RESUMO
In the hope of developing a vaccine against Clostridium difficile based on its toxin(s), we have developed a fermentation medium for the bacterium that results in the formation of Toxin A and contains no meat or dairy products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Soy Peptone A3, have been found to replace both the meat/dairy product tryptone in the preparation of working cell banks and seed media, and NZ-Soy BL4 does the same in the fermentation medium. These replacements yield even higher toxin titers.
Assuntos
Toxinas Bacterianas/biossíntese , Clostridioides difficile/metabolismo , Meios de Cultura/metabolismo , Laticínios , Proteínas/metabolismo , Animais , Reatores Biológicos , Carbono/farmacologia , Clostridioides difficile/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Glucose/farmacologia , Glicerol/farmacologia , Manitol/farmacologia , Peptonas/metabolismo , Proteínas de Soja/metabolismoRESUMO
BACKGROUND: Toxins A and B are the primary virulence factors of Clostridium difficile. Since 2002, an epidemic of C difficile-associated disease with increased morbidity and mortality has been present in Quebec province, Canada. We characterised the dominant strain of this epidemic to determine whether it produces higher amounts of toxins A and B than those produced by non-epidemic strains. METHODS: We obtained isolates from 124 patients from Centre Hospitalier Universitaire de Sherbrooke in Quebec. Additional isolates from the USA, Canada, and the UK were included to increase the genetic diversity of the toxinotypes tested. Isolate characterisation included toxinotyping, pulsed-field gel electrophoresis (PFGE), PCR ribotyping, detection of a binary toxin gene, and detection of deletions in a putative negative regulator for toxins A and B (tcdC). By use of an enzyme-linked immunoassay, we measured the in-vitro production of toxins A and B by epidemic strain and non-dominant strain isolates. FINDINGS: The epidemic strain was characterised as toxinotype III, North American PFGE type 1, and PCR-ribotype 027 (NAP1/027). This strain carried the binary toxin gene cdtB and an 18-bp deletion in tcdC. We isolated this strain from 72 patients with C difficile-associated disease (58 [67%] of 86 with health-care-associated disease; 14 [37%] of 38 with community-acquired disease). Peak median (IQR) toxin A and toxin B concentrations produced in vitro by NAP1/027 were 16 and 23 times higher, respectively, than those measured in isolates representing 12 different PFGE types, known as toxinotype 0 (toxin A, median 848 microg/L [IQR 504-1022] vs 54 microg/L [23-203]; toxin B, 180 microg/L [137-210] vs 8 microg/L [5-25]; p<0.0001 for both toxins). INTERPRETATION: The severity of C difficile-associated disease caused by NAP1/027 could result from hyperproduction of toxins A and B. Dissemination of this strain in North America and Europe could lead to important changes in the epidemiology of C difficile-associated disease.
Assuntos
Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Clostridioides difficile , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Enterocolite Pseudomembranosa/epidemiologia , Enterotoxinas/biossíntese , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Canadá/epidemiologia , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/transmissão , Humanos , Proteínas Repressoras/genética , Reino Unido/epidemiologia , Estados Unidos/epidemiologia , VirulênciaRESUMO
Rapamycin is used in medicine as an immunosuppressive agent (Sirolimus; Rapamune) although discovered as an antifungal agent. It is thought not to have antibacterial activity. Surprisingly, we found that rapamycin inhibits the germination of Bacillus brevis Nagano spores, but is inactive against Bacillus brevis Nagano vegetative cells. Surprisingly rapamycin did not show antimicrobial activity against other Bacillus strains, including other gramicidin S-producing Bacillus brevis strains such as ATCC 9999 and BI-7, whether tested as spores or vegetative cells.
Assuntos
Bacillus/efeitos dos fármacos , Bacillus/fisiologia , Sirolimo/farmacologia , Antibacterianos/farmacologia , Bacillus/classificação , Testes de Sensibilidade Microbiana , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/fisiologiaRESUMO
When tetanus toxin is made by fermentation with Clostridium tetani, the traditional source of iron is an insoluble preparation called reduced iron powder. This material removes oxygen from the system by forming FeO(2) (rust). When inoculated in a newly developed medium lacking animal and dairy products and containing glucose, soy-peptone, and inorganic salts, growth and toxin production were poor without reduced iron powder. The optimum concentration of reduced iron powder for toxin production was found to be 0.5 g/l. Growth was further increased by higher concentrations, but toxin production decreased. Inorganic iron sources failed to replace reduced iron powder for growth or toxin formation. The iron source that came closest was ferrous ammonium sulfate. The organic iron sources ferric citrate and ferrous gluconate were more active than the inorganic compounds but could not replace reduced iron powder. Insoluble iron sources, such as iron wire, iron foil, and activated charcoal, were surprisingly active. Combinations of activated charcoal with soluble iron sources such as ferrous sulfate, ferric citrate, and ferrous gluconate showed increased activity, and the ferrous gluconate combination almost replaced reduced iron powder. It thus appears that the traditional iron source, reduced iron powder, plays a double role in supporting tetanus toxin formation, i.e., releasing soluble sources of iron and providing an insoluble surface.
Assuntos
Clostridium tetani/metabolismo , Ferro/metabolismo , Toxina Tetânica/biossíntese , Clostridium tetani/crescimento & desenvolvimento , Meios de Cultura/química , Fermentação , Compostos Férricos/metabolismo , Compostos Ferrosos/metabolismo , Glucose , Oxirredução , Peptonas , Compostos de Amônio Quaternário/metabolismoRESUMO
We have developed a fermentation medium for Clostridium tetani that results in the formation of tetanus toxin and contains no meat (e.g., beef heart infusion) or dairy (e.g., casein digest) products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Quest Hy-Soy, have been found to replace both the meat extract and casein digest components of traditional tetanus toxin production media and to yield even higher toxin titers. The comparison of the traditional versus the new medium has been carried out repeatedly by us and the superiority of our medium has been consistently observed. To our knowledge, this is the first time that such a medium has been devised.
Assuntos
Clostridium tetani/metabolismo , Meios de Cultura , Toxina Tetânica/biossíntese , Animais , Química Encefálica , Caseínas/química , Bovinos , Clostridium tetani/crescimento & desenvolvimento , Fermentação , Proteínas do Leite/química , Miocárdio/química , Glycine max/química , Toxina Tetânica/análiseRESUMO
Aspergillus fumigatus NRRL 2346 is the producer of fumagillin, an antitumor antibiotic that inhibits angiogenesis. This strain is very difficult to grow reproducibly in shake flasks owing to an extreme form of pellet growth and extensive wall growth. The effects of carboxymethylcellulose (CMC) and carboxypolymethylene (Carbopol) on growth and fumagillin production by A. fumigatus were investigated. By adding the polymers to the fermentation medium, the growth form of the mold was changed from a single large glob to small reproducible pellets, and wall growth was diminished to a minimum. Carbopol, at a lower concentration, was more effective than CMC in improving both morphology and production. Small pellets were produced which favored fumagillin biosynthesis. 1.5% (wt/vol) CMC and 0.3% (wt/vol) Carbopol were found to be the optimum concentrations; higher levels increased viscosity to an unacceptable level.
Assuntos
Antibacterianos/biossíntese , Aspergillus fumigatus/efeitos dos fármacos , Carboximetilcelulose Sódica/farmacologia , Ácidos Graxos Insaturados/biossíntese , Polivinil/farmacologia , Resinas Acrílicas , Aspergillus fumigatus/citologia , Aspergillus fumigatus/metabolismo , Cicloexanos , Relação Dose-Resposta a Droga , Hifas/fisiologia , Sesquiterpenos , Esporos Fúngicos/fisiologiaRESUMO
The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L(-1) xylan and 50 g L(-1) mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L(-1) was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings.