of acetone/butanol fermentation on an industrial scale seems to be economically feasible, a view that is supported by a new pilot plant in Austria recently coming into operation. Postgrad Med J, 1998 Apr, 74(870), 216 - 9 The role of surgery in pseudomembranous enterocolitis; Viswanath YK et al.; Pseudomembranous enterocolitis is an inflammatory bowel disorder caused by Clostridium difficile toxins . Classical presentation includes abdominal pain, pyrexia, diarrhoea and leucocytes . The management is mainly conservative but in extreme cases surgery is necessary . Resectional procedures (colectomy) carry a better prognosis than diversion procedures (colostomy) . A careful history, a high index of suspicion, and early diagnosis and treatment would reduce the associated morbidity and mortality of this condition . The aetiopathogenesis, pathology, clinical presentation, diagnosis, differential diagnosis, complications, medical and surgical management are reviewed, and three case reports briefly discussed . A management algorithm is also suggested. FEMS Microbiol Lett, 1998 Jul 15, 164(2), 337 - 43 Nucleotide sequence of arfB of Clostridium stercorarium, and prediction of catalytic residues of alpha-L-arabinofuranosidases based on local similarity with several families of glycosyl hydrolases; Zverlov VV et al.; The nucleotide sequence of the alpha-L-arabinofuranosidase gene arfB from Clostridium stercorarium was determined . The deduced protein has a molecular mass of 56.2 kDa with an amino terminus identical to the N-terminal sequence of the purified mature enzyme from C . stercorarium . Its sequence is homologous to arabinofuranosidases of glycosyl hydrolase family 51 . Sequence alignment and cluster analysis reveal three new members of glycosyl hydrolase family 51, allowing for the definition of highly conserved regions . Two of these regions are remarkably similar to the most conserved regions within several other families of glycosyl hydrolases, which have in common a (beta/alpha)8-barrel as the core super-secondary structure, and allow to predict the acid/base catalyst and the nucleophile of the active site. Vet Rec, 1998 Jun 27, 142(26), 722 - 5 Protection of goats against experimental enterotoxaemia by vaccination with Clostridium perfringens type D epsilon toxoid; Uzal FA et al.; Enterotoxaemia in goats is mainly characterized by enterocolitis, and it has been suggested that the poor efficacy of commercial vaccines in preventing the disease is due to the local action of Clostridium perfringens toxin/s within the intestine, where circulating antibodies might not exert their action . Five goat kids were vaccinated with an incomplete Freund's adjuvant C perfringens type D epsilon toxoid vaccine on three occasions at three-week intervals, four similar kids were vaccinated with a commercial enterotoxaemia vaccine at the same times, and five other unvaccinated kids were used as controls . All the animals were challenged intraduodenally, one week after the last vaccination, with C perfringens type D filtered culture supernatant . At the time of challenge, the level of epsilon toxin antibodies in the serum of the Freund's adjuvant-vaccinated kids ranged between 2.45 and 230 iu/ml, while the kids that received the commercial vaccine had levels between 0.22 and 1.52 iu/ml . No clinical or postmortem changes were observed in the kids that received the Freund's adjuvant-vaccine . Three of the four kids that received the commercial vaccine developed mild, pasty diarrhoea, with a slight reddening of the colonic mucosa being observed postmortem . All the unvaccinated kids developed severe diarrhoea, respiratory distress and central nervous system signs, and were killed humanely between six and 24 hours after challenge . The postmortem changes consisted of pseudomembranous colitis, lung oedema and perivascular oedema of the brain . Moderate to high serum levels of anti-epsilon antibody appeared to protect the goats against both the systemic and the intestinal effects of C perfringens type D toxins. Vaccine, 1998 May-Jun, 16(9-10), 997 - 1003 Measurement of Clostridium perfringens beta-toxin production by surface plasmon resonance immunoassay; Hsieh HV et al.; A rapid and sensitive assay using surface plasmon resonance (SPR) immunoassay has been developed for the detection of Clostridium perfringens beta-toxin . The SPR immunoassay was conducted off-line by passing fermentation broth by a sensor chip coated with a monoclonal antibody specific for C . perfringens beta-toxin . Quantitation of toxin using SPR immunoassay was achieved by mass transport analysis; results were obtained within 20 min . The SPR immunoassay was compared with an ELISA and the traditional bioassay for C . perfringens beta-toxin . The SPR immunoassay and ELISA detected at least twofold differences in toxin levels at 95% confidence over a broad range of toxin concentrations . The traditional bioassay did not produce the resolution observed with the immunoassays . The SPR immunoassay allows for real-time monitoring of beta-toxin accumulation during production and permits the bioengineer to harvest C . perfringens fermentations when toxin is most concentrated . The SPR methodology may be applied to other fermentations to enhance and optimize toxin yields. J Am Podiatr Med Assoc, 1998 Jul, 88(7), 349 - 52 The risk of tetanus in podiatric medicine; Pouillon JM et al.; Although tetanus is a preventable disease, several cases are reported to the Centers for Disease Control and Prevention each year . Many conditions treated by podiatric physicians carry the risk of infection by Clostridium tetani, and it is advisable for podiatrists to update a patient's tetanus immunization status if the patient presents with a tetanus-prone wound. Biochem Mol Biol Int, 1998 Jun, 45(2), 401 - 7 Cloning and characterization of the upstream region of Clostridium botulinum type B neurotoxin gene; Yang GH et al.; The upstream region of the gene coding for Clostridium botulinum type B (BoNT/B) neurotoxin was cloned and sequenced . There were two open reading frames, which were identified as a nontoxic-nonhemagglutinin component (ntnh/B) and a 22 kDa adjacent open reading frame (orf22/B) . Deduced primary structure of ntnh/B showed that it was composed of 1,197 amino acid residues . Pairwise comparisons of the ntnh/B component with other botulinum toxin types showed high degree of homology to ntnh/A (82% identity) . Northern blot analysis revealed that toxin gene could be transcribed alone or co-transcribed with the ntnh gene . The orf22/B gene encoding for 178 amino acids (M.W . 21.6 kDa) was located between the 33 kDa hemagglutinin gene and the ntnh gene . Orf22/B also showed high degree of homology to orf22/A (98.9% identity) . These results suggested that the upstream region of the BoNT/B gene (containing the ntnh/B and orf22/B genes) might be evolutionarily closely related to the counterparts of the BoNT/A. J Biol Chem, 1998 Jul 31, 273(31), 19566 - 72 A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins; Busch C et al.; A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro . Here we identified several amino acid residues in C . sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment . Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity . No enzyme activity was detected with the double mutant D286A/D288A . Whereas the wild-type fragment of C . sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling . At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold . The exchange of Asp270 and Arg273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity . The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose. J Mol Biol, 1998 Jul 24, 280(4), 669 - 85 Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum; Schlessman JL et al.; The nitrogenase iron (Fe) protein performs multiple functions during biological nitrogen fixation, including mediating the mechanistically essential coupling between ATP hydrolysis and electron transfer to the nitrogenase molybdenum iron (MoFe) protein during substrate reduction, and participating in the biosynthesis and insertion of the FeMo-cofactor into the MoFe-protein . To establish a structural framework for addressing the diverse functions of Fe-protein, crystal structures of the Fe-proteins from Azotobacter vinelandii and Clostridium pasteurianum have been determined at resolutions of 2.2 A and 1.93 A, respectively . These two Fe-proteins are among the more diverse in terms of amino acid sequence and biochemical properties . As described initially for the A . vinelandii Fe-protein in a different crystal form at 2.9 A resolution, each subunit of the dimeric Fe-protein adopts a polypeptide fold related to other mononucleotide-binding proteins such as G-proteins, with the two subunits bridged by a 4Fe:4S cluster . The overall similarities in the subunit fold and dimer arrangement observed in the structures of the A . vinelandii and C . pasteurianum Fe-proteins indicate that they are representative of the conformation of free Fe-protein that is not in complex with nucleotide or the MoFe-protein . Residues in the cluster and nucleotide-binding sites are linked by a network of conserved hydrogen bonds, salt-bridges and water molecules that may conformationally couple these regions . Significant variability is observed in localized regions, especially near the 4Fe:4S cluster and the MoFe-protein binding surface, that change conformation upon formation of the ADP.AlF4- stabilized complex with the MoFe-protein . A core of 140 conserved residues is identified in an alignment of 59 Fe-protein sequences that may be useful for the identification of homologous proteins with functions comparable to that of Fe-protein in non-nitrogen fixing systems . FEMS Microbiol Lett, 1998 Jul 1, 164(1), 21 - 8 Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum; Macis L et al.; The genes encoding coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum were subcloned and expressed in Escherichia coli . The native molecular mass of the enzyme is 190,000 Da . The enzyme converts glycerol, 1,2-propanediol and 1,2-ethanediol to 3-hydroxypropionaldehyde, propionaldehyde and acetaldehyde, respectively, but glycerol is the preferred substrate . The nucleotide sequences of the dhaBCE genes encoding the three subunits of glycerol dehydratase and of orfZ whose function is unknown were determined . The deduced products of the dhaBCE genes with calculated molecular masses of 60,813, 19,549 and 16,722 Da, respectively, revealed high similarity to amino acid sequences of subunits of coenzyme B12-dependent glycerol and diol dehydratases from other organisms. Lett Appl Microbiol, 1998 May, 26(5), 382 - 6 Detection of alpha- and epsilon-toxigenic Clostridium perfringens type D in sheep and goats using a DNA amplification technique (PCR); Miserez R et al.; Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the alpha-, beta- and epsilon-toxin genes . Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the alpha- and epsilon-toxin genes but were devoid of the beta-toxin gene . These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats . In contrast, Cl . perfringens isolated from 11 of 13 healthy animals only contained the alpha-toxin gene which is typical for type A . Two of the healthy animals contained Cl . perfringens with the alpha- and epsilon-toxin genes . However, when several individual Cl . perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the epsilon-toxin gene, whereas the majority of the colonies were of type A with the alpha-toxin gene only . This is in contrast to the findings from the diseased animals which contained practically only type D Cl . perfringens . The beta-toxin gene was not found in any Cl . perfringen