Curing salts, nitrite and nitrate salts, are multifunctional additives in meat matrices, responsible for generating and maintaining desirable sensory attributes throughout storage, as well as ensuring microbiological safety, particularly against Clostridium botulinum, the causative agent of a highly lethal intoxication in humans. However, the undesirable formation of N-nitrosamines, potentially carcinogenic compounds, is also associated with nitrite addition. Consequently, regulatory reevaluations and scientific investigations have sought alternatives to these curing salts that balance food safety and sensory acceptance while mitigating health risks linked to nitrite consumption. This review presents and updates the principal strategies explored for the partial or complete replacement of nitrite in meat products, covering well-established approaches such as the use of colorants, biopreservation techniques, and the incorporation of natural nitrate sources, as well as an unprecedented perspective by examining, for the first time, the potential of S-nitrosothiols as viable substitutes.

In Kotzebue, Alaska, traditional Qikiqtaġruŋmiut food practices help reduce risks of food-borne diseases from locally harvested foods. Changes to climate and societal change affecting food systems may introduce new pathogens (e.g., avian influenza, neurotoxins from harmful algal blooms) to local food resources or increase the potential for known pathogens to cause illness (e.g., Clostridium botulinum, Brucella spp., Toxoplasma gondii). To learn more about local concerns and knowledge, we first discussed the topic with five knowledgeable Elders, then conducted formal interviews with five Qikiqtaġruŋmiut, from young adults to Elders, selected for experience gathering, processing, and preparing traditional foods. We also reviewed published literature, selected through keyword searches, on our topic to provide a preliminary assessment of how current practices reduce or exacerbate risks from food-borne pathogens. We found that traditional practices, such as inspecting animals and foods, freezing, drying, and cooking, among others, remain effective for reducing some risks, if those practices are followed carefully. We also found that some emerging pathogens (e.g., harmful algal bloom toxins) are likely to evade detection or mitigation using traditional practices, and thus may be a priority for monitoring and raising awareness. Further steps to reducing the risks from food-associated diseases include emphasizing the importance of sustaining Qikiqtaġruŋmiut knowledge and practices as the basis for food safety in the community and supporting mutual learning between traditional food practitioners, wildlife veterinarians, and public health officials. We believe our findings have relevance to other northern communities with similar food resources and practices.

NEDD8 is a ubiquitin-like molecule that is conjugated to lysine residues of protein substrates in a process known as neddylation, which affects their stability, degradation, and interactions. It was recently found that the neddylation inhibitor MLN4924 prevents endocytic uptake of herpes simplex virus (HSV) into target cells, implicating a broader role for neddylation in viral entry and infection. Because AB-type bacterial toxins share similar cell entry mechanisms with viruses, we studied the impact of neddylation in the cellular internalization of a prototypical, well-established model of bacterial AB-type toxins, namely the diphtheria toxin (DT). We found that pretreatment of HeLa cells with MLN4924 reduced morphological changes and protein biosynthesis inhibition induced by DT. Arctigenin, another neddylation inhibitor acting at a later step of the neddylation cascade, was also capable of reducing DT intoxication in HeLa cells. In combination, both neddylation inhibitors synergistically increased their inhibitory potential against DT. Further mechanistic studies with MLN4924 revealed that the neddylation inhibitor neither interfered with cell surface binding, proteolytic activation, enzymatic activity, and membrane translocation of DT, nor with endosomal acidification in HeLa cells. Finally, we could demonstrate that pharmacological inhibition of neddylation also affects further bacterial protein toxins, namely single-chain toxin Clostridioides difficile TcdB and binary toxin Clostridium botulinum C2. In conclusion, our study is the first to support a connection between neddylation and the cellular uptake of bacterial AB-type toxins, thereby establishing a foundation for future investigations into these molecular mechanisms and for the development of anti-toxin strategies.IMPORTANCENeddylation, a ubiquitin-like protein modification, is emerging as a critical regulator of viral entry into cells. Here, we demonstrate for the first time that pharmacological inhibition of neddylation impairs the intoxication of target cells with diverse bacterial AB-type toxins. Mechanistic studies suggest that neddylation most likely reduces toxin internalization, thus supporting the connection between neddylation and endocytic processes. Our study offers new opportunities to explore anti-toxin interventions and highlights the use of bacterial toxins as molecular probes to further study the role of neddylation in endocytic trafficking.

Timeliness and Diagnostic Yield of Suspected Botulism Notifications in a Tertiary Medical Center in Taiwan, 2014-2024.

Quantitative Microbial Risk Assessment of Clostridium botulinum in Chicken Mortadella under Ambient and Refrigerated Storage Conditions

The kinetics of inactivation of Clostridium sporogenes spores as a surrogate for the sterilization marker Clostridium botulinum using Pulsed Electric Field (PEF) treatment has not been previously investigated. Hence, this study aimed to evaluate and determine the kinetics of C. sporogenes spore inactivation by PEF. The spore suspensions of C. sporogenes were treated using PEF at an electric field intensity of 36 kV/cm with a residence time of 1.302 s. The spore inactivation kinetics were analyzed using several mathematical models, including log-linear, log-linear shoulder, and Weibull models. The electroporation effects were evaluated by measuring the concentration of Ca 2+ released into the suspension following PEF treatment. PEF treatment resulted in a reduction of C. sporogenes spores by up to 3.6 log cycles. The inactivation curve of C. sporogenes spores demonstrated a concave downward shape, with a shoulder pattern at the initial time and an exponential decrease as the process time increased. The spore electroporation was evidenced by an increase in Ca 2+ concentration in the suspension from 1.58 to 2.03 ppm after PEF treatment. The death rate of C. sporogenes spores ( kmax ) increased and the shoulder length ( S i ) decreased with higher electric field strengths. A concave downward curve of the inactivation curve is shown by a curve shape value ( p )> 1. The log-linear shoulder was the best model to describe C. sporogenes spore inactivation. The D PEF and Z PEF values of C. sporogenes spores in the log-linear, log-linear shoulder and Weibull models were in the ranges 2.46–4.222 s and 25–32 kV/cm. These findings demonstrate that PEF can effectively inactivate C. sporogenes spores and that kinetic modeling provides valuable parameters for predicting spore inactivation during PEF processing.

Volume 14, no. 3, e00988-24, 2025, https://doi.org/10.1128/mra.00988-24. In the Abstract, in the second sentence, “The genome of X58540 consists of 2,371,631 bp with a GC content of 28.17% and is predicted to include 2,157 coding sequences.” should read “The genome of X58540 consists of 3,815,652 bp with a GC content of 28.17% and is predicted to include 3,461 coding sequences.”

Chrysomya megacephala is a synanthropic fly with a high potential to act as a mechanical vector of pathogenic bacteria, surpassing Musca domestica in both bacterial load and diversity. Native to Asia and Africa, it has become a cosmopolitan species, successfully adapting to a wide range of environments, including natural ecosystems. In Colombia, studies on its role as a vector are limited and have largely relied on traditional culturing methods. This study aimed to characterize the pathogenic bacterial microbiota associated with C. megacephala using 16S rRNA gene sequencing in urban, rural, and forest settings of a coastal tourist city. Flies were collected using Van Someren Rydon traps with attractants and sterile materials. Bacterial identification was performed through Oxford Nanopore MinION sequencing (Manufactured by Oxford Nanopore Technologies, Oxford, UK). A total of 49 bacterial species were identified, with urban environments showing the highest taxonomic richness. The forest environment was characterized by a highly dominant community structure, led by Vagococcus carniphilus. Notably, 20 bacterial species of public health relevance were detected, including Clostridium botulinum, Clostridium perfringens, Ignatzschineria ureiclastica, Escherichia coli, and Streptococcus agalactiae. These findings indicate that bacterial community composition varies by environment and underscore the potential role of C. megacephala as a mechanical vector, highlighting the importance of surveillance for its public health implications.

The process of ensuring the safety of the food supply is dynamic. Both the possibility of contamination and the effectiveness of safety precautions are impacted by changes in the kinds of food consumed, the geographical origins of food products, and the methods by which these foods are processed. For instance, compared to earlier generations, consumers’ general understanding of safe food preparation and handling techniques has decreased due to a higher reliance on prepackaged convenience foods. Nowadays, consumers depend increasingly on other people to make sure the food they eat is safe. Growing consumption of minimally processed foods and growing imports of fresh products from other nations have resulted from changes in consumer tastes and food processing technologies. This review aims to critically synthesize existing knowledge on microbial risks in food, focusing on their sources, mechanisms of contamination, risk evaluation methodologies, and implementation of food safety measures. Major foodborne pathogens, including Salmonella, Escherichia coli, Listeria monocytogenes, and Norovirus, are discussed alongside factors influencing their survival and transmission. Today Clostridium botulinum, Staphylococcus aureus, and Salmonella spp. remain among the major foodborne pathogens, but during the last two decades food-borne diseases such as shigellosis, listeriosis, campylobacteriosis, and diseases caused by pathogenic strains of Escherichia coli have become increasingly salient. These new concerns necessitate continued investment in research and technology development to improve the safety of the food supply. The review highlights current approaches to microbiological risk assessment, regulatory frameworks, and control strategies, while also addressing emerging challenges such as antimicrobial resistance, biofilms, and ready-to-eat foods. By integrating risk evaluation with practical implementation strategies, this review provides valuable insights for researchers, regulators, and food industry stakeholders seeking to strengthen food safety systems and reduce the burden of foodborne diseases.

Introduction: Botulism is a neuroparalytic disorder caused by botulism toxin produced by Clostridium botulinum from foodborne exposure, wound contamination, or intestinal colonization. Botulism presents with bulbar weakness and descending paralysis. Early recognition is crucial for improving morbidity and mortality. We present a rare case of botulism secondary to consumption of spoiled food. Description: A 68-year-old male with a past medical history of ischemic stroke and polysubstance abuse was transferred to our tertiary care center for evaluation of acute dysphagia and neuromuscular weakness. The patient was functionally independent prior to admission but was food insecure and had eaten spoiled food. He was intubated for NIF of -10, airway protection, and management of secretions. On initial exam, he was unable to open his eyes but otherwise alert, oriented, and engaged with yes/no questions (able to nod or use thumbs up). Pupils were equal and reactive to light, but visually sluggish. Further exam revealed complete ophthalmoplegia and absent corneal, cough, and gag reflexes. Neck flexion and extension were weak at 1/5 strength, upper extremities were 2/5 proximally and 4/5 distally, and lower extremities were 2/5 proximally and 5/5 distally. Sensation was diminished at bilateral feet but otherwise intact to all modalities. Reflexes were absent throughout, with no pathological reflexes seen. The team promptly recognized that this could be botulism and was able to administer the antitoxin via the CDC. Unfortunately, the patient continued to decline with complete loss of cranial nerve function and worsened motor exam of the proximal upper extremities. The patient had a protracted course in the ICU with slow neurologic recovery and required tracheostomy and PEG tube placement. Prior to discharge, his exam was slightly improved with neck strength at 3/5 for both flexion and extension. Discussion: Botulism is a life-threatening condition presenting with descending paralysis, absence of fever, and intact mental status. Recovery may take months as nerve pathways slowly regenerate. Food insecurity is not a traditional risk factor, as compared to consumption of honey or canned goods; however, as this case exhibits, botulism should be considered in a food-insecure patient with these clinical findings.

Recent progress in typical foodborne spore-forming bacteria detection.

Chronic migraine (CM) is the most prevalent headache-related disorder that affects 1% to 2% of the world's population. OnabotulinumtoxinA (BoNT-A) is a potent neurotoxin produced by Clostridium botulinum. In this study, we explored the health-related quality of life (HRQol) and the output of BoNT-A as a preventative therapy in CM patients. To assess the HRQol of the patients with CM on BoNT-A therapy by the headache impact test (HIT-6) score. Patients with CM (n = 51) were diagnosed and enrolled for the study. The impact of CM on Qol was assessed using the HIT-6 questionnaire at the beginning, 1 month and 3 months of the therapy. A significant reduction of HIT-6 score was observed 1 and 3 months post-BoNT-A therapy (P < 0.0001) as compared to pre-BoNT-A therapy. Our findings suggest that BoNT-A therapy can improve HRQol by reducing the incidence of migraine headaches. Thus, BoNT-A can be effective in the management of CM.

Rendang is a traditional food from West Sumatera, Indonesia, made from beef, coconut milk, and spices cooked for 6-7 h. Commercial rendang products have a long shelf-life at room temperatures, but they are not always processed according to commercial sterilization designed to reduce 12 log cycles of Clostridium botulinum. It is not known whether the rendang seasoning could assist the thermal process due to its antimicrobial activity. This study used a metabolomics approach to profile the compounds acting as antimicrobials in rendang seasoning. Due to safety concerns, Clostridium sporogenes was utilized as a surrogate for C. botulinum. Three formulas of rendang seasoning with and without coconut milk were heated for 60 or 120 min, respectively. Each seasoning was macerated in n-hexane and the extracts were tested for their antimicrobial activity toward C. sporogenes using agar well diffusion method. The extracts were analyzed using gas chromatography mass spectrometry to identify the compounds and a multivariate data analysis was conducted. The study showed that rendang seasoning A, B, and C with or without coconut milk inhibited C. sporogenes with diameters of inhibition zone ranging from 4.71 mm to 18.92 mm. The metabolomics showed a well-defined clustering of Principal Component Analysis model (Q2 = 0.654, R2X = 0.853) and Orthogonal Projection to Latent Structure model (Q2 = 0.865, R2Y = 0.95). Garlic and great galangal were identified as the leading antimicrobial spices in rendang seasoning against C. sporogenes, as OPLS showed their key compounds, 2-vinyl-1,3-dithiane, 2-vinyl-4H-1,3-dithiine, and (S)-4-(1-acetoxyallyl)phenyl acetate, as well as an unknown compound.

Thermal processing of food often leads to a reduction in quality, highlighting the need to optimize heating conditions based on the kinetics of quality changes. This study investigated the physical quality of chicken meat—including cooking loss, water-holding capacity, texture, and color—during heating in a water bath (75 and 95 °C) and a pressure cooker (110 °C) for defined durations and modeled the kinetics of these changes. Heat distribution tests confirmed uniform temperatures, as indicated by minimal differences between thermocouples and the cold spot, while heat penetration tests ensured thorough heating, with lag times of 18.25 ± 2.25, 16.13 ± 4.58, and 19.25 ± 4.77 minutes at 75, 95, and 110 °C, respectively. Changes in physical quality at constant temperatures followed first-order reaction kinetics, and the temperature effect was described using the Arrhenius equation. The Arrhenius model revealed that higher temperatures accelerated the rate of quality changes, resulting in increased cooking loss, shear force, L*, and browning index, whereas water-holding capacity and cohesiveness decreased. Comparison of the D and Z values for physical quality parameters with those of Clostridium botulinum spores (D121.1 °C = 0.22 min, Z = 10 °C) suggested that high-temperature, short-time treatments could minimize detrimental changes in chicken meat while effectively inactivating target microorganisms.

The genus Clostridium encompasses phylogenetically diverse, obligately anaerobic, Gram-positive, spore-forming bacteria, yet taxonomic resolution for several members remains limited by the conservatism of the 16S rRNA gene. We reassessed four closely related species pairs: Clostridium baratii and Clostridium nitritogenes; Clostridium thermopalmarium and Clostridium colicanis; Clostridium botulinum and Clostridium combesii; and Clostridium estertheticum subsp. estertheticum and Clostridium estertheticum subsp. laramiense. A polyphasic framework was applied, integrating 16S rRNA and core-genome phylogenies, whole-genome relatedness metrics (digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity), phenotypic characterization and carbohydrate-active enzyme profiling. Concordant genomic and phenotypic evidence supported the synonymization of C. nitritogenes (Prévot 1940) Bernard et al. 2018 with C. baratii (Prévot 1938) Holdeman and Moore 1970, C. colicanis Greetham et al. 2003 with C. thermopalmarium Soh et al. 1991 and C. estertheticum subsp. laramiense (Kalchayanand et al. 1993) Spring et al. 2003 with C. estertheticum subsp. estertheticum (Collins et al. 1993) Spring et al. 2003. By contrast, despite high genomic relatedness, C. botulinum (van Ermengem 1896) Bergey et al. 1923 and C. combesii (Prévot and Laplanche 1947) Bernard et al. 2018 displayed reproducible phenotypic and functional distinctions consistent with subspecies status. We, therefore, propose C. botulinum subsp. combesii comb. nov., alongside C. botulinum subsp. botulinum comb. nov., thereby refining genome-based classification within the genus.

Bovine botulism is a lethal disease caused by Clostridium botulinum neurotoxins (BoNT). In cattle, the most frequent form of this disease in several South American countries and elsewhere is caused by BoNT type D, which has been associated with phosphorus deficiency, leading to pica and osteophagy. An outbreak of botulism occurred in a fully vaccinated 600-steer herd of cattle. The cattle were grazing on native pasture in a paddock in which several decomposing animal carcasses were found; the animals had performed osteophagy. The first 2 deaths were recorded in November 2016, and the number of fatalities increased to 84 through April of 2017. All the cases had similar clinical signs, which were consistent with botulism. The affected animals had hypophosphatemia and rear-leg weakness, ataxia, progressive flaccid paralysis of several muscles, recumbency, and death. Autopsies were performed on 3 animals; gross findings included hydropericardium, congestion of the cerebellum, and bone fragments and stones in the rumen and reticulum. Botulism was confirmed in the 3 animals by detecting BoNT type D by mouse bioassay. A large outbreak of botulism in vaccinated cattle associated with phosphorus deficiency and osteophagy has not been reported previously in Argentina, to our knowledge. Our case demonstrates that, even in vaccinated herds, rigorous carcass management is essential to reduce the risk of environmental contamination and to prevent fatal botulism outbreaks, especially in phosphorus-deficient areas. Improved reporting of similar cases is vital to refine prevention strategies and reduce the economic impact of the disease.

This study aimed to investigate the utility of Fourier-Transform Infrared Spectroscopy (FT-IRS) for differentiating Clostridium botulinum (C. botulinum) based on its botulinum neurotoxin (BoNT)-encoding gene type and its potential as an epidemiological tool for investigating botulism outbreaks. A total of 110 botulinum neurotoxin-producing clostridia (BNPC) strains, including reference, animal isolates, and human outbreak strains, were analyzed in four replicates using the IR Biotyper system (IRBT). Samples preparation was carried out according to the manufacturer's instructions. Similarity analysis was performed by hierarchical cluster analysis (HCA), principal component analysis (PCA), and linear discriminant analysis (LDA). The artificial intelligence capabilities of the IRBT software were applied to develop a classifier for C. botulinum differentiation at toxin-serotype or subtype level. HCA, PCA, and LDA showed a good clustering of strains belonging to the same type. In accordance with the lineages evidenced in whole-genome sequencing (WGS) studies, types A, B, and F BNPC appeared clearly separated from types C, CD, DC, and D. Considering only C, CD, DC, and D types, the highest discriminatory power is achieved in the wavenumber range 1,800-1,500 cm-1. Four different clusters were detected. Support vector machine algorithm with linear kernel (Linear SVM) showed the highest accuracy of discrimination at the BoNT type level with an accuracy of 97%. Although these preliminary results need to be confirmed with a higher number of strains, the IRBT system proved to be a very promising, user-friendly, and cost-effective tool for C. botulinum typing, and the application of machine learning algorithms represents a novel approach for BNPC typing.IMPORTANCEBotulism outbreaks represent a significant threat to public and animal health. Rapid and accurate typing methods are essential for effective epidemiological investigations, source tracing, and the implementation of appropriate control measures. Current methods for botulinum neurotoxin serotyping are often time-consuming, expensive, and require specialized expertise. Our research demonstrated that FT-IRS, a rapid, user-friendly, and cost-effective technique already well established in microbiology for broader bacterial characterization, can be successfully adapted for this crucial task. The use of a commercially available system like the IRBT significantly enhances the potential for widespread adoption of this methodology in routine diagnostics and surveillance.

With the increase of sporadic cases of foodborne botulism and the occurrence of several large incidents of botulinum contamination in food, botulism has received greater attention in clinical practice. Foodborne botulinum toxin poisoning is an acute foodborne toxic disease caused by ingestion of food contaminated with Clostridium botulinum exotoxin. We present two cases of foodborne botulism affecting a mother and daughter who were admitted to our department after consumption of vacuum-packed rabbit-head meat. The time to symptom onset was less than 24 h in the daughter (aged 8 years) and 1 week for the mother (aged 45 years). The main clinical manifestations included fatigue, dizziness, diplopia, blurred vision, ptosis, dysphagia, and slurred speech. Blood samples tested positive for botulinum toxin type A. The daughter developed pneumonia and respiratory failure and required mechanical ventilation. Treatment measures included the use of botulinum antitoxin, fluid replacement, anti-infection, and nutritional nerve therapy. Two patients eventually recovered. When botulinum toxin poisoning is diagnosed, early administration of the appropriate antitoxin is paramount. When necessary, life support for severe cases can improve the success rate. Considering that these cases of botulism were caused by the consumption of vacuum-packed rabbit-head meat, we recommend enhancing public awareness of food safety.

An outbreak of botulism occurred in March 2024 among horses at a Quarter Horse stud farm in Central-West Brazil. After ingesting baleage, 22 of 26 (85%) horses housed in stables and fed baleage became ill. The affected horses had dysphagia, muscular weakness, fasciculations, and progressive recumbency; 13 of 22 (59%) died within a few days. The diagnosis of type C botulism was established based on clinical and epidemiologic findings and confirmed by mouse bioassays, which indicated botulinum toxin type C in liver samples and intestinal contents. Furthermore, PCR testing identified toxigenic Clostridium botulinum in the baleage consumed by the horses.

Euphorbia hirta is a plant belonging to the family (Euphobiaceae). It is used in ethno medicine for the treatment of diarrhea, dysentery and other bacterial infections. This work was aimed at screening the phytochemical constituents and evaluating the in vitro combined effects of amoxicillin and ethanol leaves extract of Euphorbia hirta against clinical isolates of Staphylococcus aureus, Pseudomonasa eruginosa, selmonella typhi, Clostridium botulinum and klebsiella pneumonia using broth micro-dilution and Checkerboard techniques. Phytochemical screening of the extract revealed the presence of carbohydrates, flavonoids, tannins, saponins, glycosides, steroids and triterpenes. Euphorbia hirta exhibited minimum inhibitory concentration (MIC) range 6.25 mg/mL to 25 mg/mL while Amoxicillin recorded MIC range10 mg/mL to 20 mg/mL against all test organisms. The susceptible antibiotics were used in the checkerboard technique, which was found to be useful for determining antibiotic synergism. The Fractional inhibitory concentration index (FIC) were 0.13, 0.25 and 0.50, this values indicates synergism while FIC value 1.00 for additive effect against the test organisms at 37˚C over night incubation. The result of this study encourages the uses of Euphorbia hirta/amoxicillin combination for the treatment of bacterial infection.