Association between specific IgE to staphylococcal enterotoxin B and the eosinophilic phenotype of asthma.

BACKGROUND/AIMS: Sensitization to staphylococcal superantigens (SAgs) could contribute to asthma severity. However, its relevance with eosinophilic phenotype has not yet been clarified. This study aimed to investigate associations between serum specific IgE levels to SAg and eosinophilic airway inflammation in adult asthmatics. METHODS: The serum specific IgE levels to 3 SAgs, including staphylococcal enterotoxin A (SEA) and B (SEB), and toxic shock syndrome toxin-1 (TSST-1) were measured by ImmunoCAP in 230 adult asthmatic patients and 50 healthy controls (HCs). Clinical characteristics and laboratory parameters, including serum total/free IgE, and 2 eosinophil-activation markers, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), were analyzed according to blood eosinophil counts (BEC; 150 cells/µL) and serum specific IgE levels to 3 SAgs (0.35 kU/L). RESULTS: Asthmatic patients showed higher serum specific IgE levels to 3 SAgs than HCs (p < 0.05 for all). The serum total/clinfree IgE levels were significantly higher in asthmatics with positive IgE responses to 3 SAgs than those without (p < 0.05 for all). There were no significant differences in clinical parameters including age, asthma severity, comorbidities, or smoking according to IgE responses to 3 SAgs. Patients with positive IgE responses to SEB (not to SEA/TSST-1) had higher serum specific IgE levels to house dust mites and ECP/EDN as well as higher BEC with positive correlations between serum SEB-specific IgE levels and BEC/ECP/EDN (p < 0.05 for all). CONCLUSION: These findings suggest that serum SEB-specific IgE levels could contribute to eosinophil activation as well as IgE production in adult asthma.

Tofacitinib Treatment Suppresses CD4+ T-Cell Activation and Th1 Response, Contributing to Protection against Staphylococcal Toxic Shock.

Staphylococcal toxic shock syndrome (STSS) is a rare, yet potentially fatal disease caused by Staphylococcus aureus (S. aureus) enterotoxins, known as superantigens, which trigger an intense immune response. Our previous study demonstrated the protective effect of tofacitinib against murine toxin-induced shock and a beneficial effect against S. aureus sepsis. In the current study, we examined the effects of tofacitinib on T-cell response in peripheral blood using a mouse model of enterotoxin-induced shock. Our data revealed that tofacitinib suppresses the activation of both CD4+ and CD8+ T cells in peripheral blood. Furthermore, both gene and protein levels of Th1 cytokines were downregulated by tofacitinib treatment in mice with enterotoxin-induced shock. Importantly, we demonstrated that CD4+ cells, but not CD8+ cells, are pathogenic in mice with enterotoxin-induced shock. In conclusion, our findings suggest that tofacitinib treatment suppresses CD4+ T-cell activation and Th1 response, thereby aiding in protection against staphylococcal toxic shock in mice. This insight may guide the future development of novel therapies for STSS.

Identification and Removal of Pollen Spectral Interference in the Classification of Hazardous Substances Based on Excitation Emission Matrix Fluorescence Spectroscopy.

Sensitively detecting hazardous and suspected bioaerosols is crucial for safeguarding public health. The potential impact of pollen on identifying bacterial species through fluorescence spectra should not be overlooked. Before the analysis, the spectrum underwent preprocessing steps, including normalization, multivariate scattering correction, and Savitzky-Golay smoothing. Additionally, the spectrum was transformed using difference, standard normal variable, and fast Fourier transform techniques. A random forest algorithm was employed for the classification and identification of 31 different types of samples. The fast Fourier transform improved the classification accuracy of the sample excitation-emission matrix fluorescence spectrum data by 9.2%, resulting in an accuracy of 89.24%. The harmful substances, including Staphylococcus aureus, ricin, beta-bungarotoxin, and Staphylococcal enterotoxin B, were clearly distinguished. The spectral data transformation and classification algorithm effectively eliminated the interference of pollen on other components. Furthermore, a classification and recognition model based on spectral feature transformation was established, demonstrating excellent application potential in detecting hazardous substances and protecting public health. This study provided a solid foundation for the application of rapid detection methods for harmful bioaerosols.

Spores of Clostridioides difficile are toxin delivery vehicles.

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.

Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.

[Analysis of Clostridioides difficile infection characteristics and risk factors in patients hospitalized for diarrhea in 3 university hospitals in a mid-south city of China].

OBJECTIVE: To investigate the characteristics of Clostridioides difficile infection (CDI) in patients hospitalized for diarrhea and analyze the risk factors for CDI. METHODS: Stool samples were collected from 306 patients with diarrhea hospitalized in 3 university hospitals in a mid-south city of China from October to December, 2020. C. difficile was isolated by anaerobic culture, and qRT-PCR was used to detect the expressions of toxin A (tcdA) and B (tcdB) genes and the binary toxin genes (cdtA and cdtB). Multilocus sequence typing (MLST) was performed for the isolated strains without contaminating strains as confirmed by 16S rDNA sequencing. Etest strips were used to determine the drug resistance profiles of the isolated strains, and the risk factors of CDI in the patients were analyzed. RESULTS: CDI was detected in 25 (8.17%) out of the 306 patients. All the patients tested positive for tcdA and tcdB but negative for the binary toxin genes. Seven noncontaminated C. difficile strains with 5 ST types were isolated, including 3 ST54 strains and one strain of ST129, ST98, ST53, and ST631 types each, all belonging to clade 1 and sensitive to metronidazole and vancomycin. Hospitalization within the past 6 months (OR= 3.675; 95% CI: 1.405-9.612), use of PPIs (OR=7.107; 95% CI: 2.575-19.613), antibiotics for ≥1 week (OR=7.306; 95% CI: 2.274-23.472), non-steroidal anti-inflammatory drugs (OR=4.754; 95% CI: 1.504-15.031) in the past month, and gastrointestinal disorders (OR=5.050; 95% CI: 1.826-13.968) were all risk factors for CDI in the patients hospitalized for diarrhea. CONCLUSION: The CDI rate remains low in the hospitalized patients with diarrhea in the investigated hospitals, but early precaution measures are recommended when exposure to the risk factors is reported to reduce the risk of CDI in the hospitalized patients.

Nasal carriage of Staphylococcus aureus in healthy dairy cows in Algeria: antibiotic resistance, enterotoxin genes and biofilm formation.

BACKGROUND: Staphylococcus aureus can colonize and infect a variety of animal species. In dairy herds, it is one of the leading causes of mastitis cases. The objective of this study was to characterize the S. aureus isolates recovered from nasal swabs of 249 healthy cows and 21 breeders of 21 dairy farms located in two provinces of Algeria (Tizi Ouzou and Bouira). METHODS: The detection of enterotoxin genes was investigated by multiplex PCRs. Resistance of recovered isolates to 8 antimicrobial agents was determined by disc-diffusion method. The slime production and biofilm formation of S. aureus isolates were assessed using congo-red agar (CRA) and microtiter-plate assay. Molecular characterization of selected isolates was carried out by spa-typing and Multi-Locus-Sequence-Typing (MLST). RESULTS: S. aureus was detected in 30/249 (12%) and 6/13 (28.6%) of nasal swabs in cows and breeders, respectively, and a total of 72 isolates were recovered from positive samples (59 isolates from cows and 13 from breeders). Twenty-six of these isolates (36.1%) harbored genes encoding for staphylococcal enterotoxins, including 17/59 (28.8%) isolates from cows and 9/13 (69.2%) from breeders. Moreover, 49.1% and 92.3% of isolates from cows and breeders, respectively, showed penicillin resistance. All isolates were considered as methicillin-susceptible (MSSA). Forty-five (76.3%) of the isolates from cows were slime producers and 52 (88.1%) of them had the ability to form biofilm in microtiter plates. Evidence of a possible zoonotic transmission was observed in two farms, since S. aureus isolates recovered in these farms from cows and breeders belonged to the same clonal lineage (CC15-ST15-t084 or CC30-ST34-t2228). CONCLUSIONS: Although healthy cows in this study did not harbor methicillin-resistant S. aureus isolates, the nares of healthy cows could be a reservoir of enterotoxigenic and biofilm producing isolates which could have implications in human and animal health.

Contribution of MALDI-TOF mass spectrometry and machine learning including deep learning techniques for the detection of virulence factors of Clostridioides difficile strains.

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA+B+CDT+, 22 ToxA+B+CDT+ Hv+ and 105 ToxA+B+CDT-) and 50 non-toxigenic (ToxA-B-) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA-B- strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA+B+CDT- strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA+B+CDT+ strains. The classifiers' performances for Hv strain detection were linked to high specificity (≥0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

Klebsiella oxytoca inhibits Salmonella infection through multiple microbiota-context-dependent mechanisms.

The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.

Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment.

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

Janus plasmonic-aggregation induced emission nanobeads as high-performance colorimetric-fluorescent probe of immunochromatographic assay for the ultrasensitive detection of staphylococcal enterotoxin B in milk.

Herein, a colorimetric-fluorescent hybrid bifunctional nanobead with Janus structure (J-cf-HBN) was synthesized via one-pot microemulsification. Oleylamine-coated AuNPs and aggregation-induced emission luminogens (AIEgens) were suggested as building blocks to obtain high-performance colorimetric-fluorescent signals. The as-prepared J-cf-HBNs were used as a signal amplification probe to construct an immunochromatographic assay (J-cf-HBNs-ICA) platform for the ultrasensitive detection of staphylococcal enterotoxin B (SEB) in milk samples. Owing to the rational spatial distribution of AuNPs and AIEgens, the J-cf-HBNs present a highly retained photoluminescence and enhanced colorimetric signals. Combined with a pair of highly affinitive anti-SEB antibodies, the J-cf-HBN-ICA platform enabled the fast naked-eye visualization and fluorescent quantitative detection of SEB in various milk matrices. Given the advantages of the dual-mode high-performance J-cf-HBNs, the proposed strip achieved a high sensitivity for SEB qualitative determination with a visual limit of detection (LOD) of 1.56 ng mL-1 and exhibited ultrasensitivity for SEB quantitative detection with a LOD of 0.09 ng mL-1, which is 139-fold lower than that of ELISA using same antibodies. In conclusion, this work provides new insights into the construction of multimode immunochromatographic methods for food safety detection in the field.

A highly neutralizing human monoclonal antibody targeting a novel linear epitope on staphylococcal enterotoxin B.

Staphylococcal Enterotoxin B (SEB), produced by Staphylococcus aureus (S. aureus), is a powerful superantigen that induces severe immune disruption and toxic shock syndrome (TSS) upon binding to MHC-II and TCR. Despite its significant impact on the pathogenesis of S. aureus, there are currently no specific therapeutic interventions available to counteract the mechanism of action exerted by this toxin. In this study, we have identified a human monoclonal antibody, named Hm0487, that specifically targets SEB by single-cell sequencing using PBMCs isolated from volunteers enrolled in a phase I clinical trial of the five-antigen S. aureus vaccine. X-ray crystallography studies revealed that Hm0487 exhibits high affinity for a linear B cell epitope in SEB (SEB138-147), which is located distantly from the site involved in the formation of the MHC-SEB-TCR ternary complex. Furthermore, in vitro studies demonstrated that Hm0487 significantly impacts the interaction of SEB with both receptors and the binding to immune cells, probably due to an allosteric effect on SEB rather than competing with receptors for binding sites. Moreover, both in vitro and in vivo studies validated that Hm0487 displayed efficient neutralizing efficacy in models of lethal shock and sepsis induced by either SEB or bacterial challenge. Our findings unveil an alternative mechanism for neutralizing the pathogenesis of SEB by Hm0487, and this antibody provides a novel strategy for mitigating both SEB-induced toxicity and S. aureus infection.

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring.

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 µg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

Degradation of Toxins Derived from Foodborne Pathogens by Atmospheric-Pressure Dielectric-Barrier Discharge.

Foodborne diseases can be attributed not only to contamination with bacterial or fungal pathogens but also their associated toxins. Thus, to maintain food safety, innovative decontamination techniques for toxins are required. We previously demonstrated that an atmospheric-pressure dielectric-barrier discharge (APDBD) plasma generated by a roller conveyer plasma device is effective at inactivating bacteria and fungi in foods. Here, we have further examined whether the roller conveyer plasma device can be used to degrade toxins produced by foodborne bacterial pathogens, including aflatoxin, Shiga toxins (Stx1 and Stx2), enterotoxin B and cereulide. Each toxin was spotted onto an aluminum plate, allowed to dry, and then treated with APDBD plasma applied by the roller conveyer plasma device for different time periods. Assessments were conducted using a competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrate a significant time-dependent decrease in the levels of these toxins. ELISA showed that aflatoxin B1 concentrations were reduced from 308.6 µg/mL to 74.4 µg/mL within 1 min. For Shiga toxins, Stx1 decreased from 913.8 µg/mL to 65.1 µg/mL, and Stx2 from 2309.0 µg/mL to 187.6 µg/mL within the same time frame (1 min). Enterotoxin B levels dropped from 62.67 µg/mL to 1.74 µg/mL at 15 min, and 1.43 µg/mL at 30 min, but did not display a significant decrease within 5 min. LC-MS/MS analysis verified that cereulide was reduced to below the detection limit following 30 min of APDBD plasma treatment. Taken together, these findings highlight that a range of foodborne toxins can be degraded by a relatively short exposure to plasma generated by an APDBD using a roller conveyer device. This technology offers promising advancements in food safety, providing a novel method to alleviate toxin contamination in the food processing industry.

Identification of a hemorrhagic determinant in Clostridioides difficile TcdA and Paeniclostridium sordellii TcsH.

Paeniclostridium sordellii hemorrhagic toxin (TcsH) and Clostridioides difficile toxin A (TcdA) are two major members of the large clostridial toxin (LCT) family. These two toxins share ~87% similarity and are known to cause severe hemorrhagic pathology in animals. Yet, the pathogenesis of their hemorrhagic toxicity has been mysterious for decades. Here, we examined the liver injury after systemic exposure to different LCTs and found that only TcsH and TcdA induce overt hepatic hemorrhage. By investigating the chimeric and truncated toxins, we demonstrated that the enzymatic domain of TcsH alone is not sufficient to determine its potent hepatic hemorrhagic toxicity in mice. Likewise, the combined repetitive oligopeptide (CROP) domain of TcsH/TcdA alone also failed to explain their strong hemorrhagic activity in mice. Lastly, we showed that disrupting the first two short repeats of CROPs in TcsH and TcdA impaired hemorrhagic toxicity without causing overt changes in cytotoxicity and lethality. These findings lead to a deeper understanding of toxin-induced hemorrhage and the pathogenesis of LCTs and could be insightful in developing therapeutic avenues against clostridial infections. IMPORTANCE: Paeniclostridium sordellii and Clostridioides difficile infections often cause hemorrhage in the affected tissues and organs, which is mainly attributed to their hemorrhagic toxins, TcsH and TcdA. In this study, we demonstrate that TcsH and TcdA, but not other related toxins. including Clostridioides difficile toxin B and TcsL, induce severe hepatic hemorrhage in mice. We further determine that a small region in TcsH and TcdA is critical for the hemorrhagic toxicity but not cytotoxicity or lethality of these toxins. Based on these results, we propose that the hemorrhagic toxicity of TcsH and TcdA is due to an uncharacterized mechanism, such as the presence of an unknown receptor, and future studies to identify the interactive host factors are warranted.

Double-Enhanced Photothermal Lateral Flow Biosensor Based on Dual Gold Nanoparticle Conjugates.

Bacterial toxins emerge as the primary triggers of foodborne illnesses, posing a significant threat to human health. To ensure food safety, it is imperative to implement point-of-care testing methods. Lateral flow biosensors (LFBs) based on gold nanoparticles (GNPs) have been commonly used for rapid detection, but their applicationis limited by low sensitivity. Based on the localized surface plasmon resonance and photothermal effect of dual gold nanoparticle conjugates (DGNPs), we developed a smartphone-integrated photothermal LFB (PLFB) with double-enhanced colorimetric and photothermal sensitivity. Through numerical simulations, we verified that DGNPs have significantly enhanced photothermal performance compared to single 15 nm GNPs (SGNPs), and applied DGNPs in PLFB for the detection of staphylococcus enterotoxin A (SEA). The colorimetric and photothermal limits of detection of DGNPs-based PLFB for SEA were 0.091 and 0.0038 ng mL-1, respectively. Compared with the colorimetric detection of the SGNPs-based LFB, the colorimetric detection sensitivity of the DGNPs-based PLFB was increased by 10.7 times, and the photothermal detection sensitivity was further improved by 255.3 times. Moreover, the PLFB exhibits robust reproducibility and exceptional specificity and is applicable for detecting SEA in milk samples. This smartphone-integrated PLFB based on DGNPs allows users to detect toxins simply, conveniently, and quickly and has huge application potential in the field of food safety.

Bacillus cereus CwpFM induces colonic tissue damage and inflammatory responses through oxidative stress and the NLRP3/NF-κB pathway.

Bacillus cereus (B. cereus) from cow milk poses a threat to public health, causing food poisoning and gastrointestinal disorders in humans. We identified CwpFM, an enterotoxin from B. cereus, caused oxidative stress and inflammatory responses in mouse colon and colonic epithelial cells. Colon proteomics revealed that CwpFM elevated proteins associated with inflammation and oxidative stress. Notably, CwpFM induced activation of the NLRP3/NF-κB signaling, but suppressed antioxidant NFE2L2/HO-1 expression in the intestine and epithelial cells. Consistently, CwpFM exposure led to cytotoxicity and ROS accumulation in Caco-2 cells in a dose-dependent manner. Further, NAC (ROS inhibitor) treatment abolished NLRP3/NF-κB activation due to CwpFM. Moreover, overexpression of Nfe2l2 or activation of NFE2L2 by NK-252 reduced ROS production and inhibited activation of the NLRP3/NF-κB pathway. Inhibition of NF-κB by ADPC and/or suppression of NLRP3 by MCC950 attenuated CwpFM-induced inflammatory responses in Caco-2 cells. Collectively, CwpFM induced oxidative stress and NLRP3/NF-κB activation by inhibiting the NFE2L2/HO-1 signaling and ROS accumulation, leading to the development of intestinal inflammation. Our data elucidate the role of oxidative stress and innate immunity in CwpFM enterotoxicity and contribute to developing diagnostic and therapeutic products for B. cereus-related food safety issues.

Molecular detection of enterotoxins in multidrug resistant Aeromonas from ready to eat foods in North Western Himalayas: Public health significance.

Aeromonas spp. are normal inhabitants of aquatic environments and are emerging foodborne bacterial pathogens. Aeromonas spp. contamination is frequent in ready-to-eat (RTE) seafood and can also occur in products prepared from milk or meat. The study determined the enterotoxin and antimicrobial susceptibility profiles of Aeromonas spp. isolates recovered from RTE milk products (n = 105), RTE meat/fish products (n = 40) and drinking water (n = 60) samples collected from tourist places in Himachal Pradesh, India, in northwestern Himalayas. 7.3 % (16/220) samples were found contaminated with Aeromonas spp. These isolates were identified as A. hydrophila (31.3 %), A. schubertii (25.0 %), A. sobria (25.0 %) and A. veronii (18.8 %). Aeromonas spp. contamination was significantly higher (14.3 %, 15/105, p = 0.0001) in RTE milk products. The contamination levels for water samples were 1.7 % whereas none of the tested RTE meat or fish products yielded Aeromonas spp. Among RTE milk products, contamination was significantly higher in paneer (South Asian soft cheese) (26.1 %, p = 0.0027) and cream (25.0 %, p = 0.046) based RTE foods. All isolates carried alt (361 bp), encoding a cytotonic heat-labile enterotoxin. Ampicillin resistance was 100 % and high levels (>30 %) of resistance were recorded for amoxicillin/clavulanic acid, amikacin, cefotaxime and ceftazidime. Six (37.5 %) isolates were multi drug resistant (MDR), showing resistance to aminoglycosides, cephams and penicillins. Isolation of alt carrying MDR isolates from RTE foods indicates that Aeromonas spp. can be potential foodborne public health threat in northwestern Himalayas.

Enterotoxin-related genes PPFIA4 and SCN3B promote colorectal cancer development and progression.

To identify the role of enterotoxin-related genes in colorectal cancer (CRC) development and progression. Upregulated differentially expressed genes shared by three out of five Gene Expression Omnibus (GEO) data sets were included to screen the key enterotoxin-induced oncogenes (EIOGs) according to criteria oncogene definition, enrichment, and protein-protein interaction (PPI) network analysis, followed by prognosis survival, immune infiltration, and protential drugs analyses was performed via integration of RNA-sequencing data and The Cancer Genome Atlas-derived clinical profiles. We screened nine common key EIOGs from at least three GEO data sets. A Cox proportional hazards regression models verified that more alive cases, decreased overall survival, and highest 4-year survival prediction in CRC patients with high-risk score. Protein tyrosine phosphatase receptor type F polypeptide-interacting protein alpha-4 (PPFIA4), STY11, SCN3B, and SPTBN5 were shared in the same PPI network. Immune infiltration results showed that SCN3B and synaptotagmin 11 expression were obviously associated with B cell, macrophage, myeloid dendritic cell, neutrophils, and T cell CD4+ and CD8+ in both colon adenocarcinoma and rectal adenocarcinoma. CHIR-99021, MLN4924, and YK4-279 were identified as the potential drugs for treatment. Finally, upregulated EIOGs genes PPFIA4 and SCN3B were found in colon adenocarcinoma and PPFIA4 and SCN3B were proved to promote cell proliferation and migration in vitro. We demonstrated here that EIOGs promoting a malignancy phenotype was related with poor survival and prognosis in CRC, which might be served as novel therapeutic targets in CRC management.