Fed‐batch production of tetanus toxin by Clostridium tetani

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed‐batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ₀) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50–60 L_f/mL) for NZ₀ ≥ 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield (～60 L_f/mL) about 33% higher than those of fed‐batch runs with addition at fixed concentration (～45 L_f/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

破伤风杆菌产毒培养基主要原材料的替代研究

目的探索用干粉状的肝浸粉、胰酪胨、月示胨代替传统破伤风杆菌产毒培养基中使用的猪肝水透析外液、胰酶酪蛋白消化液、浓胨水,以满足规模化生产的需求。方法与传统的破伤风杆菌产毒培养基同时用于破伤风类毒素的生产过程,由小批量生产逐步转化到规模化批量生产,经多批次试验,以检测不同组分制备的培养基各生化指标和接种细菌后培养的产毒水平进行比对。先确定肝浸粉可以取代猪肝水透析外液,在此基础上筛选出代替浓胨水的月示胨,再以待用的胰酪胨配合肝浸粉、月示胨制备多批次不同生产量的破伤风杆菌产毒培养基。结果经过多批次小批量扩大到批量规模化生产的试验,用肝浸粉、胰酪胨、月示胨配制的破伤风杆菌产毒培养基与传统工艺制备的破伤风杆菌产毒培养基相比较,无统计学意义(P0.05)。结论肝浸粉、胰酪胨、月示胨可以替代传统破伤风杆菌产毒培养基中的猪肝水透析外液、胰酶酪蛋白消化液、浓胨水,适用于破伤风类毒素的规模化生产。     

Insights in metabolism and toxin production from the complete genome sequence of Clostridium tetani

The decryption of prokaryotic genome sequences progresses rapidly and provides the scientific community with an enormous amount of information. Clostridial genome sequencing projects have been finished only recently, starting with the genome of the solvent-producing Clostridium acetobutylicum in 2001. A lot of attention has been devoted to the genomes of pathogenic clostridia. In 2002, the genome sequence of C. perfringens, the causative agent of gas gangrene, has been released. Currently in the finishing stage and prior to publication are the genomes of the foodborne botulism-causing C. botulinum and of C. difficile, the causative agent of a wide spectrum of clinical manifestations such as antibiotic-associated diarrhea. Our team sequenced the genome of neuropathogenic C. tetani, a Gram-positive spore-forming bacterium predominantly found in the soil. In deep wound infections it occasionally causes spastic paralysis in humans and vertebrate animals, known as tetanus disease, by the secretion of potent neurotoxin, designated tetanus toxin. The toxin blocks the release of neurotransmitters from presynaptic membranes of interneurons of the spinal cord and the brainstem, thus preventing muscle relaxation. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid, a formaldehyde-treated tetanus toxin, but nevertheless, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The World Health Organization has stated that neonatal tetanus is the second leading cause of death from vaccine preventable diseases among children worldwide. This minireview focuses on an analysis of the genome sequence of C. tetani E88, a vaccine production strain, which is a toxigenic non-sporulating variant of strain Massachusetts. The genome consists of a 2,799,250 bp chromosome encoding 2618 open reading frames. The tetanus toxin is encoded on a 74,082 kb plasmid, containing 61 genes. Additional virulence-related factors as well as an insight into the metabolic strategy of C. tetani with regard to its pathogenic phenotype will be presented. The information from other clostridial genomes by means of comparative analysis will also be explored.     

The immunochemistry of the antigenic fractions of Clostridium tetani toxin

The localization of tetanospasmin and tetanolysin in C. tetani cells has been determined by the cytochemical method in the dynamics of the development of the microbial population. As shown in this investigation, tetanolysin, similarly to exotoxins, is released from microbial cells into the culture fluid mainly during the first 24 hours of cultivation, while tetanospasmin, similarly to endotoxins, is released only in the process of the destruction of these cells at the phase of the death of the microbial population.     

Effect of Lactobacillus fermentum on beta2 toxin production by Clostridium perfringens

Clostridium perfringens, although a member of the normal gut flora, is also an important cause of intestinal disease in animals and, to a lesser extent, in humans. Disease is associated with the production of one or more toxins, and little is known about environmental influences on the production of these toxins. One of the health-promoting effects of lactic acid bacteria (LAB) is the establishment and maintenance of a low pH in the intestine since an acidic environment inhibits the growth of many potentially harmful bacteria. Here, the effect of the LAB Lactobacillus fermentum on beta2 toxin production by C. perfringens is described. Coculturing of C. perfringens with L. fermentum showed that under in vitro conditions, L. fermentum was capable of silencing beta2 toxin production by C. perfringens without influencing bacterial viability. The reduction in toxin production was shown to be most likely a result of the decline in pH. Quantitative PCR showed that the reduction in beta2 toxin production was due to a decrease in cpb2 mRNA. These results suggest that in the intestine, the production of beta2 toxin by C. perfringens might be regulated by other members of the normal intestinal flora.     

Effect of fermentation conditions on toxin production by Clostridium botulinum type B.

To obtain high yields of toxin for the preparation of purified neurotoxoids, we examined the time of appearance and the quantity of toxin produced by the Bean strain of Clostridium botulinum type B under various conditions by using a fermentor system. The medium employed consisted of 2.0% casein hydrolylsate and 1.5% yeast extract plus an appropriate concentration of glucose. The maximum toxin concentration (4 x 10(5) to 5 x 10(5) mouse median lethal doses per ml) was attained within 48 h under the following fermentation conditions: an initial glucose concentration of 0.5 or 1.0%, a temperature of 35 degrees C, a nitrogen overlay at a rate of 5 liters/min, and an agitation rate of 50 rpm.     

Immunoenzimatic detection of the Clostridium tetani bacterial toxin: an alternative to mice bioassays

Cell-free extracts from 20 strains of Clostridium tetani isolated from soil samples, were tested for tetanus toxin production using an enzyme immunoassay. All the extracts were classified as positive for the toxin presence, and eight of them showed absorbance values corresponding to tetanus toxin concentrations between 3.2 and 88 ng/ml; thus, they fell within the linear absorbance range (0.135-0.317). All dilutions of toxin used to obtain the calibration curve (0.0071 to 1.1 ng) were lethal for mice.     

Effect of medium composition on amylase production by some starch-degrading yeasts

Abstract The production of extracellular amylase activity by a number of recently described amyloytic yeast species, viz. Candida homilentoma, C. silvanorum, C. tsukubaensis, Cryptococcus flavus, Leucosporidium capsuligenum, Filobasidium capsuligenum and Trichosporon pullulans , was investigated. The effects on amylase secretion of pH, different carbon sources (glucose, maltose, dextrin, soluble starch) and of various nitrogen sources [yeast nitrogen base, yeast extract, corn steep liquor (CSL)] were compared for these yeasts.     

Phage-like particles produced by Clostridium tetani]

Two C. tetani strains used for toxin production spontaneously produce two varieties of phage-like particles with isometric heads. Type A has a contractile tail, whereas type B shows a non-contractile tail with a long, wavy tail fiber. No relationship between the presence of these particles and the amount of toxin produced was found.