Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani.

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a "fermentation map", which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.

The Role of the Gastrointestinal Tract in Toxigenic Clostridium tetani Infection: A Case-Control Study.

Tetanus arises from wound contamination with Clostridium tetani, but approximately one fifth of patients have no discernable entry wound. Clostridium tetani is culturable from animal feces, suggesting the gastrointestinal tract could be an endogenous reservoir or direct-entry portal, but human data are lacking. In this study of 101 Vietnamese adults with tetanus and 29 hospitalized control subjects, admission stool samples were cultured for C. tetani. Anti-tetanus toxin antibodies were measured by ELISA. Clostridium tetani toxigenicity was evaluated using polymerase chain reaction and sequencing. Toxigenic C. tetani was cultured from stool samples in 50 of 100 (50%) tetanus cases and 12 of 28 (42.9%) control subjects (P = 0.50), and stool samples of 44 of 85 (52.4%) tetanus cases with clinically identified wounds compared with 6 of 15 (47.6%) patients without clinically identified wounds (P = 0.28). Nine of 12 (75%) control subjects with toxigenic C. tetani in their stool samples lacked protective antibody concentrations. These findings fail to show evidence of an association between gastrointestinal C. tetani and tetanus infection, but emphasize the importance of increasing vaccination coverage.

Tetanus vaccine-induced human neutralizing antibodies provide full protection against neurotoxin challenge in mice.

Clostridium tetani causes life-threatening disease by producing tetanus neurotoxin (TeNT), one of the most toxic protein substances. Toxicosis can be prevented and cured by administration of anti-TeNT neutralizing antibodies. Here, we identified a series of monoclonal antibodies (mAbs) derived from memory B cells of a healthy adult immunized with the C-terminal domain of TeNT (TeNT-Hc). Thirteen mAbs bound to both tetanus toxoid (TT) and TeNT-Hc, while two mAbs recognized only TT. VH3-23 was the most frequently used germline gene in these TT-binding mAbs, and the pairwise identity values of the VH gene sequences ranged from 27% to 69%. Three of these mAbs-T3, T7, and T9-6-completely protected mice from challenge with 2× LD50 of TeNT, and two (T2 and T18) significantly prolonged the survival time. The five neutralizing mAbs recognized distinct epitopes on TT, with binding affinities ranging from 0.123 to 11.9 nM. Our study provides promising therapeutic candidates for tetanus.

The population structure of Clostridium tetani deduced from its pan-genome.

Clostridium tetani produces a potent neurotoxin, the tetanus neurotoxin (TeNT) that is responsible for the worldwide neurological disease tetanus, but which can be efficiently prevented by vaccination with tetanus toxoid. Until now only one type of TeNT has been characterized and very little information exists about the heterogeneity among C. tetani strains. We report here the genome sequences of 26 C. tetani strains, isolated between 1949 and 2017 and obtained from different locations. Genome analyses revealed that the C. tetani population is distributed in two phylogenetic clades, a major and a minor one, with no evidence for clade separation based on geographical origin or time of isolation. The chromosome of C. tetani is highly conserved; in contrast, the TeNT-encoding plasmid shows substantial heterogeneity. TeNT itself is highly conserved among all strains; the most relevant difference is an insertion of four amino acids in the C-terminal receptor-binding domain in four strains that might impact on receptor-binding properties. Other putative virulence factors, including tetanolysin and collagenase, are encoded in all genomes. This study highlights the population structure of C. tetani and suggests that tetanus-causing strains did not undergo extensive evolutionary diversification, as judged from the high conservation of its main virulence factors.

Coxofemoral luxation in a border collie as a complication of a Clostridium tetani infection.

A four-month-old male, entire, border collie was presented to the Queen Mother Hospital for Animals with a two day history of muscular spasms and "Risus sardonicus". Tetanus was diagnosed, and the dog was treated with tetanus antitoxin, antibiotics and supportive therapy. Coxofemoral luxation resulted as a complication of the tetanus and was successfully managed by performing a femoral head and neck excision. This is the first report of joint luxation associated with Clostridium tetani infection in a dog.

Genomic insights into the evolution and ecology of botulinum neurotoxins.

Clostridial neurotoxins, which include botulinum neurotoxins (BoNTs) and tetanus neurotoxins, have evolved a remarkably sophisticated structure and molecular mechanism fine-tuned for the targeting and cleavage of vertebrate neuron substrates leading to muscular paralysis. How and why did this toxin evolve? From which ancestral proteins are BoNTs derived? And what is, or was, the primary ecological role of BoNTs in the environment? In this article, we examine these questions in light of recent studies identifying homologs of BoNTs in the genomes of non-clostridial bacteria, including Weissella, Enterococcus and Chryseobacterium. Genomic and phylogenetic analysis of these more distantly related toxins suggests that they are derived from ancient toxin lineages that predate the evolution of BoNTs and are not limited to the Clostridium genus. We propose that BoNTs have therefore evolved from a precursor family of BoNT-like toxins, and ultimately from non-neurospecific toxins that cleaved different substrates (possibly non-neuronal SNAREs). Comparison of BoNTs with these related toxins reveals several unique molecular features that underlie the evolution of BoNT's unique function, including functional shifts involving all four domains, and gain of the BoNT gene cluster associated proteins. BoNTs then diversified to produce the existing serotypes, including TeNT, and underwent repeated substrate shifts from ancestral VAMP2 specificity to SNAP25 specificity at least three times in their history. Finally, similar to previous proposals, we suggest that one ecological role of BoNTs could be to create a paralytic phase in vertebrate decomposition, which provides a competitive advantage for necrophagous scavengers that in turn facilitate the spread of Clostridium botulinum and its toxin.

[Diagnostic image (320). A new mother with opisthotonus and extensor spasms]. / Diagnose in beeld (320). Een kraamvrouw met opisthotonus en strekkrampe.

A 21-year-old woman was admitted to a rural hospital in Tanzania after a home delivery one week before. She had signs of general body rigidity due to maternal tetanus.

Comparative pathogenomics of Clostridium tetani.

Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP) and four distinct constituents of the mobile genome (mobilome): a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a common ancestor.

Adjuvants for Clostridium tetani and Clostridium diphtheriae vaccines updating.

It's known that diphtheria and tetanus are a contagious lethal diseases over the years, they caused by pathogenic microbes corynebacterium diphtheria and Clostridium tetani, respectively. The diseases result from the production of bacterial toxin. Vaccination with bacterial toxoid vaccines adsorbed on particulates adjuvants still are the best way to prevent this epidemic diseases from spread. The particulate vaccines have been shown to be more efficient than soluble one for the induction of the immune responses. Nanoparticles can be engineered to enhance the immune responses. As well known the immune response to inactivate killed and subunit vaccine enhances by alum adjuvants. The adjuvants examined and tested after reducing its size to particle size, thus mimic size of viruses which is considered smallest units can derive the immune system. The major issue is minimizing the adjuvant particles, to gain insight of resulting immunity types and impact on immune response. The adjuvant effect of micro/nanoparticles appears to largely be a consequence of their uptake into antigen presenting cells.

Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

BACKGROUND: Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. OBJECTIVE: In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. MATERIAL AND METHODS: Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. RESULTS: All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. CONCLUSION: Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

Tetanus complicating snakebite in northern Nigeria: clinical presentation and public health implications.

BACKGROUND: There is inequality in vaccination coverage, and adult farmers in tropical rural communities could have missed tetanus toxoid as campaigns target children. Snakebite is not uncommon and partly because of lack of effective antivenom, management in inaccessible areas could be complicated with tetanus in unimmunized victims. PATIENTS AND RESULTS: Four snakebite victims who developed tetanus were seen in northern Nigeria. Three were bitten while farming and patients took 10-25 days before presenting to hospital. All patients had incised bite sites and applied local medicinal herbs and in one case, a black 'snake' stone. Bites were by the cobra (Naja nigricollis) and carpet viper (Echis ocellatus) in the extremities and victims had swollen limbs and bleeding with incoagulable blood. Three of the patients were given anti-tetanus serum (ATS) after development of tetanus symptoms in a primary facility and only one recalled receipt of one dose of tetanus toxoid prior to bite. Patients had trismus, rigidity, backache, spasms and one had autonomic dysfunction and was managed with antispasmodics, ATS, wound care, antibiotics and supportive measures. Two patients with envenoming and severe tetanus received antivenom, but died. One of the two surviving patients developed osteomyelitis with prolonged hospital stay, while the other recovered with residual disability. CONCLUSION: Tetanus could follow snakebite in inaccessible rural agricultural communities with inadequate health care provision. Clinical presentation is typical but late and is confounded by snakebite complications leading to considerable morbidity and mortality. The cases highlight the triple problems of inadequacy, inaccessibility and inequality in health care and calls for measures aimed at improvement.

Autism and Clostridium tetani.

Autism is a severe developmental disability believed to have multiple etiologies. This paper outlines the possibility of a subacute, chronic tetanus infection of the intestinal tract as the underlying cause for symptoms of autism observed in some individuals. A significant percentage of individuals with autism have a history of extensive antibiotic use. Oral antibiotics significantly disrupt protective intestinal microbiota, creating a favorable environment for colonization by opportunistic pathogens. Clostridium tetani is an ubiquitous anaerobic bacillus that produces a potent neurotoxin. Intestinal colonization by C. tetani, and subsequent neurotoxin release, have been demonstrated in laboratory animals which were fed vegetative cells. The vagus nerve is capable of transporting tetanus neurotoxin (TeNT) and provides a route of ascent from the intestinal tract to the CNS. This route bypasses TeNT's normal preferential binding sites in the spinal cord, and therefore the symptoms of a typical tetanus infection are not evident. Once in the brain, TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein. This inhibition of neurotransmitter release would explain a wide variety of behavioral deficits apparent in autism. Lab animals injected in the brain with TeNT have exhibited many of these behaviors. Some children with autism have also shown a significant reduction in stereotyped behaviors when treated with antimicrobials effective against intestinal clostridia. When viewed as sequelae to a subacute, chronic tetanus infection, many of the puzzling abnormalities of autism have a logical basis. A review of atypical tetanus cases, and strategies to test the validity of this paper's hypothesis, are included.

Chronic ulcers and myasis as ports of entry for Clostridium tetani.

Evaluating tetanus immune status is not yet the usual clinical practice regarding patients with chronic ulcers or myasis. However, of 858 tetanus patients at Hospital Couto Maia (Salvador, Bahia, Brazil) aged 1 year or above, 2 had pressure ulcers and 17 had chronic ulceration of the lower limbs where these skin lesions were the ports of entry for Clostridium tetani. In these 19 cases, the following predisposing factors were described: venous insufficiency (n=6), sickle cell anemia (n=2), Hansen s disease (n=1), malnutrition (n=1), diabetes mellitus (n=1), trauma (n=1) and unknown factors (n=7). In 6 other cases, in addition to the Hansen s disease patient, the port of entry for tetanus was the site of extraction of Tunga penetrans larvae. In these 25 cases, the majority of patients (68%) were over 40 years old (17/25) and all of these patients stated that they had either not followed a tetanus toxoid vaccination regimen (19/25), or had partially completed such a regimen, or did not give precise information (6/25). Among the same series studied, over half (52%) of the patients died (13/25). We conclude that tetanus prevention must be included in the treatment of chronic skin ulcer patients, vaccination coverage should be increased among older people, and strategies aimed at improving coverage for all age groups must be reviewed.

Management and prevention of tetanus.

The World Health Organization was committed to eliminating neonatal tetanus by 1995. Three years after this date, the infection killed over 400,000 babies a year, even though a safe, effective vaccine had been available for most of this century. The frequency of tetanus in the developing world epitomizes the healthcare disparity between the developed and the developing world. Consequently, the priority of the medical profession must be prevention, with the development of simpler immunization schedules with longer protection. Consequently, the purpose of this collective review is to provide an overview to the management of tetanus as well as to review the immunization strategy that will prevent this potentially deadly illness. Tetanus is caused by Cloistridium tetani, which is an obligate anaerobic, gram-positive rod that is motile and readily forms endospores. Although C. tetani is located everywhere, the disease is encountered largely in underdeveloped, overcrowded, and economically disadvantaged countries. C. tetani is widespread in the feces of domestic animals and humans, while spores of C. tetani are abundant in soil and in the environment surrounding the habitation of humans and animals. Tetanus usually follows deep penetrating wounds where anaerobic bacterial growth is facilitated. Three basic forms of tetanus may be distinguished: local, cephalic, and generalized. At least 80% of the cases are the generalized form. In the adult patient, the most characteristic sign of generalized tetanus is lockjaw, or trismus. The diagnosis of tetanus is most frequently made on clinical manifestations, rather than on bacteriologic findings. The three objectives of management of tetanus are: (1) to provide supportive care until the tetanospasmin that is fixed in tissue has been metabolized; (2) to neutralize circulating toxin; and (3) to remove the source of tetanospasmin. Because there is essentially no immunity to tetanus toxoid, the only effective way to control tetanus is by prophylactic immunization.

[Guinea pig resistance to infection by the spores of the tetanus causative agent at various levels of tetanus antitoxin and delayed hypersensitivity]. / Rezistentnost' morskikh svinok k zarazheniiu sporami vozbuditelei stolbniaka pri raznykh urovniakh stolbniachnogo antitoksina i giper-chuvstvitel'nosti zamedlennogo tipa.

Comparative experimental investigations on guinea pigs immunized against tetanus, carried out with the use of different methods for the determination of immunity to tetanus, have demonstrated the existence of a relationship between the results of the tests used in these investigations (the neutralization test, the passive hemagglutination test and the allergic skin test) and the resistance of the animals to challenge with C. tetani spores in Dcl and Dlm50. The mechanisms of not only humoral, but also cell-mediated immunity play an important role in protection from tetanus infection. The possibility of using, on principle, the allergic skin test with tetanin for the evaluation of immunity to tetanus in individual patients with the aim of differential approach to protection against tetanus in traumas has been shown.

[Use of a synthetic medium for cultivating pathogenic anaerobes]. / Primenenie sinteticheskoi sredy dlia kul'tivirovaniia patogennykh anaerobov

It was shown that a synthetic medium suggested by the authors earlier was useful for the growth and toxin formation of Cl. tetani, Cl. botulinum and Cl. perfringens, types B and E. A study of the character of growth and toxinogensis, microscopic examination of morphology of culture cells and results of passages showed the suggested synthetic medium to be of value; a possibility of its application for studying the nutrient requirements and the role of individual components of the nutrient media in the process of growth and toxinogenesis was also demonstrated.

[Tetanus prophylaxis after an injury; check the need for vaccination and immunoglobulin]. / Tetanusprofylaxe na verwonding: check de indicatie voor vaccinatie én immunoglobuline.

Tetanus can occur after an injury and is caused by the exotoxin of Clostridium tetani. Characteristics of generalised tetanus include spasms in the back and other muscles, trismus, risus sardonicus and difficulty in breathing caused by laryngospasms. Vaccination through the National Vaccination Programme of the Netherlands has resulted in 94% of the population being protected against tetanus; certain groups, however, have a low rate of vaccination. In the Netherlands, 5 patients were reported to have generalised tetanus in 2011. This figure is relatively high in comparison with previous years. Of these 5 patients, 3 did not receive post-exposure-prophylaxis (PEP) after their injuries, or received it incompletely. PEP may be comprised of 1 or more vaccinations with the tetanus toxoid and/or the administration of tetanus immunoglobulin. Patients who have sustained an injury should be evaluated in accordance with the guideline 'Tetanus' by the Landelijke Coördinatie Infectieziekten (National Coordination Centre for communicable disease control), and to assess whether PEP is indicated.

In silico exploration of novel phytoligands against probable drug target of Clostridium tetani.

Though tetanus is an old disease with well known medicines, its complications are still a serious issue worldwide. Tetanus is mainly due to a powerful neurotoxin, tetanolysin-O, produced by a Gram positive anaerobic bacterium, Clostridium tetani. The toxin has a thiol-activated cytolysin which causes lysis of human platelets, lysosomes and a variety of subcellular membranes. The existing therapy seems to have challenged as available vaccines are not so effective and the bacteria developed resistance to many drugs. Computer aided approach is a novel platform to screen drug targets and design potential inhibitors. The three dimensional structure of the toxin is essential for structure based drug design. But the structure of tetanolysin-O is not available in its native form. Moreover, the interaction and pharmacological activities of current drugs against tetanolysin-O is not clear. Hence, there is need for three dimensional model of the toxin. The model was generated by homology modeling using crystal structure of perfringolysin-O, chain-A (PDB ID: 1PFO) as the template. The modeled structure has 22.7% α helices, 27.51% ß sheets and 41.75% random coils. A thiol-activated cytolysin was predicted in the region of 105 to 1579, which acts as a functional domain of the toxin. The hypothetical model showed the backbone root mean square deviation (RMSD) value of 0.6 Å and the model was validated by ProCheck. The Ramachandran plot of the model accounts for 92.3% residues in the most allowed region. The model was further refined by various tools and deposited to Protein Model Database (PMDB ID: PM0077550). The model was used as the drug target and the interaction of various lead molecules with protein was studied by molecular docking. We have selected phytoligands based on literatures and pharmacophoric studies. The efficiency of herbal compounds and chemical leads was compared. Our study concluded that herbal derivatives such as berberine (7, 8, 13, 13a-tetradehydro-9,10-dimethoxy-2,3 [methylenebis(oxy)] berbinium), curcumin ((1E,6E)-1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), coumarin (2H-chromen-2-one), catechol (Benzene-1,2-diol) and diosphenol (2-hydroxy-3-methyl-6-propan-2-ylcyclohex-2-en-1-one) are the best inhibitors compared to known chemicals. Hence, these leads can be used as potential inhibitors against tetanolysin.