This study describes, through a retrospective analysis, the epidemiological and clinicopathological findings of gas gangrene in horses in the Amazon biome. The study includes observations made in 20 animals from 2018 to 2023 by reviewing the clinical records of horses with muscle and mobility injuries treated at proprieties in the Amazon biome. The affected horses exhibited a sudden onset of severe lameness, swelling, and crepitus in the affected limb. The rapid progression of symptoms, including intense pain, fever, and toxemia, led to the clinical suspicion of gas gangrene. Immediate treatment was crucial, involving aggressive surgical debridement to remove necrotic tissue and high doses of broad-spectrum antibiotics to combat the infection. Despite these efforts, the condition of the horses deteriorated rapidly due to the aggressive nature of the disease and the challenging environment of the Amazon, which complicates timely veterinary intervention. The age range of the affected horses was from four months to 12 years, with a greater frequency in those over 12 months (80%, 16/20). Necropsy revealed histopathological lesions characterized by intense muscle necrosis, liquefaction, and gas formation, suggestive of Clostridium infection. The diagnosis was confirmed by multiplex PCR.

Several genetic tools have been developed for Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. A method based on the integration, by single crossing-over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing-over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows the modification of the genome without any marker or scar left in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties inducing mutations in the strain’s genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the bacteria became completely resistant to 5FU in the presence of the upp gene, resulting in failure in the selection for the second crossing-over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. We developed two plasmids, one overexpressing the native pyrR gene and a suicide plasmid carrying a non-mutated and optimized pyrR gene (pyrR*) and upp, which allowed us to restore the 5FU sensitivity of the strain. We also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1mM to 5 µM using a define synthetic medium.

Phenotypic complementation of gene knock-outs is an essential step in establishing function. Here we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9, but could be designed for any CRISPR/Cas system. For proof of concept, 9 bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by incorporation of ‘watermark’ sequences into the complementing genes.

Clostridioides (Clostridium) difficile is an enteric pathogen of neonatal pigs with zoonotic transmission remaining a significant problem in humans, including nosocomial resurgence, following oral administration of broad-spectrum antibiotics. To date, the immune response to C. difficile has mostly focused on neutrophils and cytokine/chemokines, particularly in human infection. We show that porcine monocytes respond to C. difficile differently compared with many other bacterial infections. Infection of porcine monocytes with human C. difficile strains CD630 (Ribotype 078) or R20291 (Ribotype 027) for 3 or 24h post-infection (pi) resulted in a lack of oxidative burst or nitrite ion production when compared to uninfected controls (P >0.05). The survival dynamics of both CD630 and R20291 in monocytes were similar with intracellular bacterial numbers being similar at 3h pi and 24h pi (P >0.05). However, we show that porcine monocytes entrap C. difficile via extracellular DNA traps. This process began as early as 3h pi and at 24h pi the nuclei appeared to be depleted of DNA, although extracellular DNA was associated with the cell membrane. Our preliminary study also suggests that entrapment of C. difficile by extracellular DNA may occur via a process of monocyte etosis.

Gas gangrene refers to severe necrotizing syndromes in soft tissues of animals caused by Clos-tridium. Five species of histotoxic clostridia are responsible for myonecroses. These agents require a port of entry, such as skin punctures with needles contaminated with bacterial spores. The present case report describes the clinicopathological findings of gas gangrene in a male Quarter horse living in the Brazilian Amazon biome. Clinical examination revealed clinical signs of toxemia and an enlarged left limb. The anamnesis found the administration of medication to this limb. The animal died five hours after its arrival at the veterinary hospital. At necropsy, histopathological lesions characterized by intense muscle necrosis, liquefaction, and gas formation were observed, suggestive of Clostridium lesions, which were confirmed by multiplex PCR. C. septicum was the etiological agent identified.

The genus Clostridium is an important group of pathogenic and nonpathogenic Gram-positive anaerobic bacteria with sporulation capacity and wide distribution in different environments, including the gastrointestinal tract of healthy and diseased animals and humans. Among the pathogenic species of the genus, Clostridium chauvoei stands out as a histotoxic agent. It causes important myonecroses such as blackleg, a disease with high lethality and mortality, especially in young cattle, and is responsible for significant losses to livestock worldwide. The pathogenic-ity of the disease is complex and has not yet been fully elucidated. The hypotheses cover pro-cesses from the initial absorption to the transport and deposition of the agent in the affected tis-sues. The virulence factors of C. chauvoei were divided into somatic and flagellar antigens and soluble antigens/toxins, which are the main antigens involved in vaccines against blackleg in Brazil and worldwide. This review provides important information on the first and current ap-proaches to the agent C. chauvoei and its virulence factors, as well as provides a compilation of data on Brazilian studies related to blackleg.

United States is the largest producer and the second largest exporter of broiler meat in the world. In the U.S, broiler production is largely converting to antibiotic-free programs which has caused an increase in morbidity and mortality within broiler farms. Escherichia coli and Clostridium perfringens are two important pathogenic bacteria readily found in the broiler environment and result in annual billion-dollar losses from colibacillosis, gangrenous dermatitis, and necrotic enteritis. Broiler industry is in search of non-antibiotic alternatives including novel vaccines, prebiotics, probiotics, and housing management strategies to mitigate production losses due to these diseases. This review provides an overview of the broiler industry and antibiotic free production, current challenges, and emerging research on antibiotic alternatives to reduce pathogenic microbial presence and improve bird health.

The induction of macrophage death is regarded as a potential mechanism by which components secreted by Clostridium septicum are used to evade the innate immune response and cause tissue damage. This study aimed to determine the effect of partially purified fractions of extracellular proteins secreted by C. septicum on the death of mouse peritoneal macrophages. Elicited mouse peritoneal macrophages were incubated with partially purified fractions of proteins secreted by C. septicum into culture medium. After incubation, we found that the protein fraction with a molecular weight ≥ 100 kDa caused significant cell death in macrophages, changed cell morphology, increased markers of apoptosis and autophagy, and increased the expression (protein and mRNA) of IL-1 and TNFα. Our data suggest that the proteins secreted by C. septicum (MW ≥100kDa) induce cell death in macrophages by promoting autophagy-triggered apoptosis. This may contribute to our understanding of the molecular mechanism of immune evasion by C. septicum at the infection site.

Botulinum neurotoxins (BoNTs) are emerging as multipurpose therapeutic compounds for the treatment of several different syndromes involving peripheral and central nervous systems, and muscular and musculoskeletal disorders both in human and veterinary medicine. Therefore, the studies of BoNTs are rapidly developing and identifying newly produced BoNT variants. Efforts should be made to clarify the biological and pharmacological characteristics of these novel BoNTs as well as the natural ones. The high potential of BoNTs as a therapeutic compound for medical syndromes lies on its ability to reach a specific cell type bypassing other cells, thus having mild or no side effects. In this paper the recent developments on BoNTs are reviewed with the aim to analyze the current knowledge on BoNTs biological mechanisms of action, immunogenicity, formulations, and therapeutic applications in the veterinary field, highlighting advantages and drawbacks and identifying the gaps to be filled in order to address research priorities.

The production of butanol, acetone and ethanol by Clostridium acetobutylicum is a biphasic fer-mentation process. In the first phase the carbohydrate substrate is metabolized to acetic and bu-tyric acid, in the following second phase the product spectrum is shifted towards the economi-cally interesting solvents. Here we present a cascade of six continuous stirred tank reactors (CCSTR), which allows performing the time dependent metabolic phases of an ace-tone-butanol-ethanol (ABE) batch fermentation in a spatial domain. Experimental data of steady states under four operating conditions - with variations of the pH in the first bioreactor between 4.3 and 5.6 as well as the total dilution rate between 0.042 1/h and 0.092 1/h - were used to optimize and validate a corresponding mathematical model. Beyond a residence time distribution representation and substrate, biomass and product kinetics this model also includes the differen-tiation of cells between the metabolic states. Model simulations predict a final butanol product concentration of 8.2 g/L and a butanol productivity of 0.75 g/(L h) in the CCSTR operated at a pH in bioreactor 1 of 4.3 and D = 0.092 1/h, while 31 % of the cells are differentiated to the solventogenic state. Aiming at an enrichment of solvent-producing cells, a feedback loop was introduced into the cascade - sending cells from a later state of the process (bioreactor 4) back to an early stage of the process (bioreactor 2). In agreement with the experimental observations, the model accurately predicted an increase of butanol formation rate in bioreactor stages 2 and 3, resulting in an overall butanol productivity of 0.76 g/(L h) for the feedback loop cascade. The here presented CCSTR and the validated model will serve to investigate further ABE fermentation strategies for a controlled metabolic switch.

In the recent past about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons, but are also recognized pharmaceuticals for efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here we report the primary structure of a ninth subtype of BoNT/F. Its amino acid sequence diverges by at least 8.4% at the holotoxin and 13.4% on the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q⁵⁸/K⁵⁹ bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6 and vary by up to factor eight. K_M values increase in the order F1 > F9 > F6 ≈ F7, whereas k_cat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.

A novel binary toxin-positive non-027, non-078 Clostridium difficile strain designated LC693 whose sequence type was ST201 was isolated from the fecal sample of a patient with severe diarrhea in China. To understand the pathogenesis basis of C. difficile ST201, this recently recovered isolate LC693 was then chosen for whole genome sequencing. The project finally generated an estimated genome size of approximately 4.07 Mbp. The genome sequence was then analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. Genome size of the three ST201 strains ranged from 4.07 Mb~4.16 Mb, with an average GC content between 28.5%~28.9%. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance, suggesting that they may have a strong antibiotic resistance. The ST201 strains contained a typical clade 3 specific PaLoc with a Tn6218 element inserted, and those genes harbored on their PaLoc that participated in the toxin expression and regulation were highly homologues to the epidemic 027 and 078 strains, with the exception of tcdC. A truncated TcdC was found in the ST201 strains, which is suggestive to have a contribution to the toxin production of the ST201 strains. In addition, the ST201 strains contained intact binary toxin coding and regulation genes, which is also proposed to contribute to the virulence. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for the C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The identification of those genes also contributes to the pathogenesis of the ST201 strain. To our knowledge, this is the first study that the genome sequence of C. difficile ST201 was discussed in detail, and the present study would have a contribution to understanding the pathogenesis basis of C. difficile ST201.

Avian necrotic enteritis is an enteric disease of broiler chickens caused by certain pathogenic strains of Clostridium perfringens in combination with predisposing factors. A vaccine offering complete protection against the disease has not yet been commercialized. In a previous study, we produced five recombinant proteins predicted to be surface-exposed and unique to necrotic enteritis-causing C. perfringens and the immunogenicity of these potential vaccine candidates was assessed in broiler chickens. In the current work, the relative contribution of the antibodies raised by these putative antigens to protect broiler chickens was evaluated using an experimental necrotic enteritis induction model. Additionally, the link between the immune response elicited and the gut microbiota profiles in immunized birds subjected to infection with virulent C. perfringens was studied. ELISA results showed that the IgY antibody titers in vaccinated birds on days 21 and 33 were significantly higher than those on days 7 and 14 and those in birds receiving the adjuvant alone, while the relative contribution of the specific immunity attributed to these antibodies could not be precisely determined using this experimental NE induction model. Besides, 16S rRNA gene amplicon sequencing showed that immunization of birds with recombinant proteins had a low impact on the chicken caecal microbiota.

Providing simultaneously autotrophic and heterotrophic carbon sources is a promising strategy to overcome the limits of autotrophic syngas fermentations. D-xylose and L-arabinose are particularly interesting as they can be obtained by the hydrolysis of lignocellulosic biomass. The individual conversion of varying initial concentrations of these pentoses and D-fructose as reference was studied with C. autoethanogenum in fully-controlled stirred-tank reactors with a continuous syngas supply. All mixotrophic batch processes showed increased biomass and product formation compared to an autotrophic reference process. Simultaneous CO and D-xylose or L-arabinose conversion was observed in contrast to D-fructose. In the mixotrophic batch processes with Larabinose or D-xylose, the simultaneous CO and sugar conversion resulted in high final alcohol to acid ratios of up to 58 g g-1. L-arabinose was superior as a mixotrophic carbon source because biomass and alcohol concentrations (ethanol and 2,3-butanediol) were highest, and significant amounts of meso-2,3-butanediol (>1 g L-1) in addition to D-2,3-butanediol (>2 g L-1) were solely produced with L-arabinose. Furthermore, C. autoethanogenum could not produce meso-2,3 butanediol under purely heterotrophic conditions. The mixotrophic production of meso-2,3-butanediol from Larabinose and syngas, both available from residual lignocellulosic biomass, is very promising for use as a monomer for bio-based polyurethanes or as an antiseptic agent.

This study aimed to investigate the efficacy of probiotics in elderly hospitalized tube-fed patients undergoing antibiotic therapy for Clostridium difficile-associated diarrhea (CDAD). Probiotics were administered to 29 patients, with twice-daily doses for 7 days following the completion of antibiotic treatment. In the probiotic group, while stool form showed no significant change, there was a significant decrease in stool frequency. Conversely, the control group exhibited improved stool form but not frequency. The probiotic group maintained a significant difference in stool form during the follow-up period. Additionally, the control group showed a positive correlation between stool form, frequency, antibiotic therapy, and intensive care unit admission, which was not observed in the probiotic group. These findings suggest that supplementing with probiotics can effectively normalize stool form in elderly hospitalized tube-fed patients undergoing antibiotic treatment for CDAD, highlighting the potential benefits of probiotic intervention in this specific population.

Increasing clinical and preclinical evidences implicate gut microbiome (GM) dysbiosis as a key susceptibility factor for neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s diseases (PD). In recent years, neurodegenerative diseases have not been viewed as driven solely by defects in brain, and the role of GM in modulating the central nervous system function via gut-brain axis has attracted considerable interest. Encouraged by GM researches, the next-generation probiotics may lead to tangible impacts on the treatment of neurodegenerative disorders. This review summarizes the current understandings of GM composition and characteristics associated with neurodegenerative diseases, and the demonstrations of molecules from GM affecting neurodegeneration. Particularly, applications of next-generation probiotics such as Clostridium butyricum, Akkermansia muciniphila, Faecalibacterium prausnitzii, and Bacteroides fragilis for remediation of neurodegenerative diseases are discussed.

Extracellular electron transfer (EET) is key to the success of microbial fuel cells (MFCs). Clostridium sp. often occurs in MFC anode communities, but its ability to perform EET remains controversial. We have employed Clostridium pasteurianum DSM 525 as biocatalyst in a glycerol-fed MFC, designated MFC_DSM. We have also followed the EET of this biocatalyst in the presence of a mediator, namely soluble neutral red (NR), soluble methyl viologen (MV), neutral red film (FNR), or methyl viologen film (FMV). MFC_DSM provided power and current densities (j) of 0.39 mW.cm^-2 and 2.47 mA.cm^-2, respectively, which evidenced that the biocatalyst performs direct electron transfer (DET). Introducing 150.0 µM NR or MV into the MFC_DSM improved the current density by 7.0- and 3.7-fold (17.05 and 8.45 mA.cm^-2), respectively. After 20 cyclic voltammetry (CV) cycles, the presence of FNR in the MFC_DSM anodic chamber provided almost twofold higher current density (30.76 µA.cm^-2) compared the presence of NR in the MFC_DSM. Introducing MV or FMV into the MFC_DSM anodic chamber gave practically the same current density after 10 CV cycles. The MFC_DSM anodic electrode might interact with FMV weakly than with FNR, so FNR is more promising to enhance C. pasteurianum DSM 525 EET within MFC_DSM.

Solventogenic Clostridium species are important for establishing sustainable industrial bioproduction of fuels and important chemicals. The inherent versatility of these species in substrate utilization and the range of solvents produced during acetone butanol-ethanol (ABE) fermentation make solventogenic Clostridium an attractive choice for biotechnological applications such as production of fuels and chemicals. The functional qualities of these microbes have thus been identified to be due to complex regulatory networks that play essential roles in modulating the metabolism of this group of bacteria. Genomes of solventogenic Clostridium species have relatively greater prevalence of genes that are intricately controlled by various regulatory molecules than most other species. Consequently, the use of genetic or metabolic engineering strategies that do not consider the underlying regulatory mechanisms will not be effective. Several regulatory factors involved in substrate uptake/utilization, sporulation, solvent production, and stress responses (Carbon Catabolite Protein A, Spo0A, AbrB, Rex, CsrA) have been identified and characterized. In this review, the focus is on newly identified regulatory factors in solventogenic Clostridium species, the interaction of these factors with previously identified molecules, and potential implications on substrate utilization, solvent production, and resistance/tolerance to lignocellulose-derived microbial inhibitory compounds.

Natural products have long played a major role in medicine and science. The garden snail Cornu aspersa is a rich source of biologically active natural substances which might be an important source for new drugs to treat human disease. Based on our previous studies seven fractions containing compounds with Mw <3 kDa, <10 kDa, <20 kDa, >20 kDa, and between 3-5 kDa, 5-10 kDa, and 10-30 kDa were purified from the mucus of C. aspersa and analyzed by tandem mass spectrometry (MALDI-TOF/TOF). Seventeen novel peptides with potential antibacterial activity have been identified by de novo MS/MS sequencing using tandem mass spectrometry. The different fractions were tested for antibacterial activity against Gram─ (Pseudomonas aureofaciens and Escherichia coli) and Gram+ (Brevibacillus laterosporus) bacterial strains as well anaerobic bacterium Clostridium perfringens. These results revealed that the peptide fractions exhibit a predominant antibacterial activity against B. laterosporus, the fraction with Mw 10 – 30 kDa against E. coli, another peptide fraction <20 kDa against P. aureofaciens, and the protein fraction >20 kDa against the bacterial strain C. perfringens. The discovery of new antimicrobial peptides (AMPs) from natural sources is of great importance for public health due to their effective antimicrobial activities and low resistance rates.

The presence of potentially pathogenic bacteria on veterinary clinic surfaces may be problematic. In this study we collected swab samples (Fisherbrand, double transport swabs with Stuart’s liquid medium) and water samples from five veterinary rehabilitation clinics. Swabs and water samples were transported to a microbiology lab for processing. At the lab swabs were used to inoculated Hardy’s Cdiff Banana broth (for Clostridium difficile [Cdiff]), and five different types of bacterial growth media, including: Hardy CHROM MRSA agar (methicillin resistant Staphylococcus aureus [MRSA], and S. pseudintermedius [SIM]), Mannitol Salt Agar (S. aureus), Eosin Methylene Blue agar (enterics [ENT]), Pseudomonas Isolation agar (Pseudomonas sp.), and Tryptic Soy Agar [TSA] (non-specific). The most prominent species cultured was Cdiff (on nearly 55% of swabs). Bacillus sp. and Enterics were encountered on nearly 35% of swabs, with MRSA and SIM on just over 10% of swabs. The most contaminated sample site was harnesses/life jackets used with the underwater treadmill (33% of swabs). Underwater treadmill water had total bacterial counts from 1.6 to 2.7 e3 cfu/ml. Of all bacterial species detected SIM tends to be more pathogenic for dogs. Targeted cleaning/disinfecting in these clinics could help reduce risks for both animals and caregivers utilizing these clinics.