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Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 led to the coronavirus infection diseases 2019 (COVID-19) pandemic, significantly impacting global public health and the economy. Numerous COVID-19 vaccines based on the receptor binding domain (RBD) of SARS-CoV-2 spike protein have been developed, utilizing various protein expression platforms and adjuvant systems. In a previous study, we reported using the direct fusion of the A subunit of type IIb E. coli heat-labile enterotoxin with the SARS-CoV-2 RBD protein (RBD-LTA) as an intranasal vaccine candidate (Hsieh et al., 2023). In this study, we investigated the effects of an intranasal booster of RBD-LTA/RBD mixture proteins after one or two doses of intramuscular bivalent BA.4/5 mRNA vaccination over 17 and 35 weeks. Our results indicate that the intranasal RBD-LTA/RBD mixture proteins booster maintains high levels of anti-RBD IgG and neutralizing antibodies, comparable to those elicited by a two-dose mRNA vaccination regimen. An additional RBD-LTA/RBD mixture proteins booster significantly increased antibody titers, demonstrating the potential of this approach for long-term immunity against SARS-CoV-2. Our findings suggest that combining primary mRNA vaccination with an intranasal RBD-LTA/RBD mixture proteins booster can effectively sustain antibody levels over extended periods, providing a promising strategy for long-term protection against SARS-CoV-2 and its variants. •RBD-LTA as an intranasal booster after intramuscular mRNA vaccination.•Intranasal RBD-LTA booster prolongs antibody levels.•Combining mRNA vaccination with intranasal booster for long-term immunity.•Early boosting enhances primed immunity.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea in humans and animals, including pigs. Enterotoxins are important virulence factors for ETEC. Although much is known about the mechanism of enterotoxin-induced diarrhoea, less is known about its effects on innate immune cells such as monocytes. Monocytes can differentiate into macrophages and dendritic cells and play a pivotal role in bridging the innate and adaptive immune systems. Understanding the interaction between ETEC enterotoxins and monocytes can help in the development of more effective preventive and therapeutic strategies to combat this disease. In this study, we aimed to investigate the effects of heat labile enterotoxin (LT) and heat stable enterotoxin a (STa) produced by ETEC on porcine monocytes. Our results showed that STa did not affect the viability or effector functions of monocytes. LT, on the other hand, decreased the viability of monocytes. While LT did not alter the production of reactive oxygen species (ROS) by monocytes, it significantly reduced the production of ROS induced by phorbol 12-myristate 13-acetate (PMA). In addition, LT decreased the phagocytosis of E. coli by monocytes and enhanced the survival of intracellular ETEC. Furthermore, LT triggered the production of the cytokines IL-1β, IL-6 and TNF-α as well as the chemokines CCL-3 and CXCL-8. Together, our results show that, in contrast to STa, LT can cause monocyte death and disrupt monocyte immune effector functions, potentially acting as an immune evasion strategy to establish infection.

Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes (pvl) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa-VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl-positive clinical isolates in Myanmar. A pvl-positive, ST2250 nasal isolate was identified as S. argenteus, a novel coagulase-positive staphylococcus species. Toxic shock syndrome toxin-1 (TSST-1) gene was detected in five pvl-negative isolates. All of the 144 isolates harbored at least one of the 21 enterotoxin(-like) gene(s). The most prevalent enterotoxin(-like) gene was selw (98%), followed by selx (97%), sei (28%), sely (28%), sem (26%), sel (24%), and sea and sec (22% each). Considerable genetic diversity with five groups was detected for selw. The present study revealed the relatively high rate of pvl, as well as the wide distribution of enterotoxin(-like) genes among colonizing S. aureus in Myanmar.

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

Introduction Raw milk is a nutrient‐rich food, but it may harbour harmful bacteria, such as enterotoxigenic Staphylococcus aureus (S. aureus), which can cause staphylococcal food poisoning. Antibiotic resistance of S. aureus in raw milk can increase the risk of such infections, particularly among susceptible individuals. Objective This study aimed to investigate the prevalence of enterotoxin genes a, d, g, i and j and the antibiotic resistance of S. aureus isolated from raw milk samples. Methods During a 6‐month sampling period, 60 raw milk specimens were obtained from diverse locations in Yazd province, Iran. Antibiogram profiling was conducted via the disc diffusion method. In addition, staphylococcal enterotoxin (SE) genes a, d, g, i, and j were detected through real‐time PCR analysis. Results Bacteriological assays confirmed the presence of S. aureus in 11 samples (18.3%). All isolates demonstrated 100% resistance to penicillin G but exhibited sensitivity to vancomycin, while resistance to other antibiotics ranged from 36.4% to 45.5%. The prevalence of enterotoxin genes in these strains showed variable distribution, with sea being the predominant SE (45.5%), followed by sed (36.4%), seg (18.2), sej and sei (9.1% each). Conclusions This study discovered the presence of multiple enterotoxins in S. aureus strains obtained from raw milk samples. These strains also demonstrated resistance to a variety of antibiotics. Since enterotoxigenic S. aureus is known to cause human food poisoning, monitoring food hygiene practices, especially during raw milk production, is critical. In this study culture, biochemical tests, real‐time PCR, and the Kirby‐Bauer disk diffusion method were used to identify and characterise the isolates. Enterotoxin genes and antibiotic resistance were detected in 11 (18.3%) S. aureus strains from raw milk samples. The strains had the sea, sed, seg, sei and sej genes, with the sea being the most frequent. All strains resisted penicillin G but were susceptible to vancomycin. Resistance to other antibiotics ranged from 36.4% to 45.5%.

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.

Staphylococcal enterotoxins (SEs), particularly enterotoxin C (SEC), are potent superantigens primarily known for causing food poisoning, but recent studies have highlighted their potential role in immune-mediated intestinal diseases. Despite the widespread use of food preservatives, their influence on SEC production-especially from coagulase-negative staphylococci (CNS)-remains poorly understood. In this study, we evaluated the effects of commonly used preservatives, including sodium chloride, potassium nitrate, and sorbic acid, on the expression and production of SEC and SEC in and , respectively. Using ELISA and RT-qPCR, we analyzed toxin levels at both the protein and mRNA levels. Proliferation assays on human PBMCs assessed the mitogenic potential of culture supernatants. While sodium chloride and potassium nitrate did not significantly alter SEC levels or bacterial growth, only sorbic acid at 0.07% consistently inhibited both mRNA expression and protein production of SEC and SEC . Furthermore, supernatants from sorbic acid-treated cultures induced significantly lower PBMC proliferation. These results suggest that even sub-emetic concentrations of enterotoxins may have immunomodulatory effects, and sorbic acid could be a promising agent in mitigating such risks.

Staphylococcus aureus stands out as one of the most virulent pathogens in the genus Staphylococcus. This characteristic is due to its ability to produce a wide variety of staphylococcal enterotoxins (SEs) and exotoxins, which in turn can cause staphylococcal food poisoning (SFP), clinical syndromes such as skin infections, inflammation, pneumonia, and sepsis, in addition to being associated with the development of inflammation in the mammary glands of dairy cattle, which results in chronic mastitis and cell necrosis. SEs are small globular proteins that combine superantigenic and emetic activities; they are resistant to heat, low temperatures, and proteolytic enzymes and are tolerant to a wide pH range. More than 24 SE genes have been well described (SEA-SEE, SEG, SEH, SEI, SEJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElR, SElS, SElT, SElU, SElV, SElW, SElX, SElY, and SElZ), being a part of different SFP outbreaks, clinical cases, and isolated animal strains. In recent years, new genes (sel26, sel27, sel28, sel31, sel32, and sel33) from SEs have been described, as well as two variants (seh-2p and ses-3p) resulting in a total of thirty-three genes from Ses, including the nine variants that are still in the process of genetic and molecular structure evaluation. SEs are encoded by genes that are located in mobile genetic elements, such as plasmids, prophages, pathogenicity islands, and the enterotoxin gene cluster (egc), and housed in the genomic island of S. aureus. Both classical SEs and SE-like toxins (SEls) share phylogenetic relationships, structure, function, and sequence homology, which are characteristics for the production of new SEs through recombination processes. Due to the epidemiological importance of SEs, their rapid assessment and detection have been crucial for food security and public health; for this reason, different methods of identification of SEs have been developed, such as liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), molecular methods, and whole-genome sequencing; providing the diagnosis of SEs and a better understanding of the occurrence, spread, and eradication of SEs. This review provides scientific information on the enterotoxins produced by S. aureus, such as structural characteristics, genetic organization, regulatory mechanisms, superantigen activity, mechanisms of action used by SEs at the time of interaction with the immune system, methods of detection of SEs, and recent biocontrol techniques used in food.

Bacillus cereus has long been recognized as an important pathogen in foodborne poisoning worldwide. Fresh vegetables are often contaminated with enterotoxigenic B. cereus and have been implicated as a vehicle for the transmission of this bacterium. This study reports on the occurrence, virulence gene profile, and antibiotic resistance of B. cereus in fresh vegetables. Of 102 examined samples, 48 (47%) of the samples were contaminated with B. cereus (>1 log CFU/g) and 7 (6.8%) of the samples showed more than 3 log CFU/g. In total, 118 B. cereus isolates were examined for the virulence genes nheA, nheB, nheC, hblA, hblC, hblD, cytK, and entFM and for resistance to antibiotics. Of these B. cereus isolates, 70% harbored nheA, nheB, nheC, and cytK. Eighteen (80%) of 21 isolates from bell peppers possessed eight enterotoxin genes. B. cereus isolates were susceptible to imipenem, vancomycin, gentamicin, erythromycin, ciprofloxacin, and chloramphenicol, whereas 22.4% of isolates from garlic chives, 48.7% from perilla leaf, and 40.5% from romaine lettuce showed antibiotic resistance to rifampin and 6% of isolates from garlic chives exhibited resistance to tetracycline. Three isolates from garlic chives were resistant to both tetracycline and rifampin. Raw vegetables were revealed to be major sources of B. cereus containing multiple toxin genes and exhibiting antibiotic resistance. Therefore, the potential health risks of consuming these vegetables raw or undercooked should not be underestimated. This study provides basic information for monitoring the antibiotic resistance and toxigenicity of B. cereus in the food chain during vegetable distribution and for developing food safety management to reduce the contamination with and transmission of B. cereus.