Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

Clinical Safety and Tolerability of A2NTX, a Novel Low-Molecular-Weight Neurotoxin Derived from Botulinum Neurotoxin Subtype A2, in Comparison with Subtype A1 Toxins

All the botulinum type A neurotoxins available for clinical use are of the A1 subtype. We developed a subtype A2 low-molecular-weight (150 kD (kilo Dalton)) neurotoxin (A2NTX) with less spread and faster entry into the motor nerve terminal than A1 in vitro and in vivo. Preliminary clinical studies showed that its efficacy is superior to A1 toxins. We conducted an open study exploring its safety and tolerability profile in comparison with A1LL (LL type A1 toxin, or onabotulinumtoxinA) and a low-molecular-weight (150 kD) A1 neurotoxin (A1NTX). Those who had been using A1LL (n = 90; 50–360 mouse LD50 units) or A1NTX (n = 30; 50–580 units) were switched to A2NTX (n = 120; 25–600 units) from 2010 to 2018 (number of sessions ~27, cumulative doses ~11,640 units per patient). The adverse events for A2NTX included weakness (n = 1, ascribed to alcoholic polyneuropathy), dysphagia (1), local weakness (4), and spread to other muscles (1), whereas those for A1LL or A1NTX comprised weakness (n = 2, A1NTX), dysphagia (8), ptosis (6), local weakness (7), and spread to other muscles (15). After injections, 89 out of 120 patients preferred A2NTX to A1 for the successive sessions. The present study demonstrated that A2NTX had clinical safety up to the dose of 500 units and was well tolerated compared to A1 toxins.

Clinical Safety and Tolerability of A2NTX, a Novel Low Molecular Weight Neurotoxin Derived From Botulinum Toxin Subtype A2, in Comparison with Subtype A1 Toxins

All the available botulinum type A neurotoxins for clinical uses are of A1 subtype. We developed a subtype A2 low molecular weight (150kD) neurotoxin (A2NTX), with less spread and faster entry into the motor nerve terminal than A1 in vitro and in vivo. Preliminary clinical studies showed its efficacy superior to A1 toxins. We conducted an open study exploring its safety and tolerability profile in comparison with A1LL (onabotulinumtoxinA) and low molecular weight (150kD) A1 neurotoxin (A1NTX). Those who had been using A1LL (n=90; 50-360 mouse LD50 units) or A1NTX (n=30; 50-580 units) were switched to A2NTX (n=120; 25-600 units) from 2010 till 2018 (number of sessions ~ 27, cumulative doses ~11,640 units per patient). Adverse events for A2NTX included weakness (n=1, ascribed to alcoholic polyneuropathy), dysphagia (1), local weakness (4), spread to other muscles (1), whereas those for A1LL or A1NTX comprised weakness (n=2, A1NTX), dysphagia (8), ptosis (6), local weakness (7) and spread to other muscles (15). After injections, 89 out of 120 patients preferred A2NTX to A1 for the successive sessions. The present study demonstrated that A2NTX had the clinical safety up to the dose of 500 units, and was well tolerated compared to A1 toxins.

Clostridia in Insect Processed Animal Proteins—Is an Epidemiological Problem Possible?

The aim of this study was the evaluation of the insect processed animal protein (IPAP) contamination level by Clostridium spp. Particularly, we screened for the occurrence of pathogenic species of Clostridia. The samples of IPAP were derived from yellow mealworm (Tenebrio molitor) and black soldier fly (Hermetia illucens) available in the Polish market. The IPAPs were added to experimental feeds for poultry. The differences between the contamination levels of the control (without the addition of IPAP) and experimental (with the addition of IPAP) groups were monitored. The samples were also examined by culture and PCR-based methods to detect 16S rDNA and genes determining botulinum toxin (BoNT) production. Statistical significance was noticed among the feed with the IPAP addition, as well as an increase of contamination by Clostridium spp. In one sample of IPAP, the occurrence of ntnh and bont/D genes determining the production of BoNT/D was noticed. However, a positive result was noticed only at the step of the liquid culture; the Clostridium botulinum type D strain was not isolated. Phenotypically, and according to the 16S rDNA analysis, genetically similar strains to C. botulinum species were isolated. Considering the microbiological safety of IPAP and expanding possibility of its use in livestock animal feed, it seems to be reasonable to provide complex risk assessment on the potential transfer of Clostridia into feed compounds, to assure the safety and sustainable development of insect PAP industry.

Experience of Botulinum Toxin A Injections for Chronic Migraine Headaches in a Pediatric Chronic Pain Clinic

OBJECTIVES Prevalence of chronic migraine in children can reach 7.7%, causing decreased school performance, difficulty with extracurricular activities (including sports, theater, or music), and changes in sleep and mood. Many studies confirm that botulinum toxin type A injections effectively alleviate chronic migraines in adults; however, the literature regarding children is sparse. This study aims to analyze the safety and effectiveness of botulinum type A injections in a group of pediatric patients diagnosed with chronic migraines in a pediatric pain clinic. METHODS In this retrospective (2013–2018) study, the effects of botulinum toxin type A injections were analyzed using data from 65 pediatric patients diagnosed with chronic migraines. The study group ranged from 11 to 18 years of age. A pediatric pain management physician administered the botulinum using the Phase 3 Research Evaluating Migraine Prophylaxis Therapy program protocol and followed the pain pattern. Dosages, tolerance, and side effects were measured. RESULTS In this study, 74% of the patients tried more than 6 medications before the injections. There was a decrease in the visual analog scale score of 5.2 ± 2.2 points upon 6-week follow-up. The mean amount of medication used was 173.2 ± 35 units, and patients received an average of 2.8 ± 1.1 units/kg. Adverse events include one patient who developed dizziness and another who had low-grade fevers with enlarged cervical lymph nodes; both resolved within few minutes. CONCLUSIONS This study supports the use of botulinum type A for chronic migraines in pediatric patients. Multicentered, randomized studies with larger population are needed to evaluate the long-term safety and efficacy of this therapy.

Individual Factors of Botulinum Type A in Treatment of Gummy Smile: A Prospective Study

Background Botulinum type A (BTX-A) injection is a promising corrective method for gummy smile (GS). However, its effect among patients is varied and inconsistent. Objective To explore the effect of individual factors on BTX-A treatment for GS and the degree of their influence, and to establish the indications of average-dose BTX-A injection for GS treatment. Methods In this prospective clinical study, a standardized BTX-A injection technique comprising bilateral single-point injections of 2 U BTX-A (total, 4 U) was administered to all GS patients. Data were collected at baseline and 4, 12, and 32 weeks of follow-up. Twenty-nine potential individual factors were analyzed using correlation and regression analysis to exclude confounding bias. Results In all, 94 patients completed the BTX-A injection. After adjusting for potential confounding factors such as exposed medial incisor, medial incisor length, width-to-length ratio of the medial incisor length, overbite and overjet of the anterior teeth, the correlation and regression analysis confirmed the following formula (adjusted R 2 = 0.617, P ≤ 0.001): anterior gingival exposure (GE) at 4 weeks = 1.44 + (0.94 × baseline anterior gingival exposure) – (1.88 × sex) (where male = 1 and female = 2). The confidence interval(CI) of the prediction showed that for all female participants with baseline anterior GE <5.3 mm, the 95%CI of anterior GE was 0.3–3.0 mm after 4 weeks of this average dose of BTX-A treatment, and it was 3.0–8.9 mm for all female participants with baseline anterior GE ≥6 mm. This value would likely be between 1.5 mm and 3.3 mm for male patients with a baseline anterior GE of 3 mm, which was between 3.2 mm and 8.9 mm for male patients with baseline anterior GE ≥4.6 mm. Conclusion The individual effect of the average dose of BTX-A treatment for GS was GS severity and patient’s sex, rather than GS etiology and other individual factors. Further, female participants with baseline anterior GE<5.3 mm were more likely to show complete improvement after 4 weeks of this average dose of BTX-A treatment. However, female participants with baseline anterior GE ≥6.0 mm or male participants were less likely to show complete improvement at 4 weeks.