Development of protective egg yolk immunoglobulins (IgY) targeting CfaB, LTB, and EtpA recombinant proteins of Enterotoxigenic Escherichia coli (ETEC) for inhibiting toxin activity and bacterial adherence.

Enterotoxigenic Escherichia coli (ETEC) stands as a prevalent bacterial cause of global diarrheal incidents. ETEC's primary virulence factors encompass the B subunit of the Heat Labile Enterotoxin, along with the adhesion factors CfaB and EtpA. In this study, we isolated IgY antibodies against the three virulence factors individually, in pairs, and as triple cocktails. The in vitro efficacy of these IgY antibodies was examined, focusing on inhibiting heat-labile enterotoxin (LT) toxin cytotoxicity and impeding ETEC adherence to HT29 cells. Assessing the impact of IgY-treated bacteria on intestinal epithelial cells utilized the standard ileal loop method. Results demonstrated that the anti-LTB IgY antibody at 125 µg/ml and IgY antibodies from double and tertiary cocktails at 200 µg/ml effectively inhibited LT toxin attachment to the Y1 cell line. Pre-incubation of HT29 intestinal cells with specific IgYs reduced bacterial attachment by 59.7%. In the ileal loop test, toxin neutralization with specific IgYs curtailed the toxin's function in the intestine, leading to a 74.8% reduction in fluid accumulation compared to control loops. These findings suggest that egg yolk immunoglobulins against recombinant proteins LTB, CfaB, and EtpA, either individually or in combination, hold promise as prophylactic antibodies to impede the functioning of ETEC bacteria.

Intranasal booster with SARS-CoV-2 RBD protein fused to E. coli enterotoxin a subunit after primary mRNA vaccination in mice.

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.

Prevalence, Cross Contamination, Virulence, and Multidrug Resistance Profiles of Staphylococcus aureus Isolates from Four Middle-Scale Dairy Farms in Bareilly, Northern India.

Milk, a nutritious global important food commodity, serves as an excellent medium for microbial growth as well. The foodborne pathogen Staphylococcus aureus is a commensal member of human microflora that enters the food chain through poor hygienic practices and cross contamination and causes various clinical manifestations in humans. During this study, raw milk and swab samples (milker's hand, udder, towel, milking bucket, and farm floor) were collected from four middle-scale buffalo dairy farms. The results revealed S. aureus presence in 11.6% (n = 56/448) bucket raw milk samples and 2.6% (n = 12/448) udder raw milk samples. Contrarily, S. aureus prevalence was significantly higher in farm floors (100%, n = 84/84), towel (35.7%, n = 10/28), milking bucket (11.6%, n = 56/448), milker's hand (10.7%, n = 3/28), and udder swab samples (4.0%, n = 18/448). The chi-square test yielded p values of 0.000, 0.005, and 0.0011 for udder raw milk, udder swab, and milker's hand swab, respectively. The p values of the milking bucket (p = 0.048) and farm floors (p = 0.0183) confirmed their possible role in S. aureus cross contamination. Gene amplifications of nuclease and enterotoxin A indicate potential virulence of S. aureus isolates in collected samples. Antibiotic susceptibility testing revealed multidrug resistance in 44% (n = 239) of S. aureus isolates with the highest resistance of 61% against penicillin. Resistance against ampicillin, streptomycin, and lincomycin was observed. Fewer S. aureus isolates were resistant to kanamycin and erythromycin, whereas the lowest number of resistant isolates was observed against chloramphenicol. A high prevalence of S. aureus in the farm environment and milking equipment suggested the cross contamination of potentially enterotoxin-producing and multidrug-resistant S. aureus to raw milk. Therefore, good hygiene practices should be enforced to avoid foodborne and zoonotic infections.

Ciclesonide exhibits lung-protective effects in neonatal rats exposed to intra-amniotic enterotoxin.

Introduction: Despite the advances in perinatal care, bronchopulmonary dysplasia (BPD) continues to be a highly prevalent chronic lung disease that affects newborns, especially affecting premature newborns. There is no specific cure for BPD, and treatments aimed at reducing the risk of developing BPD focus mainly on lung-protective ventilation strategies, surfactant therapy, and/or corticosteroid administration. Our objective was to evaluate whether systemic postnatal administration of a new glucocorticoid, ciclesonide, can attenuate the alteration of lung structure and pulmonary hypertension in a rat model of chorioamnionitis-induced BPD, with minimal adverse effects on the developing brain. Methods: Endotoxin (ETX) or saline was administered to pregnant rats by intra-amniotic (i.a.) injection on day 20 of pregnancy, and pups were delivered by cesarean section on day 22. Ciclesonide (0.5 mg/kg) was administered postnatally for five consecutive days to pups previously exposed to i.a. ETX. On postnatal day 14, we assessed lung function (compliance), lung structure (radial alveolar count, mean linear intercept, pulmonary vessel density), pulmonary hypertension, and brain histology (edema, inflammation, apoptosis, hemorrhage, and infarction). Result: On postnatal day 14, the effects of i.a. ETX administration were evident in neonatal rats not receiving treatment; these animals showed impaired lung compliance, disrupted lung structure, and developing pulmonary hypertension compared to those receiving i.a. saline. Postnatal administration of ciclesonide for 5 days was associated with significantly better outcomes in terms of lung compliance, alveolarization, lung vascular growth, and pulmonary hypertension, without affecting the brain histological parameters evaluated. Conclusion: Postnatal ciclesonide administration preserved lung function and structure and prevented pulmonary hypertension in a BPD model induced by antenatal i.a. ETX administration, without causing any adverse effects on brain development. These findings suggest that the new glucocorticoid, ciclesonide, may provide a novel strategy for the prevention of BPD; however, more long-term studies are required.

Characterization of Staphylococcus aureus isolated from milk samples for their virulence, biofilm, and antimicrobial resistance.

The Staphylococcus aureus (S. aureus) one of the important food borne pathogen from milk, which was investigated in this study. The isolates were screened for antimicrobial resistance, enterotoxin genes, biofilm formation, spa typing, coagulase gene polymorphism and accessory gene regulator types. The prevalence of S. aureus in milk samples was 34.4% (89/259). Methicillin resistant S. aureus (MRSA) was found at 27% (24/89) of the isolates, were classified as community acquired based on SCCmec typing. The 24.71% (22/89) isolates demonstrated multiple antimicrobial resistance (MAR) pattern. However, none of the isolates carried vancomycin and mupirocin resistance genes. The isolates were positive for sea and sed enterotoxin genes and exhibited high frequency of biofilm formation. The High-Resolution Melting and conventional spa typing revealed that the isolates had both animal and community-associated S. aureus clustered origins. Coagulase gene polymorphism and agr typing demonstrated variable genotypic patterns. The finding of this study establishes the prevalence of community associated, enterotoxigenic, biofilm forming and antimicrobial resistance among S. aureus from milk in Chennai city. This emphasizing a potential threat to public health which needs a continuous monitoring system and strategies to mitigate their spread across the food chain and achieve food safety.

A tetravalent peptide efficiently inhibits the intestinal toxicity of heat-labile enterotoxin by targeting the receptor-binding region of the B-subunit pentamer.

Infection by enterotoxigenic Escherichia coli (ETEC) causes severe watery diarrhea and dehydration in humans. Heat-labile enterotoxin (LT) is a major virulence factor produced by ETEC. LT is one of AB5-type toxins, such as Shiga toxin (Stx) and cholera toxin (Ctx), and the B-subunit pentamer is responsible for high affinity binding to the LT-receptor, ganglioside GM1, through multivalent interaction. In this report, we found that Glu51 of the B-subunit plays an essential role in receptor binding compared with other amino acids, such as Glu11, Arg13, and Lys91, all of which were previously shown to be involved in the binding. By targeting Glu51, we identified four tetravalent peptides that specifically bind to the B-subunit pentamer with high affinity by screening tetravalent random-peptide libraries, which were tailored to bind to the B-subunit through multivalent interaction. One of these peptides, GGR-tet, efficiently inhibited the cell-elongation phenotype and the elevation of cellular cAMP levels, both induced by LT. Furthermore, GGR-tet markedly inhibited LT-induced fluid accumulation in the mouse ileum. Thus, GGR-tet represents a novel therapeutic agent against ETEC infection.

Integrating lateral flow device with controllable gold in situ growth for sensitive detection of staphylococcal enterotoxin A in milk.

Gold nanoparticle-based lateral flow immunoassays (AuNP-LFIA) are widely used for pathogen monitoring to prevent foodborne illness outbreaks. However, conventional AuNP-LFIA exhibits poor sensitivity and limited quantitative capacity due to the low colorimetric signal intensity of AuNPs. Herein, we introduced a low-background gold in situ growth (GISG) strategy by lowering the pH of the growth solution to weaken the reducibility of hydroxylamine, thereby enhancing the sensitivity of AuNP-LFIA. Additionally, we developed a universal and manufacturable lateral flow device to streamline the GISG process. We applied this device to detect staphylococcal enterotoxin A (SEA), an exotoxin produced by Staphylococcus aureus. Under optimal conditions, the proposed device demonstrated superior practicality and excellent sensitivity for SEA detection, achieving a detection limit of 0.061 ng/mL with the total detection time of 37 min, showing 311 times more sensitive than the unamplified AuNP-LFIA. Furthermore, SEA detection in milk samples showed a strong correlation (R2 = 0.8845) with results obtained from a conventional ELISA kit. Therefore, this promising LFIA device offers a novel strategy with high sensitivity and practicality for in-field detection of Staphylococcus aureus and can be easily adapted for screening other foodborne pathogens.

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.

Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania Donavani.

AIM: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin [LTB] in the functional activity of L. donovani. BACKGROUND: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease [gp63] has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani has fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. RESULTS: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin [LTB] of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. CONCLUSION: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein [LTB] could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

Lateral flow immunoassay device to detect staphylococcal enterotoxin B (SEB) in durian candy.

Staphylococcal enterotoxin B (SEB), a potent enterotoxin produced by Staphylococcus aureus, has been implicated in incidences of Staphylococcal food poisoning in the Philippines. The use of lateral flow immunoassay devices to detect this toxin in solid food samples, like durian candy, at the point of sampling is constrained by the requirement for sample purification (e.g. centrifugation). This problem is also true with the other applications of LFIA devices on food samples. To overcome this challenge, a lateral flow immunoassay (LFIA) device capable of detecting SEB in unpurified durian candy sample was developed in this study. A modified LFIA device was assembled with three layers of glass fiber pads functioning as sample pads instead of a conventional cellulose fiber pad. Unlike with the cellulose fiber pad, the glass fiber sample pads acted as filter and allowed the flow of a 1:5 dilution of durian candy. The LFIA device applied to spiked 1:5 diluted durian candy samples achieved a visual limit of detection of 5 ng/mL for SEB, which is twofold lower than reported for previous LFIA devices designed to detect SEB in food samples.

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.

Heat resistance differences are common between both vegetative cells and spores of Clostridium perfringens type F isolates carrying a chromosomal vs plasmid-borne enterotoxin gene.

Clostridium perfringens type F isolates utilize C. perfringens enterotoxin (CPE) to cause food poisoning (FP) and nonfoodborne gastrointestinal diseases. The enterotoxin gene (cpe) can be located on either the chromosome or plasmids, but most FP isolates carry a chromosomal cpe (c-cpe) gene. Our 2000 article in Applied and Environmental Microbiology (66:3234-3240, 2000, https://doi.org/10.1128/aem.66.8.3234-3240.2000https://doi.org/10.1128/AEM.66.8.3234-3240.2000) determined that vegetative cells and spores of c-cpe isolates are more heat resistant than those of plasmid cpe (p-cpe) isolates, which is favorable for their survival in improperly cooked or held food. However, that 2000 article was recently retracted (90:e00249-24, 2024, https://doi.org/10.1128/aem.00249-24). To our knowledge, the 2000 article remains the only study reporting that heat resistance differences are common between both vegetative cells and spores of type F c-cpe isolates vs type F p-cpe isolates. To confirm and preserve this information in the literature, the heat resistance portion of the 2000 study has been repeated. The 2024 results reproduced the 2000 results by indicating that, relative to the surveyed type F p-cpe isolates, the vegetative cells of surveyed type F c-cpe isolates are ~2-fold more heat resistant and the spores of most surveyed c-cpe isolates are ~30-fold more heat resistant. However, consistent with several reports since our 2000 paper, one surveyed type F c-cpe isolate (which did not appreciably sporulate in 2000 but sporulated in 2024) produced spores with intermediate heat sensitivity, confirming that spores of some type F c-cpe isolates lack exceptional heat resistance.IMPORTANCEClostridium perfringens type F food poisoning (FP), which is the second most common bacterial cause of FP, involves the production of C. perfringens enterotoxin. While the enterotoxin gene (cpe) can be located on either the chromosome or plasmids in type F isolates, most FP cases are caused by chromosomal cpe isolates. The current results support the conclusion that the vegetative cells and spores of type F chromosomal cpe isolates are often more heat resistant than vegetative cells and spores of type F plasmid cpe isolates. Greater heat resistance should favor the survival of the spores and vegetative cells of those chromosomal cpe isolates in temperature-abused food, which may help explain the strong association of type F chromosomal cpe strains with FP.

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.

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.

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.

Targeted Therapy with a Novel Superantigen-based Fusion Protein Against Interleukin-13 Receptor α2-overexpressing Tumor Cells: An In-silico Study.

Background & Objective: Superantigens are bacterial toxins that induce a massive immune response in the host. Superantigen staphylococcal enterotoxin B (SEB) can form a ternary complex with its receptors, MHC class II (MHCII) and TCR, and can be used in tumor-targeting therapy, particularly when cooperating with a specific vector. In this study, SEB was fused to interleukin-13 (IL13), which forms a complex with IL13 receptor α2 (IL13Rα2) overexpressed in glioblastoma multiforme (GBM) cells for therapeutic goals. Methods: We designed four fusion proteins based on the arrangement of SEB (N- or C-terminal domain) and provided a flexible inter-domain linker (no or yes), resulting in the formation of SEB-IL13, SEB-L-IL13, IL13-SEB, and IL13-L-SEB, respectively. These fusion proteins were then evaluated for their various physicochemical properties and structural characteristics. Bioinformatics tools were employed to predict, refine, and validate the three-dimensional structure of the fusion proteins. In addition, the fusion proteins were docked with IL13Rα2, MHCII, and TCR receptors through the HADDOCK 2.4 server. The candidate fusion protein was subjected to molecular dynamics simulation. Results: There were differences among the designed fusion proteins. The model with the N-terminal domain of IL13 and containing an inter-domain linker (IL13-L-SEB) was stable and had a long half-life. The docking analysis revealed that the IL13-L-SEB fusion protein had a higher binding affinity to the IL13Rα2, MHCII, and TCR receptors. Finally, using molecular dynamics simulation through iMODS, acceptable results were obtained for the IL13-L-SEB docked complexes. Conclusion: The results suggest IL13-L-SEB is a promising novel fusion protein for cancer therapeutic application.

Coupling enterotoxigenic Escherichia coli heat-stable peptide toxin with 8-arm PEG enhances immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh.

Molecular characterization of Methicillin-resistant Staphylococcus aureus isolated in ready-to-eat food sold in supermarkets in Bobo-Dioulasso: case of charcuterie products.

BACKGROUND: Staphylococcus aureus (S. aureus) is one of the most widespread bacterial pathogens in animals and humans, and its role as an important causative agent of food poisoning is well-documented. The aim of this study was to highlight and characterize the resistance patterns of methicillin-resistant S. aureus (MRSA) in charcuterie products sold in selected supermarkets (SM) in Bobo-Dioulasso, Burkina Faso. METHODS: In this study, 72 samples including ham (n = 19), merguez (n = 22), sausage (n = 15) and minced meat (n = 16) were collected from 3 supermarkets. Standard microbiology methods were utilised to characterise S. aureus isolates. Phenotypic resistance patterns were investigated using the disk diffusion method on Mueller-Hinton agar. Genotypic testing using polymerase chain reaction (PCR) was performed on the isolates to detect the 16S-23S gene. Using specific primers, the following genes PVL, TSST-1, mecA, gyrA, gyrB, qnrA, intI1 and aac(6')-Ib-cr were identified from purified DNA by PCR. RESULTS: Among the 72 ready-to-eat food samples, S. aureus was present in 51, (70.83%). The yield was highest in both the ham and merguez food products, 15/51 (29.41%) each, followed by minced meat 12/51 (23.53%) and sausage 9/51 (17.65%). A total of 35 isolates (68.63%) were confirmed as S. aureus after molecular characterization using 16-23 S primers with 05 (14.29%) strains identified as MRSA. All of the MRSA and majority of the methicillin-sensitive S.aureus (MSSA) isolates were resistant to penicillin G, ampicillin, tetracycline and erythromycin, whereas one isolate from minced meat was found in SM3-harbouring PVL, TSST-1, mecA, gyrA, gyrB and Int1 genes. CONCLUSIONS: Our study revealed a high prevalence of S. aureus in chacuterie products in Bobo-Dioulasso with antimicrobial profiles that show resistance to most antibiotics. These findings should inform and augment efforts to raise awareness among local supermarket owners on adequate food manufacturing practices as well as promoting food safety and hygiene.

Emergence of plasmid-mediated tigecycline resistance tet(X4) gene in Enterobacterales isolated from wild animals in captivity.

Background: Over the past few decades, antimicrobial resistance (AMR) has emerged as a global health challenge in human and veterinary medicine. Research on AMR genes in captive wild animals has increased. However, the presence and molecular characteristics of tet(X)-carrying bacteria in these animals remain unknown. Methods: Eighty-four samples were collected from captive wild animals. tet(X) variants were detected using polymerase chain reaction and the isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. All isolated strains were subjected to antimicrobial susceptibility testing and whole-genome sequencing. The virulence of an Escherichia coli strain carrying enterotoxin genes was assessed using a Galleria mellonella larval model. Results: We isolated two tet(X4)-positive E. coli strains and one tet(X4)-positive Raoultella ornithinolytica strain. Antimicrobial susceptibility tests revealed that all three tet(X4)-carrying bacteria were sensitive to the 13 tested antimicrobial agents, but exhibited resistance to tigecycline. Notably, one tet(X4)-carrying E. coli strain producing an enterotoxin had a toxic effect on G. mellonella larvae. Whole-genome sequencing analysis showed that the two tet(X4)-carrying E. coli strains had more than 95% similarity to tet(X4)-containing E. coli strains isolated from pigs and humans in China. Conclusion: The genetic environment of tet(X4) closely resembled that of the plasmid described in previous studies. Our study identified tet(X4)-positive strains in wildlife and provided valuable epidemiological data for monitoring drug resistance. The identification of enterotoxin-producing E. coli strains also highlights the potential risks posed by virulence genes.