Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Molecular identification of Staphylococcus aureus-related enterotoxin genes in cheese samples.

Background: Dairy products are considered some important sources of various nutritional compounds; however, pathogenic bacterial growth is a critical destructive factor to these products leading to consumer health and system financial crises. Aim: The current study was carried out to identify if there is any presence of Staphylococcus aureus-related enterotoxin genes in cheese samples. Methods: The research included the collection of 35 samples. The samples passed through conventional cultivation processes and a PCR method to detect the presence of icaA, sea, hla, and fnbA enterotoxin genes in these samples. Results: The conventional identification revealed the growth of S. aureus from the cheese samples. The PCR findings recorded the presence of the icaA, sea, hla, and fnbA in 31 (88.5%), 27 (77%), 19 (54%), and 12 (34%), respectively, of cheese samples. The sequencing revealed close similarities with global isolates, which reached up to 98.5% of identity. Conclusion: The current results indicate the presence of enterotoxin genes of S. aureus in high rates in the dairy products examined, which reveals critical problems of food safety due to the possible presence of enterotoxins in consumer dairy products.

Sudden death due to enterotoxemia among Arabian camels (Camelus dromedaries) and associated risk factors.

Background: Sudden death is defined as an unexpected death occurring with no observed antecedent clinical signs. Aim: The current study was performed to notice the tangible causes of sudden death among 51 out of 340 she-camels on a private farm in the eastern region of El Khafgi, Saudi Arabia. Methods: A retrospective cohort study design was conducted to investigate the sudden death of camels through microscopic examination of fecal matter to identify the gastrointestinal parasites, analysis of whole blood thin films to diagnose blood parasites, blood culturing to recognize bacterial infection as Pasteurella multicida, and macroscopic postmortem examination to identify the gastrointestinal adult worm. The quantity and composition of feed were also analyzed. Afterward, a commercial multiscreen Ag-ELISA kit technique determined the toxins of Clostridium perfringens (C. perfringens). Results: The results revealed that the incidence rate of sudden death was 15%. The sudden death occurred due to C. perfringens enterotoxins detected in the rumen, intestinal content, and intestinal wall. The enterotoxins and Alpha toxins were noticed, but the other toxin types, including Beta and Epsilon, could not be detected. All C. perfringens toxins were discovered to be negative in fecal matter. A significant association was reported between sudden death, she-camels age, and feeding habits as risk factors (p = 0.020 and 0.028, respectively). Risk factor assessment by relative risk (RR) revealed that the odds of RR of sudden death occurring among she-camels aged over two years were higher than those less than two years (2.24 CI 95%, 1.093-4.591). Furthermore, the odds RR of sudden death occurring due to exposure of she-camels to a concentrated ration of 18% were higher twice than those not exposed (2.346 CI 95%, 1.039-5.296). Conclusion: Clostridium perfringens enterotoxaemia should be listed as a cause of sudden death in camels and the alteration in diet with 18% concentration feed changes the intestinal environment, which leads to C. perfringens proliferating and yielding potent toxins. More observations and interferences like regular immunization are recommended to reduce the disease and increase the awareness of the farmers of the importance of risk factors.

Enterotoxigenic Escherichia coli heat labile enterotoxin affects neutrophil effector functions via cAMP/PKA/ERK signaling.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.

Purified CDT toxins and a clean deletion within the CDT locus provide novel insights into the contribution of binary toxin in cellular inflammation and Clostridioides difficile infection.

Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1ß and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.

Helper T Cells are Hyperactive and Contribute to the Dysregulation of Antibody Production in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease, mediated by a complex interaction between B cells and various subsets of T cells. Dysfunction of helper T (Th) and regulatory T (Treg) cells may contribute to the breakdown of self-tolerance and the progression of autoimmune disease. In this study, we investigated the activity of Th and Treg cells on the differentiation of autologous B cells in vitro using cell cultures from the peripheral blood of healthy controls (HCs) and RA patients. The expressions of programmed death 1 (PD-1) and IL-21 were monitored as activation markers for Th cells. Unstimulated Th cells from RA patients showed remarkably higher PD-1 expression than HC samples. Stimulation of Th cells from RA patients with Staphylococcus enterotoxin B (SEB) in the presence of B cells significantly induced their PD-1 and IL-21 expression at a considerably higher level in RA compared to HCs, and Treg cells did not affect IL-21 production. When monitoring B-cell differentiation, a significantly higher frequency of plasma cells was observed, even in unstimulated samples of RA patients compared to HCs. In the SEB-stimulated co-cultures of the RA samples, plasma cell frequency and IgG production were considerably higher than in HCs and were not significantly affected by Tregs. These findings demonstrate that Th cells are constitutively active in RA, and their hyperactivity upon interaction with diseased B cells may lead to uncontrolled antibody production.

Host-derived CEACAM-laden vesicles engage enterotoxigenic Escherichia coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here, however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

Generation of Novel Tumour-Selective SEA Superantigen-Based Peptides with Improved Safety and Efficacy for Precision Cancer Immunotherapy.

Bacterial superantigens are T-cell-stimulatory protein molecules which produce massive cytokines and cause human diseases. Due to their ability to activate up to 20% of resting T-cells, they have effectively killed T-cell-dependent tumours in vivo. However, the intrinsic toxicity of whole SAg molecules highlights the urgent need to develop more effective and safer SAg-based immunotherapy. With its unique approach, our study is a significant step towards developing safer tumour-targeted superantigen peptides (TTSP). We identified the T-cell activation function regions on the SEA superantigen and produced variants with minimal lethality, ensuring a safer approach to cancer treatment. This involved the creation of twenty 50-amino-acid-long overlapping peptides covering the full-length SEA superantigen (P1-P20). We then screened these peptides for T-cell activation, successfully isolating two peptides (P5 and P15) with significant T-cell activation. These selected peptides were used to design and synthesise tumour-targeted superantigen peptides, which were linked to a cancer-specific third loop (L3) of transforming growth factor-α (TGF-α), TGFαL3 from either a C' or N' terminal with an eight-amino-acid flexible linker in between. We also produced several P15 variants by changing single amino acids or by amino acid deletions. The novel molecules were then investigated for cytokine production and tumour-targeted killing. The findings from our previous study and the current work open up new avenues for peptide-based immunotherapy, particularly when combined with other immunotherapy techniques, thereby ensuring effective and safer cancer treatment.

Electrochemical aptasensor for sensitive detection of staphylococcal enterotoxin type A in milk and fruit juice.

An aptamer-based electrochemical sensor for the sensitive detection of staphylococcal enterotoxin type A (SEA) is presented. The truncated aptamer AptSEA1.4 used in this work was screened using computational techniques, which reduced the cost of the SELEX screening process. The aptamer-SEA interactions were confirmed by employing circular dichroism (CD) and fluorescence spectroscopy. Afterwards, for developing an electrochemical aptasensor, a fabricated GNR/FTO aptasensor was prepared and characterized using scanning electron microscopy-energy-dispersive X-ray analysis (SEM-EDX), atomic force microscopy (AFM), cyclic voltammetry (CV), and square wave voltammetry (SWV). A detailed investigation of aptamer and SEA interaction in the presence of various experimental conditions was also conducted through SWV and electrochemical impedance spectroscopy (EIS). The aptamer exhibits a strong affinity for SEA, with a dissociation constant (Kd) of 19.93 nM. The aptasensor is sensitive, with a lower limit of detection of 12.44 pg mL-1. It has good stability, repeatability, and specificity and has displayed highly specific and sensitive detection SEA in spiked packaged mixed fruit juice and milk, with a recovery of 95-110%. The aptasensor has high promise for detecting SEA in other food items.

Development of two recombinant vaccines against Clostridioides difficile infection and immunogenicity in pregnant sows and neonatal piglets.

INTRODUCTION: Clostridioides difficile is the main cause of antibiotic-associated diarrhea in humans and is a major enteropathogen in several animal species. In newborn piglets, colonic lesions caused by C. difficile A and B toxins (TcdA and TcdB, respectively) cause diarrhea and significant production losses. OBJECTIVE: The present study aimed to develop two recombinant vaccines from immunogenic C-terminal fragments of TcdA and TcdB and evaluate the immune response in rabbits and in breeding sows. Two vaccines were produced: bivalent (rAB), consisting of recombinant fragments of TcdA and TcdB, and chimeric (rQAB), corresponding to the synthesis of the same fragments in a single protein. Groups of rabbits were inoculated with 10 or 50 µg of proteins adjuvanted with aluminum or 0.85 % sterile saline in a final volume of 1 mL/dose. Anti-TcdA and anti-TcdB IgG antibodies were detected in rabbits and sows immunized with both rAB and rQAB vaccines by ELISA. The vaccinated sows were inoculated intramuscularly with 20 µg/dose using a prime-boost approach. RESULTS: Different antibody titers (p ≤ 0.05) were observed among the vaccinated groups of sows (rAB and rQAB) and control. Additionally, newborn piglets from vaccinated sows were also positive for anti-TcdA and anti-TcdB IgGs, in contrast to control piglets (p ≤ 0.05). Immunization of sows with the rQAB vaccine conferred higher anti-TcdA and anti-TcdB responses in piglets, suggesting the superiority of this compound over rAB. CONCLUSION: The synthesized recombinant proteins were capable of inducing antibody titers against C. difficile toxins A and B in sows, and were passively transferred to piglets through colostrum.

Symbiotic biofilms formed by Clostridioides difficile and bacteroides thetaiotaomicron in the presence of vancomycin.

Vancomycin (VAN) treatment in Clostridioides difficile infection (CDI) suffers from a relatively high rate of recurrence, with a variety of reasons behind this, including biofilm-induced recurrent infections. C. difficile can form monophyletic or symbiotic biofilms with other microbes in the gut, and these biofilms protect C. difficile from being killed by antibiotics. In this study, we analyzed the ecological relationship between Bacteroides thetaiotaomicron and C. difficile and their formation of symbiotic biofilm in the VAN environment. The production of symbiotic biofilm formed by C. difficile and B. thetaiotaomicron was higher than that of C. difficile and B. thetaiotaomicron alone in the VAN environment. In symbiotic biofilms, C. difficile was characterized by increased production of the toxin protein TcdA and TcdB, up-regulation of the expression levels of the virulence genes tcdA and tcdB, enhanced bacterial cell swimming motility and c-di-GMP content, and increased adhesion to Caco-2 cells. The scanning electron microscope (SEM) combined with confocal laser scanning microscopy (CLSM) results indicated that the symbiotic biofilm was elevated in thickness, dense, and had an increased amount of mixed bacteria, while the fluorescence in situ hybridization (FISH) probe and plate colony counting results further indicated that the symbiotic biofilm had a significant increase in the amount of C. difficile cells, and was able to better tolerate the killing of the simulated intestinal fluid. Taken together, C. difficile and B. thetaiotaomicron become collaborative in the VAN environment, and targeted deletion or attenuation of host gut B. thetaiotaomicron content may improve the actual efficacy of VAN in CDI treatment.

A highly sensitive dual-mode lateral flow immunoassay based on plasmonic hollow Ag/Au nanostars enhancing light absorption.

The conventional lateral flow immunoassay (LFIA) based on gold nanoparticles (Au NPs) is limited by low sensitivity due to the insufficient brightness of Au NPs. To address this problem, noble metal nanomaterials with localized surface plasmon resonance (LSPR) and synthetic tunability are potential signal outputs for LFIA, which can achieve better optical properties by adjusting the preparation conditions. Herein, this study prepared the hollow silver/gold nano-stars (HAg/Au NSts) as LFIA signal output via the galvanic replacement method. HAg/Au NSts with anisotropic hollow alloy nanostructures exhibit a wide visible light absorption band and great NIR thermal conversion efficiency (η = 37.32 %), which endows them with enhanced colorimetric and photothermal signals. Further, we constructed a colorimetric-photothermal (CM-PT) dual-signal HAg/Au NSts-LFIA and chose staphylococcal enterotoxin B as the target analyte. The linear range of HAg/Au NSts-LFIA is 0.19-100 ng mL-1, and the limit of detection (LOD) is up to 0.29 ng mL-1 and 0.09 ng mL-1 in the colorimetric and photothermal modes respectively. Compared with the conventional Au NPs-LFIA, HAg/Au NSts-CM/PT-LFIA effectively improved the detection performance of LFIA. In addition, HAg/Au NSts-LFIA also showed satisfactory sensitivity (vLOD = 0.78 ng mL-1) and recovery (89.06-114.74 %) in milk and pork samples. Therefore, this work provides a new shape design idea for noble metal nanomaterials in biosensor applications.

Enhanced immunochromatographic assay using multifunctional gold@iridium nanoflower with colorimetric photothermal catalytic activity for the detection of staphylococcal enterotoxin B.

The development of a rapid, sensitive, and accurate screening method for staphylococcal enterotoxin B (SEB) in food is urgently needed because trace amounts of SEB can pose a serious threat to human health. Here, we developed a ultrasensitive triple-modal immunochromatographic assay (ICA) for SEB detection. The AuNFs@Ir nanoflowers exhibited enhanced colorimetric, photothermal, and catalytic performance by modulating the sharp branching structure of the gold nanoflowers and depositing high-density Ir atoms. Subsequently, the combination of AuNFs@Ir and ICA promoted colorimetric, catalytic amplified colorimetric, and photothermal-assisted quantitative detection. The results showed detection limits of 0.175, 0.0188, and 0.043 ng mL-1 in the colorimetric/photothermal/catalytic mode, which increased the sensitivity by 16.5-fold, 153.7-fold, and 67.2-fold, respectively, compared with the AuNPs-ICA. Furthermore, the proposed strategy was verified in milk, milk powder, pork, and beef successfully. This strategy improves significantly the sensitivity, accuracy, flexibility and offers an effective insight for foodborne bacterial toxin monitoring.

A Clostridioides difficile endolysin modulates toxin secretion without cell lysis.

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404.

Heterogeneous IL-9 Production by Circulating Skin-Tropic and Extracutaneous Memory T Cells in Atopic Dermatitis Patients.

Interleukin (IL)-9 is present in atopic dermatitis (AD) lesions and is considered to be mainly produced by skin-homing T cells expressing the cutaneous lymphocyte-associated antigen (CLA). However, its induction by AD-associated triggers remains unexplored. Circulating skin-tropic CLA+ and extracutaneous/systemic CLA- memory T cells cocultured with autologous lesional epidermal cells from AD patients were activated with house dust mite (HDM) and staphylococcal enterotoxin B (SEB). Levels of AD-related mediators in response to both stimuli were measured in supernatants, and the cytokine response was associated with different clinical characteristics. Both HDM and SEB triggered heterogeneous IL-9 production by CLA+ and CLA- T cells in a clinically homogenous group of AD patients, which enabled patient stratification into IL-9 producers and non-producers, with the former group exhibiting heightened HDM-specific and total IgE levels. Upon allergen exposure, IL-9 production depended on the contribution of epidermal cells and class II-mediated presentation; it was the greatest cytokine produced and correlated with HDM-specific IgE levels, whereas SEB mildly induced its release. This study demonstrates that both skin-tropic and extracutaneous memory T cells produce IL-9 and suggests that the degree of allergen sensitization reflects the varied IL-9 responses in vitro, which may allow for patient stratification in a clinically homogenous population.

Phylogenetic analysis of Staphylococcus aureus enterotoxin A gene in Iraqi breed cows with bovine mastitis: Implications for disease management.

Background: Although milk is nutritionally valuable, it also serves as a significant medium for the transmission of pathogens and their toxins. Aim: This study aimed to investigate the role of enterotoxin gene A (SEA) in the development of bovine mastitis. We accomplished this by examining milk through polymerase chain reaction (PCR) testing, amino acid substitution analysis, and phylogenetic analysis. Methods: A total of fifty milk samples were collected from locally bred dairy cows in Al-Diwaniyah, located in southern Iraq. We employed the VITEK-2 platform to validate the diagnosis of Staphylococcus aureus and confirm the results of routine tests (culturing and biochemical tests). Subsequently, the genetic mutation and phylogeny analysis were achieved utilizing DNA sequencing to 16S rRNA and enterotoxin A genes. Results: 66% (33/50) of the milk samples found to be contain S. aureus by the VITEK-2 system. Furthermore, 25/33 of the samples were positive by the PCR test. While 60% (15 out of 25) tested positive for the SEA gene. After genomic analysis, we identified amino acid substitutions of serine, glutamine with arginine, tyrosine with cysteine, and aspartic acid with glycine at positions 9, 101, 119, 187, and 191. The phylogenetic investigation demonstrated a genetic relationship between our isolates (Iraqi isolates) and isolates from Indian and the United States. Conclusion: Our study indicated the widespread distribution of the enterotoxin gene A (SEA) of S. aureus among dairy cows. The molecular study revealed significant changes in key amino acids that could play an important role in the bacterium's pathogenesis. The phylogenetic similarities among S. aureus samples from various countries suggest that the bacteria has spread globally.

Machine learning powered detection of biological toxins in association with confined lateral flow immunoassay (c-LFA).

Biological weapons, primarily dispersed as aerosols, can spread not only to the targeted area but also to adjacent regions following the movement of air driven by wind. Thus, there is a growing demand for toxin analysis because biological weapons are among the most influential and destructive. Specifically, such a technique should be hand-held, rapid, and easy to use because current methods require more time and well-trained personnel. Our study demonstrates the use of a novel lateral flow immunoassay, which has a confined structure like a double barbell in the detection area (so called c-LFA) for toxin detection such as staphylococcal enterotoxin B (SEB), ricinus communis (Ricin), and botulinum neurotoxin type A (BoNT-A). Additionally, we have explored the integration of machine learning (ML), specifically, a toxin chip boosting (TOCBoost) hybrid algorithm for improved sensitivity and specificity. Consequently, the ML powered c-LFA concurrently categorized three biological toxin types with an average accuracy as high as 95.5%. To our knowledge, the sensor proposed in this study is the first attempt to utilize ML for the assessment of toxins. The advent of the c-LFA orchestrated a paradigm shift by furnishing a versatile and robust platform for the rapid, on-site detection of various toxins, including SEB, Ricin, and BoNT-A. Our platform enables accessible and on-site toxin monitoring for non-experts and can potentially be applied to biosecurity.

Bacillus cereus containing nheA, hblC and cytk enterotoxin genes is associated with acute childhood gastroenteritis in Nigeria.

Bacillus cereus is rarely implicated when diarrheal cases in children are diagnosed in developing countries due to the lack of molecular methods to identify its enterotoxigenic genes. We report that out of 62 enterobacteria isolated from 70 stool samples collected from children hospitalized at the Mile 4 Hospital, Ebonyi State, Nigeria, 24 isolates were identified as B. cereus based on 16SrRNA gene sequence. The enterotoxins genes nheA and cytK2 were detected in 23 out of the 24 isolates, while hblC was detected in 19 isolates. B. cereus may be responsible for greater number of yearly incidences of acute childhood gastroenteritis in Nigeria.

Structural insights into the binding of nanobodies to the Staphylococcal enterotoxin B.

Staphylococcal Enterotoxin Type B (SEB), produced by Staphylococcus aureus bacteria, is notorious for inducing severe food poisoning and toxic shock syndrome. While nanobody-based treatments hold promises for combating SEB-induced diseases, the lack of structural information between SEB and nanobodies has hindered the development of nanobody-based therapeutics. Here, we present crystal structures of SEB-Nb3, SEB-Nb6, SEB-Nb8, SEB-Nb11, and SEB-Nb20 at resolutions ranging from 1.59 Å to 2.33 Å. Crystallographic analysis revealed that Nb3, Nb8, Nb11, and Nb20 bind to SEB at the T-cell receptor (TCR) interface, while Nb6 binds at the major histocompatibility complex (MHC) interface, suggesting their potential to inhibit SEB function by disrupting interactions with TCR or MHC molecules. Molecular biological analyses confirmed the thermodynamic and kinetic parameters of Nb3, Nb5, Nb6, Nb8, Nb11, Nb15, Nb18, and Nb20 to SEB. The competitive inhibition was further confirmed by cell-based experiments demonstrating nanobody neutralization. These findings elucidate the structural basis for developing specific nanobodies to neutralize SEB threats, providing crucial insights into the underlying mechanisms and offering significant assistance for further optimization towards future therapeutic strategies.

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.