Embracing the Versatility of Botulinum Neurotoxins in Conventional and New Therapeutic Applications.

Botulinum neurotoxins (BoNTs) have been used for almost half a century in the treatment of excessive muscle contractility. BoNTs are routinely used to treat movement disorders such as cervical dystonia, spastic conditions, blepharospasm, and hyperhidrosis, as well as for cosmetic purposes. In addition to the conventional indications, the use of BoNTs to reduce pain has gained increased recognition, giving rise to an increasing number of indications in disorders associated with chronic pain. Furthermore, BoNT-derived formulations are benefiting a much wider range of patients suffering from overactive bladder, erectile dysfunction, arthropathy, neuropathic pain, and cancer. BoNTs are categorised into seven toxinotypes, two of which are in clinical use, and each toxinotype is divided into multiple subtypes. With the development of bioinformatic tools, new BoNT-like toxins have been identified in non-Clostridial organisms. In addition to the expanding indications of existing formulations, the rich variety of toxinotypes or subtypes in the wild-type BoNTs associated with new BoNT-like toxins expand the BoNT superfamily, forming the basis on which to develop new BoNT-based therapeutics as well as research tools. An overview of the diversity of the BoNT family along with their conventional therapeutic uses is presented in this review followed by the engineering and formulation opportunities opening avenues in therapy.

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Intra-species diversity of Clostridium perfringens: A diverse genetic repertoire reveals its pathogenic potential.

Clostridium perfringens is the causative agent of many enterotoxic diseases in humans and animals, and it is present in diverse environments (soil, food, sewage, and water). Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) have provided a general approach about genetic diversity of C. perfringens; however, those studies are limited to specific locations and often include a reduced number of genomes. In this study, 372 C. perfringens genomes from multiple locations and sources were used to assess the genetic diversity and phylogenetic relatedness of this pathogen. In silico MLST was used for typing the isolates, and the resulting sequence types (ST) were assigned to clonal complexes (CC) based on allelic profiles that differ from its founder by up to double-locus variants. A pangenome analysis was conducted, and a core genome-based phylogenetic tree was created to define phylogenetic groups. Additionally, key virulence factors, toxinotypes, and antibiotic resistance genes were identified using ABRicate against Virulence Factor Database (VFDB), TOXiper, and Resfinder, respectively. The majority of the C. perfringens genomes found in publicly available databases were derived from food (n = 85) and bird (n = 85) isolates. A total of 195 STs, some of them shared between sources such as food and human, horses and dogs, and environment and birds, were grouped in 25 CC and distributed along five phylogenetic groups. Fifty-three percent of the genomes were allocated to toxinotype A, followed by F (32%) and G (7%). The most frequently found virulence factors based on > 70% coverage and 99.95% identity were plc (100%), nanH (99%), ccp (99%), and colA (98%), which encode an alpha-toxin, a sialidase, an alpha-clostripain, and a collagenase, respectively, while tetA (39.5%) and tetB (36.2%), which mediate tetracycline resistance determinants, were the most common antibiotic resistance genes detected. The analyses conducted here showed a better view of the presence of this pathogen across several host species. They also confirm that the genetic diversity of C. perfringens is based on a large number of virulence factors that vary among phylogroups, and antibiotic resistance markers, especially to tetracyclines, aminoglycosides, and macrolides. Those characteristics highlight the importance of C. perfringens as a one of the most common causes of foodborne illness.

Clostridium perfringens Associated with Foodborne Infections of Animal Origins: Insights into Prevalence, Antimicrobial Resistance, Toxin Genes Profiles, and Toxinotypes.

Several food-poisoning outbreaks have been attributed to Clostridium perfringens (C. perfringens) worldwide. Despite that, this crisis was discussed in a few studies, and additional studies are urgently needed in this field. Therefore, we sought to highlight the prevalence, antimicrobial resistance, toxin profiles, and toxinotypes of C. perfringens isolates. In this study, 50 C. perfringens isolates obtained from 450 different animal origin samples (beef, chicken meat, and raw milk) were identified by phenotypic and genotypic methods. The antimicrobial susceptibility results were surprising, as most of the isolates (74%) showed multidrug-resistant (MDR) patterns. The phenotypic resistance to tetracycline, lincomycin, enrofloxacin, cefoxitin/ampicillin, and erythromycin was confirmed by the PCR detections of tet, lnu, qnr, bla, and erm(B) genes, respectively. In contrast to the toxinotypes C and E, toxinotype A prevailed (54%) among our isolates. Additionally, we found that the genes for C. perfringens enterotoxin (cpe) and C. perfringens beta2 toxin (cpb2) were distributed among the tested isolates with high prevalence rates (70 and 64%, respectively). Our findings confirmed that the C. perfringens foodborne crisis has been worsened by the evolution of MDR strains, which became the prominent phenotypes. Furthermore, we were not able to obtain a fixed association between the toxinotypes and antimicrobial resistance patterns.

Clostridium perfringens Types A and D Involved in Peracute Deaths in Goats Kept in Cholistan Ecosystem During Winter Season.

Enterotoxemia is a severe and peracute disease caused by Clostridium perfringens (C. perfringens) rendering high mortality leading to huge economic losses, especially in small ruminants. The bacterium induces peracute death in animals based on the rapid production of different lethal toxins. Mortality occurred three private herds of two breeds, i.e., Makhi Cheeni and Beetal, and one non-descriptive (Teddy) herds reared in the desert area of Bahawalpur, Pakistan. At necropsy, tissue samples for histopathology and intestinal contents for bacterial isolation and culture were collected. Following the standard procedure, tissue slides were prepared. Multiplex PCR was used to identify toxinotypes using specific primers. Morbidity, mortality, and case fatality in Makhi Cheeni, Beetal, and Teddy goats caused by enterotoxemia were 87.58, 75.81, and 76.11%, respectively. Based on toxinotypes in the present outbreaks, C. perfringens type A (cpα = 20.7%; cpα + cpß2 = 11.2%) and C. perfringens type D (cpα + cpß2 + etx = 47.7%; cpα + etx = 20.7%) were detected. Deaths due to C. perfringens type D (68.10%) were significantly higher (p < 0.001) compared with deaths by C. perfringens type A (34.90%). Petechiation of serosal surfaces, hemorrhage of intestines, lungs, and liver were seen. Kidneys were soft, and under the microscope, tubules were studded with erythrocytes. There was stunting and fusion in the intestinal villi. From this study, we concluded that endotoxemia can occur in any season; thus, a proper vaccination schedule must be followed for the protection of small ruminants' health.

Phenotypic detection and genotyping of Clostridium perfringens associated with enterotoxemia in sheep in the Qassim Region of Saudi Arabia.

BACKGROUND AND AIM: Enterotoxemia caused by Clostridium perfringens toxinotypes is an often fatal disease of sheep of all ages, with a substantial economic loss to the sheep industry. This study was conducted to isolate C. perfringens from suspected cases of enterotoxemia in sheep in the central part of the Qassim Region, Saudi Arabia, and to determine the prevalent toxinotype by detecting alpha (cpA), beta (cpB), and epsilon (etX) toxin genes, which might help control this disease locally. MATERIALS AND METHODS: A total of 93 rectal swabs and intestinal content samples were collected from diseased and animals suspected of having died of enterotoxemia in early 2020. Samples were subjected to bacteriological examination, biochemical analysis of isolates by VITEK 2, and molecular toxinotyping of isolates by LightCycler® real-time polymerase chain reaction (RT-PCR). RESULTS: Our results revealed that only 14 isolates were confirmed by VITEK 2 as being C. perfringens, with excellent identification (probability of 95% and 97%). According to the toxinotyping of isolates by RT-PCR, all 14 isolates possessed both the cpA and etX toxin genes, while the cpB toxin gene was not detected in any of the isolates. CONCLUSION: Our findings demonstrated that C. perfringens type D was the only toxinotype found in the central part of the Qassim Region in 2020; moreover, according to the culture method, only 15% (14/93) of the suspected cases of enterotoxemia were confirmed to be caused by C. perfringens infection, which highlighted the importance of clinical and laboratory differential diagnosis of enterotoxemia in sheep.

Comparative genomics of Clostridioides difficile toxinotypes identifies module-based toxin gene evolution.

Clostridioides difficile is a common cause of nosocomial diarrhoea. Toxins TcdA and TcdB are considered to be the main virulence factors and are encoded by the PaLoc region, while the binary toxin encoded in the CdtLoc region also contributes to pathogenicity. Variant toxinotypes reflect the genetic diversity of a key toxin-encoding 19 kb genetic element (the PaLoc). Here, we present analysis of a comprehensive collection of all known major C. difficile toxinotypes to address the evolutionary relationships of the toxin gene variants, the mechanisms underlying the origin and development of variability in toxin genes and the PaLoc, and the relationship between structure and function in TcdB variants. The structure of both toxin genes is modular, composed of interspersed blocks of sequences corresponding to functional domains and having different evolutionary histories, as shown by the distribution of mutations along the toxin genes and by incongruences of domain phylogenies compared to overall C. difficile cluster organization. In TcdB protein, four mutation patterns could be differentiated, which correlated very well with the type of TcdB cytopathic effect (CPE) on cultured cells. Mapping these mutations to the three-dimensional structure of the TcdB showed that the majority of the variation occurs in surface residues and that point mutation at residue 449 in alpha helix 16 differentiated strains with different types of CPE. In contrast to the PaLoc, phylogenetic trees of the CdtLoc were more consistent with the core genome phylogenies, but there were clues that CdtLoc can also be exchanged between strains.

Molecular Characterization of Clostridium perfringens Strains Isolated in Italy.

Clostridium (C.) perfringens is the causative agent of several diseases and enteric infections in animals and humans. The pathogenicity of the bacterium is largely mediated by the production of a wide range of toxins. Individual C. perfringens strains produce only subsets of this toxin repertoire, which permits the classification in seven toxinotypes (A-G). In addition, a variety of minor toxins further characterizes the single strains. The aim of this work was to evaluate, using Polymerase Chain Reaction (PCR) assays, the diversity of 632 C. perfringens strains isolated in Italy over 15 years. The genotyped strains were analyzed to determine the presence of major and minor toxins (cpe, consensus, and atypical cpb2), their geographical origins, and the source of isolation (animal species or food). Our study shows that toxinotype A had the greatest representation (93%) and correlated mainly with consensus cpb2 in a variety of animal species, as well as with atypical cpb2 in the five food samples. Type D, associated with cpe and atypical cpb2 minor toxins, was identified in 3% of the cases, and type F was identified in 2.5%. Seven type C isolates (1.1%) were detected in cattle, whereas the only type B atypical cpb2 isolated in Italy was detected in a goat, and one type E cpe+atypical cpb2 was detected in a sheep. Type G was not detected.

Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period.

Digestate produced by agricultural biogas plants (BGPs) may contain pathogenic bacteria. Among them, Clostridium perfringens deserves particular attention due to its ability to grow under anaerobic conditions and persist in amended soil. The aim of this study was to examine the potential pathogenicity and the antimicrobial resistance of C. perfringens in manure and digestate collected from three agricultural biogas plants (BGPs). A total of 157 isolates (92 from manure, 65 from digestate) were screened for genes encoding seven toxins (cpa, cpb, etx, iapcpe, netB, and cpb2). The 138 cpa positive isolates were then screened for tetA(P), tetB(P), tet(M), and erm(Q) genes and tested for antimicrobial susceptibility. The toxinotypes identified in both manure and digestate were type A (78.3% of the isolates), type G (16.7%), type C (3.6%), and type D (1.4%), whereas none of the isolates were type F. Moreover, half of the isolates carried the cpb2 gene. The overall prevalence of tetA(P) gene alone, tetA(P)-tetB(P) genes, and erm(Q) gene was 31.9, 34.8, and 6.5%, respectively. None of the isolates harbored the tet(M) gene. Multiple antimicrobial resistant isolates were found in samples that were collected from all the manure and digestates. Among them, 12.3% were highly resistant to some of the antibiotics tested, especially to clindamycin (MIC ≥ 16 µg/mL) and tilmicosin (MIC > 64 µg/mL). Some isolates were highly resistant to antibiotics used in human medicine, including vancomycin (MIC > 8 µg/mL) and imipenem (MIC > 64 µg/mL). These results suggest that digestate may be a carrier of the virulent and multidrug resistant C. perfringens.

Multilocus sequence typing of Clostridium perfringens strains from neonatal calves, dairy workers and associated environment in India.

Clostridium perfringens is a globally recognized zoonotic pathogen. We report isolation and genotyping of C. perfringens from neonatal calves, dairy workers and their associated environment in India. A total of 103 fecal samples from neonatal calves, 25 stool swabs from the dairy workers and 50 samples from their associated environment were collected from two dairy farms. C. perfringens was detected in 26 out of 103 (25.2%) neonatal calf samples, 7 out of 25 (28%) human stool samples and 17 out of 50 (34%) environmental samples. C. perfringens type A strains were predominant in neonatal calves (24/26; 92.3%) and associated environment (15/17; 88.2%). In contrast, strains from dairy workers mostly belonged to type F (5/7; 71.4%), which also carried the beta2 toxin gene. Seventeen strains were analyzed by multilocus sequence typing (MLST) for studying genotypic relationship along with 188 C. perfringens strains available from public databases. A total of 112 sequence types (STs) were identified from 205 C. perfringens strains analyzed. A Clonal complex (CC) represented by three STs (ST 98, ST 41 and ST 110) representing predominantly type F (18/20 strains) were mostly associated with human illnesses. Among predominant STs, ST 54 was associated with enteritis cases in foals and dogs and ST 58 associated with necrotic enteritis in poultry. Seventeen Indian strains were assigned to 13 STs. Genetic relatedness among strains of calves, dairy worker and associated environments indicate inter-host transfers and zoonotic spreads.

Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia.

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.

Caracterización molecular y resistencia antimicrobiana de aislamientos de Clostridium perfringens de diferentes orígenes en Costa Rica

Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica. Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins α, β, ε and ι, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80°C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils,20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only α toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments. Rev. Biol. Trop. 59 (4): 1479-1485. Epub 2011 December 01.

Clostridium perfringens es un bacilo Gram positivo, esporulado, anaerobio, ampliamente distribuido en la naturaleza, que produce cuatro toxinas principales α, β, ε y ι, las cuales permiten su clasificación en cinco toxinotipos (A-E). Algunas cepas producen una enterotoxina (CPE), codificada por el gen cpe, que causa diarrea en seres humanos y en algunos animales. La presencia de los genes de estas toxinas y la sensibilidad a los antibióticos se determinó en 81 cepas de C. perfringens previamente aisladas y que habían sido mantenidas a -80°C; 20 de suelos, 20 de origen animal, 20 de origen humano y 21 de alimentos cocidos no relacionados con brotes alimentarios. De acuerdo con los resultados de PCR, todas las cepas fueron clasificadas como C. perfringens tipo A, debido a que solo se les detectó el gen de la toxina α, mientras que el gen de la enterotoxina (cpe) se detectó en dos cepas (2.5%) aisladas de alimentos, tal como ha sido descrito en otras regiones del mundo. El 44% de las cepas fue resistente a algún antibiótico; clindamicina (41%), cloranfenicol (25%), penicilina (22%) y metronidazol (20%). En general, las cepas provenientes de suelos presentaron los mayores porcentajes de resistencia a casi todos los antibióticos. El 40% de las cepas de suelo presentó multiresistencia (a tres o más grupos de antibióticos), el 30% de las de origen humano, el 14% de las de alimentos y el 5% de las de origen animal. Las altas tasas de resistencia encontradas podrían deberse al amplio uso de antibióticos como promotores de crecimiento de plantas y animales y esas cepas resistentes podrían actuar como reservorio de genes de resistencia que pueden transferirse entre bacterias de diversos ambientes.