Tetanus toxin production is triggered by the transition from amino acid consumption to peptides.

Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings.

Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors.

AIMS: To further improve the soy-based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale-up the Clostridium tetani process into small fermentors. METHODS AND RESULTS: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l-tyrosine and l-cysteine could be eliminated from our soy-based medium without effect. Seven carbon sources were compared with glucose in the soy-based medium, but none was found to be superior to glucose. The process was successfully scaled-up into 250-ml bottles, 1-l bottles and 1-l fermentors. CONCLUSIONS: Quite remarkably, when comparing the tetanus production process in our soy-based medium with the traditional animal/dairy-containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l-tyrosine and l-cysteine components could be eliminated, making the medium (Hy-Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale-up from test tubes into 1-l fermentors allows us to predict that further scale-up into large fermentors will be successful. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow's disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable-based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.

Tetanus-induced acute kidney injury in a renal transplant recipient.

A 48-year-old renal transplant recipient who developed tetanus 6 years after transplantation is described. His immunosuppressive protocol was mofetil mycophenolate, sirolimus, and prednisone. The patient presented symptoms of severe tetanus with autonomic dysfunction, requiring ICU care and mechanical ventilation. His clinical course was marked by development of tetanus-induced acute kidney injury and sepsis. He was discharged after 37 days of hospitalization with recovered renal function. Tetanus is a preventable disease associated with a high fatality rate. Its treatment is difficult and requires specialized and intensive care. This case highlights the crucial importance of following adequate immunization guidelines in transplant recipients.

Menstrum for culture preservation and medium for seed preparation in a tetanus toxin production process containing no animal or dairy products.

AIMS: To completely eliminate animal and dairy products from the lyophilization menstrum and the seed medium used to produce tetanus toxin with Clostridium tetani. METHODS AND RESULTS: Tetanus toxin production in a recently developed fermentation medium lacking animal and dairy products was studied with different seed media. It was found that soy peptone could completely replace the beef heart infusion plus animal peptone previously used as seed medium. In addition, we found that cells lyophilized in soy milk could replace the usual type of cells lyophilized in cow's milk. CONCLUSIONS: We have now developed a complete tetanus toxin production process containing no animal and dairy products. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (Mad Cow's Disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. The new vegetable-based process described here avoids such unfortunate possibilities.

The role of reduced iron powder in the fermentative production of tetanus toxin.

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.

Molecular characterization of Clostridium tetani strains by pulsed-field gel electrophoresis and colony PCR.

Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.

Effective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infusion) and dairy proteins or digests (e.g., casein hydrolysates).

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.

Tetanus in a camel (Camelus dromedarius)--a case report.

Twenty days after an open castration, a 5-year-old dromedary was presented to the Dubai Camel Hospital with severe central nervous symptoms. The dromedary showed the following signs: off feed, stiff gait with extended neck, external swelling of the preputial sheath and groin region, and foamy saliva drooling from the mouth. The dromedary was unable to swallow. Three days after admission, the camel developed lockjaw, and on the fifth day it was unable to stand owing to paralysis of the hindquarters. Because of the severity of the disease and because it did not respond to treatment, the camel was euthanized 26 days after the operation and submitted to the Central Veterinary Research Laboratory for further investigation. Both castration wounds were closed and spermiducts were filled with necrotic masses from which Clostridium tetani was isolated. Two mice, which were injected with the filtrate of the thioglycolate broth, developed typical signs of tetanic spasm of the hind leg. Faecal samples from camel and horse paddocks that were only 50 metres apart were negative for C. tetani. However, C. tetani was isolated from two soil samples of the horse paddock. It is recommended that camels should be vaccinated against tetanus prior to castration.

[Study on the effect of a biostimulant on the growth and toxigenic function of Clostridium tetani strain Copenhagen-471]. / Izuchenie vliianiia biostimuliatora na rost i toksinoobrazuiushchuiu funktsiiu Clostridium tetani "Kopenhagen-471."

Bakstim, a new biostimulating preparation obtained from the organs of the immune system of animals, was developed. The impact of Bakstim on the growth and toxigenic function of C. tetani production strain Copenhagen-471 was evaluated. The addition of the preparation to Gluzman commercial medium for obtaining tetanus toxoid led to an increase in the yield of bacterial biomass from 1.9 to 4-fold and an increase in the toxoid production from 2 to 2.8-fold. The optimum concentration of this biostimulant ensuring the maximum yield of tetanus toxin from the production culture was determined (1,000 mg/l). Bakstim will supposedly be used as additive to nutrient media for the production of tetanus toxoid.

Nitrogen-gas bubbling during the cultivation of Clostridium tetani produces a higher yield of tetanus toxin for the preparation of its toxoid.

We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid. N2 was bubbled through nine 10-liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling. We found that treatment of the C. tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.

Beef allergy and the Persian Gulf syndrome.

It is suggested that the Persian Gulf Syndrome (PGS) is caused by beef allergy. In the first symptomless phase, as a result of an energetic US Army immunizing program, using sera with adjuvants to produce detectable antibody levels, the subjects not only developed immunity to the targeted substances, but also became sensitized to one or more of the other substances in the immunizing sera, and specifically to beef protein. The subjects remained healthy while in the war zone on a restricted diet essentially free from beef, but developed PGS after they came home, and were again able to obtain steaks and hamburgers.

[The possibility of the germination of spores of pathogenic clostridia and bacilli in soil]. / Vozmozhnost' prorastaniia spor patogennykh klostridii i batsill v pochve.

A possibility of germination of clostridia (Cl. tetani and Cl. perfringens) and bacilli (Bac. anthracis, STI vaccine strain) has been studied in model experiments with native soil. Mature spores did not germinate upon contact with native soil of deferent agrochemical types. Addition of meat-pepton medium and other protein, amino acid, and sugar-containing media led only to "swelling" of spores. The data obtained support the conclusions drawn by many researches that pathogenic clostridia and bacilli do not germinate in soil.

Production of toxic antigens in dialyzed cultures of microorganisms.

Data on the production of clostridial toxins in dialyzed cultures are summarized. Principal modifications of this cultivation technique suitable for both research and production are shown. If toxins are released from the cells by autolysis (neurotoxins of Clostridium tetani, C. botulinum; lethal factors of C. novyi and C. sordellii), a 10-fold increase of the antigen concentration in filtrates of dialyzed cultures is found in comparison with normal cultures. If toxins are excreted already during growth (lethal factors of C. perfringens type A, C. septicum), the positive effect of the technique is less significant. A dialyzed culture ensures a well-balanced production of toxic filtrates that contain highly concentrated, relatively pure and strongly immunogenic antigens.

The role of cell population kinetics in the efficacy of penicillin--an experimental analysis and stochastic modeling of the tumour tetanus and wound tetanus of the mouse.

When investigating tetanus lethality summation curves of mice under comparable quantitative conditions following a temporarily limited administration of penicillin, the curves obtained can be calculated by the kinetics of tumour cells or wound fibroblasts. In particular, it has been shown that the optimal efficacy of penicillin, after short-time usage as compared with a long-time administration schedule, is determined by the generation time of the tetanus rods as a function of the mitotic cycle of the "pace-making" tumour cells or wound fibroblasts. Further variables of the mathematical model imply the pharmacokinetics of penicillin and the recovery process of the "hit" tetanus rods. From these results some basic experimental and clinical tetanus issues can be elucidated; thus, the mitosis theory of tetanus is being verified for the stage of incubation and of clinical manifestation, while the classical necrosis theory of the pathogenesis of tetanus infection should be valid only for the final stage.

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.