Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I–III. We propose two distinct sporulation strategies used by C. botulinum Groups I–III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

USO DA TOXINA BOTULÍNICA COMO TERAPIA COADJUVANTE EM PACIENTES COM DOENÇA DE PARKINSON, DISFUNÇÕES TEMPOROMANDIBULARES E BRUXISMO

Introdução: A toxina botulínica é uma neurotoxina produzida pelo Clostridium botulinum, causador do botulismo, doença neuroparalítica grave(1,3). Esta, é uma proteinase de zinco que realiza a clivagem de proteínas associadas a vesículas neuronais, responsáveis pela liberação de acetilcolina na junção neuromuscular(1). A doença de Parkinson (DP), afeta significativamente a vida dos pacientes, alguns não respondem às opções terapêuticas aplicadas costumeiramente, prejudicando ainda mais essa situação(2). Ademais, disfunções temporomandibulares (DTMs) e bruxismo do sono (BS), são condições que prejudicam significativamente os pacientes, causando dores e movimentos mandibulares restritos(4). A utilização da toxina botulínica, é vista ligada principalmente a fins estéticos, contudo sua utilidade vai muito além disso. Objetivos: Este resumo possui como objetivo avaliar e correlacionar o uso da toxina botulínica à doença de Parkinson, DTMs e Bruxismo como uma possível alternativa terapêutica. Métodos: Foi realizada uma pesquisa de artigos científicos nas plataformas Pubmed e Sciencedirect, buscando por meio dos descritores “botulinum toxin AND parkinsonism, botulinum toxin AND bruxism”, onde foi encontrado os artigos utilizados. Resultados: O uso de toxina botulínica mostrou diversos benefícios quando avaliado em pacientes com DP, DTMs e BS. Isso foi evidente ao avaliar a sialorréia, que acomete 86% dos pacientes com DP, caracterizando a incapacidade de controlar as secreções orais, que após a aplicação da toxina botulínica nas glândulas salivares houve a inibição da transmissão colinérgica parassimpática e simpática pósganglionar, promovendo a diminuição da secreção salivar(2). Além disso, a disfunção urinária ligada a DP também mostrou bons prognósticos quando submetidos à aplicação da toxina, agindo no músculo detrusor podendo trazer benefício por até 6 meses(2).Em pacientes com DTMs e BS, submetidos a uma sessão de injeção da toxina nos músculos temporal e masseter, foi possível observar uma melhora considerável nos níveis de dor, como também nos movimentos mandibulares, avaliados no pós-operatório, em um, três e seis meses após o tratamento(4). Conclusões: Dessa forma, é possível concluir que o uso da terapia com toxina botulínica nos distúrbios de parkinsonismo, disfunções temporomandibulares e bruxismo tem uma grande importância terapêutica coadjuvante auxiliando de forma significativa no tratamento dos sintomas dessas síndromes. Porém, mais estudos são necessários para melhor compreender os mecanismos dessa toxina em cada síndrome, bem como buscar a padronização de doses nas mesmas, além de sua utilização em outras enfermidades.

Investigation of Botulinum Neurotoxin Types from Clostridium botulinum Causing A Recent Outbreak in Vietnam

Clostridium botulinum is one of the causes of undiagnosed sudden deaths in humans due to the lethal botulinum neurotoxins (BoNTs). Foodborne botulism rarely occurs in developed countries because of being closely monitored, in opposite to developing countries including Vietnam. In the August 2020 food poisoning outbreak in Vietnam, presence of Clostridium botulinum and BoNTs was identified by culture and mouse bioassay, however, information regarding the possible toxin types was unclear. To examine the types of toxin, we designed primers for specific amplification of gene regions encoding the light chain (LC) domains for both BoNT/A and BoNT/B. After optimization, the expected PCR products were sent for sequencing. The results showed that the sequence of gene encoding BoNT/A LC was 99.2% identical to the CB-27 strain. The sequence of gene encoding BoNT/B LC was approximately 98.8% identical to reference strains. Additionally, we analyzed the sequences of the inferred proteins and identified a substitution that resulted in an early stop codon as previously found in a defective form of BoNT/B. Collectively, we provided the first evidence for C. botulinum strain possessing A(b) type in this studied outbreak. Further enzyme activity and neutralization assays are necessary to validate this preliminary toxin typing.

APLICAÇÃO DA TOXINA BOTULÍNICA NA TERAPÊUTICA DA ENXAQUECA

A proteína produzida pela bactéria Clostridium botulinum é chamada de toxina botulínica, que após ser ingerida em altas quantidades impede os sinais nervosos do cérebro para a musculatura causando uma paralisia generalizada que se denomina botulismo. Apesar disso, quando quantidades pequenas são injetadas na musculatura facial específica, apenas o impulso que o orienta será bloqueado, causando o relaxamento do local, podendo ser utilizada em procedimentos estéticos e também de forma terapêutica como exemplo para tratar enxaqueca que é um transtorno do Sistema Nervoso Central que vem associada a dor e náuseas. Os pacientes que sofrem com essa síndrome geralmente utilizam analgésicos para alivio rápido da dor, porém a casos tão graves que os pacientes precisam procurar apoio médico. A toxina vai agir de forma que o paciente não tenha os estímulos para o aparecimento das crises e também tem a função de ajudar a liberar substâncias analgésicas para alivio da dor. O tratamento tem trazido bons resultados aos pacientes tem tido redução na intensidade e frequência dos episódios de dor.

Extensive Genome Exploration of Clostridium botulinum Group III Field Strains

In animals, botulism is commonly sustained by botulinum neurotoxin C, D or their mosaic variants, which are produced by anaerobic bacteria included in Clostridium botulinum group III. In this study, a WGS has been applied to a large collection of C. botulinum group III field strains in order to expand the knowledge on these BoNT-producing Clostridia and to evaluate the potentiality of this method for epidemiological investigations. Sixty field strains were submitted to WGS, and the results were analyzed with respect to epidemiological information and compared to published sequences. The strains were isolated from biological or environmental samples collected in animal botulism outbreaks which occurred in Italy from 2007 to 2016. The new sequenced strains belonged to subspecific groups, some of which were already defined, while others were newly characterized, peculiar to Italian strains and contained genomic features not yet observed. This included, in particular, two new flicC types (VI and VII) and new plasmids which widen the known plasmidome of the species. The extensive genome exploration shown in this study improves the C. botulinum and related species classification scheme, enriching it with new strains of rare genotypes and permitting the highest grade of discrimination among strains for forensic and epidemiological applications.

Glycan detecting tools developed from the Clostridium botulinum whole hemagglutinin complex

Lectins are proteins with the ability to recognize and bind to specific glycan structures. These molecules play important roles in many biological systems and are actively being studied because of their ability to detect glycan biomarkers for many diseases. Hemagglutinin (HA) proteins from Clostridium botulinum type C neurotoxin complex; HA1, HA2, and HA3 are lectins that aid in the internalization of the toxin complex by binding to glycoproteins on the cell surface. HA1 mutants have been previously reported, namely HA1 W176A/D271F and HA1 N278A/Q279A which are specific to galactose (Gal)/N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) sugars, respectively. In this study, we utilized HA1 mutants and expressed them in complex with HA2 WT and HA3 WT to produce glycan detecting tools with high binding affinity. Particularly, two types were made: Gg and Rn. Gg is an Alexa 488 conjugated lectin complex specific to Gal and GalNAc, while Rn is an Alexa 594 conjugated lectin complex specific to Neu5Ac. The specificities of these lectins were identified using a glycan microarray followed by competitive sugar inhibition experiments on cells. In addition, we confirmed that Gg and Rn staining is clearly different depending on cell type, and the staining pattern of these lectins reflects the glycans present on the cell surface as shown in enzyme treatment experiments. The availability of Gg and Rn provide us with new promising tools to study Gal, GalNAc, and Neu5Ac terminal epitopes which can aid in understanding the functional role of glycans in physiological and pathological events.

Outbreak of avian botulism in a backyard poultry farming / Surto de botulismo aviário em criação de aves domésticas

The purpose of this study is to report an outbreak of avian botulism in backyard poultry farming. In 2019, a botulism outbreak in a flock of laying hens was investigated in Brazil. In the flock of 30 hens, clinical signs of botulism occurred after they ate decaying vegetables. A type C botulism outbreak was confirmed using the mouse lethality assay for detection of botulinum toxin in serum and ELISA test to detect Clostridium botulinum in intestinal contents and serum. Botulism in laying hens has rarely been reported. The chickens developed cyanotic comb and wattle, dyspnea, different degrees of flaccid paralysis in the neck, and detachment of feathers. No macroscopic lesions were observed, as were microscopic findings. The chicken's serum was neutralized by C antitoxin, confirming the botulism diagnosis, and also toxin was detected in intestinal contents.