Search Results (295)

Background: Molecular syndromic stool panels are increasingly used in paediatric diarrheal syndromes; however, interpretation of Clostridioides difficile (C. difficile) detection remains challenging because colonisation is common in younger children. We aimed to assess the frequency of C. difficile detection using a syndromic gastrointestinal panel in a paediatric tertiary-care centre and to describe the subsequent microbiological work-up and CDI-directed treatment. Methods: We conducted a retrospective single-centre study of all BioFire FilmArray Gastrointestinal (GI) panels performed at San Marco Hospital (University Hospital “G. Rodolico-San Marco”, Catania, Italy) from 1 January 2023 to 31 December 2025. Only the first C. difficile-positive result per patient was included; repeat positives within 30 days were excluded. Index-positive episodes were stratified by age (<1 year, 1 to <2 years, and ≥2 years). Data collected included co-detected pathogens, toxin A/B enzyme immunoassay (EIA) results, GeneXpert PCR findings, and CDI-directed therapy. Results: Among the 714 GI panels performed during the study period, 112 (15.7%) were positive for C. difficile. After exclusion of repeat positives, 91 index-positive episodes were analysed. Median age was 1.0 years (IQR 0.75–4.0), and 48/91 cases (52.7%) occurred in children younger than two years. Toxin A/B EIA was positive in 11/82 tested episodes (13.4%), whereas GeneXpert tcdB was positive in 75/84 episodes (89.3%). Co-detection of at least one additional enteric pathogen occurred in 40/91 cases (44.0%). CDI-directed therapy was administered in 9/91 episodes (9.9%), mainly in children aged ≥2 years. Conclusions: Detection of C. difficile by syndromic molecular panels was relatively frequent in our paediatric cohort but rarely associated with toxin positivity or the need for specific treatment. These findings suggest that many positive Nucleic Acid Amplification Test (NAAT) results may represent colonisation rather than true infection, particularly in younger children. Careful clinical interpretation of syndromic panel results is therefore essential to avoid overdiagnosis and unnecessary antimicrobial therapy. Full article

Background/objectives. Clostridioides difficile infection (CDI) remains a major cause of healthcare-associated infectious colitis, particularly among elderly and multimorbid patients. Disease severity and clinical evolution are influenced by the host’s systemic inflammatory response. This study aimed to evaluate the hematological and inflammatory profile of hospitalized CDI patients and to explore the prognostic value of routine laboratory parameters for prolonged hospitalization. Methods. A retrospective observational study was conducted on 50 adult patients hospitalized with laboratory-confirmed CDI (positive glutamate dehydrogenase, antigen and toxins A/B). Hematological parameters (WBC, hemoglobin, RDW) and inflammatory markers (CRP, fibrinogen) were analyzed at admission and discharge. Prolonged hospitalization was defined as length of stay (LOS) > 8 days (cohort median). Multivariable logistic regression was performed to assess admission predictors of prolonged hospitalization, and model discrimination was evaluated using leave-one-out cross-validation (LOOCV). Results. At admission, patients exhibited marked inflammatory activation accompanied by reduced hemoglobin and elevated RDW. Significant correlations were observed between inflammatory markers. All inflammatory and hematologic parameters improved at discharge. In multivariable analysis, lower admission hemoglobin and higher log-transformed CRP showed exploratory associations with prolonged hospitalization. The internally validated model demonstrated moderate discriminative performance (AUC = 0.65). Conclusions. CDI is associated with substantial systemic inflammatory activation and hematologic alterations. While no individual predictor reached statistical significance, the observed effect sizes provide hypothesis-generating estimates to inform future prospective validation studies. Full article

Background: Persistence represents a critical evolutionary reservoir for the development of antimicrobial resistance in Acinetobacter baumannii (Ab). Understanding the basal mechanisms that enable this survival strategy is crucial for elucidating how high-risk clones evolve resistance during therapy. Methods: High-dose colistin time-kill assays were performed in ten ST2 clinical colistin-susceptible (COL-S) Carbapenem-Resistant Ab (CRAB) developing in vivo stable and full-colistin resistance to detect persisters. Genomics and basal transcriptomics of chromosomal/plasmid toxin–antitoxin systems (T/As) were performed, as duplicates for each sample, in two ST2 COL-S CRAB to investigate the genomics and basal T/A transcriptomic backgrounds. Results: Phenotypically, all strains showed a persistent subpopulation (~1% survival at 8 h) under 5× COL MIC exposure. Genomics identified 10 type-II and one type-IV T/A systems. Basal transcriptomics revealed active expression patterns mainly of GNAT superfamily T/A systems, with consistently low toxin mRNA levels associated with toxin- or antitoxin-directed asRNAs in chromosomal modules. This architecture defined new dual-combined regulatory models in which asRNAs acted as primary T/A mRNA balance modulators, putatively impacting on the T/A mRNA ratio. Conversely, the plasmid-encoded BrnT/A module showed a highly balanced expression. Conclusions: Our findings revealed, for the first time, the role of the type-II GNAT T/A superfamily as putative molecular switchers via a fine-tuning transcript balance regulation, impacting the transition from a metabolically active cell state to a dormant one in developing colistin persistence and in vivo resistance CRAB. Full article

Pertussis toxin (PTx) is a major virulence factor of Bordetella pertussis and an AB5-type exotoxin that disrupts host signaling. Its enzymatic A subunit ADP-ribosylates the α-subunit of inhibitory G proteins (Gα_i), preventing them from mediating receptor-induced inhibition of adenylyl cyclase (AC). This leads to unrestrained cAMP accumulation in host cells, a canonical mechanism underlying many pertussis disease manifestations. PTx works in concert with the bacterium’s adenylate cyclase toxin (ACT) to subvert immune defenses and establish infection. Interestingly, PTx exerts both cAMP-dependent and cAMP-independent effects. In addition to the well-known cAMP-mediated pathway, PTx’s B oligomer can engage host cell surface receptors to trigger signaling cascades independent of the A subunit’s catalytic activity. Such B oligomer-mediated pathways modulate cellular responses in the absence of ADP-ribosylation. This review provides a comprehensive analysis of PTx’s dual functionality, distinguishing its G_i protein-dependent elevation of cAMP from the noncanonical activities of the B oligomer. It also highlights how disruption of constitutive G_i signaling and the interplay between PTx and ACT shape host–pathogen interaction in pertussis pathogenesis. Full article

Background: bacterial pathogens and their toxins present analytical challenges for rapid and specific detection, contributing to over 600 million cases of illness annually and worsening antimicrobial resistance (AMR). Conventional detection methods are useful but limited. Single-domain antibodies (sdAbs) offer alternative recognition elements with unique biochemical and engineering benefits, enabling the development of nanobody-based immunoassays and biosensing platforms that provide fast, highly selective, and reliable detection of bacterial pathogens and toxins in both food and clinical environments. Objectives: this systematic review assesses the analytical and functional performance of nanobody-based immunoassays and sensing formats for detecting bacteria and toxins across food and clinical samples. Methods: following PRISMA guidelines, major scientific databases were used to gather research, resulting in 32 eligible studies published between 2011 and 2025. Results: data collected included assay platforms, target bacteria and toxins, limit of detection, sensitivity, specificity, matrix recovery, and practicality. Risk of bias was evaluated using an adapted QUADAS-2 framework. The review shows that nanobody-based immunoassays have achieved high performance, thermostability, compatibility with genetic engineering, and versatile assay design. When combined with advanced transduction and signal amplification strategies, these systems contribute to the development of highly sensitive and user-friendly bioanalytical platforms for detecting bacteria and toxins. Conclusions: however, most studies relied on spiked samples and lacked large-scale validation, emphasizing the need for standardized benchmarking and real-world testing. Full article

Insecticidal proteins derived from Bacillus thuringiensis (Bt) have been effectively employed in controlling lepidopteran pests, notably in transgenic crops targeting Spodoptera species. However, concerns have arisen regarding the long-term efficacy due to the emergence of tolerant and resistant insect populations. Prior research suggested that repeated exposures to Bt, which contains a mixture of spores and crystals, may contribute to the development of tolerance; however, the specific effects of sequential exposure to purified Cry1 and Vip3Aa proteins remain unclear. This study aimed to assess whether prior exposure of Spodoptera exigua neonate larvae to sublethal concentrations of Cry1Ab, Cry1Ca or Vip3Aa proteins would heighten their tolerance upon subsequent exposure, and whether such effects would extend to their offspring. Pre-exposure to Cry1Ab or Vip3Aa did not affect larval responses to the toxin. For Cry1Ca, a slight increase was observed under one treatment condition, but the effect was not considered biologically relevant. Transgenerational analysis revealed no enhancement of tolerance; rather, there was a negative impact on the offspring’s response in some cases. These findings indicate that although previous studies have documented that sublethal contact with bacterial preparations may significantly affect insect tolerance, exposure to sublethal doses of purified Cry1 and Vip3Aa proteins is unlikely to lead to the development of tolerance in S. exigua. Full article

Botulinum toxin type A (BoNT-A) is an established preventive therapy for chronic migraines; however, uncertainty remains regarding its comparative efficacy and safety. Thus, we aimed to summarize current evidence from high-quality systematic reviews of the therapeutic effects of BoNT-A in migraine management. An umbrella review was conducted following PRISMA guidelines and registered in PROSPERO. High-quality systematic reviews with meta-analysis evaluating BoNT-A efficacy were identified through five databases up to August 2024. Primary outcomes included monthly headache frequency and severity. Methodological quality and risk of bias were assessed using the umbrella review checklist. Fourteen articles were included. Overall, quantitative evidence indicated favorable effects of BoNT-A compared with placebo for chronic migraines, across headache frequency, headache severity, and acute medication use, but less efficacy than topiramate and the CGRP monoclonal antibodies (CGRPmAbs) galcanezumab and fremanezumab. Though the adverse events were frequent, BoNT-A was generally well-tolerated. Comparative data suggested superior tolerability versus topiramate and a safety profile like CGRPmAbs. Although botulinum toxin type A is widely used as a preventive treatment for chronic migraines, the available evidence supports its efficacy at a moderate level. Further head-to-head and long-term analyses are needed to clarify its comparative role alongside newer biologic treatments. Full article

Bacillus anthracis has three main virulence factors: an extracellular capsule and two binary toxins (lethal toxin—consists of a lethal factor and a protective antigen, and edema toxin—consists of an edema factor and a protective antigen). In the Russian Federation, the epidemiological situation regarding anthrax infection remains unfavorable. In the late stages of an anthrax infection, antibiotic therapy becomes ineffective and the patient dies within 24 h as a large amount of lethal toxin accumulates in the patient’s blood. Antibodies capable of neutralising lethal toxin (LT) can be an effective treatment for these patients. The objective of the study was to construct a chimeric monoclonal antibody targeting the protective antigen of the LT and to elucidate its mechanism of toxin neutralization. In this work, a chimeric monoclonal antibody (xi1E10) directed against the protective antigen was successfully produced. Both in vitro and in vivo experiments demonstrated the capacity of xi1E10 to neutralize lethal toxin. Confocal microscopy revealed that xi1E10 effectively suppresses the formation of a functional pore, thereby blocking the translocation of the lethal factor into the cytosol. These findings indicate that the monoclonal antibody xi1E10 represents a promising candidate for the development of a therapeutic drug. Full article

Bradysia difformis is a notorious pest in mushroom production in China. Biological control using Bacillus thuringiensis (Bt) offers an environmentally friendly and effective strategy against this pest. Here, we show that the complete genome of strain JW-1 consists of one circular chromosome and seven circular plasmids. JW-1-Plasmid 4 comprises 127,921 bp with a GC content of 33.9%, and is predicted to contain 131 genes, including six insecticidal genes: cry4Aa, cry4Ba, cry10Ab, cry11Aa, cyt1Aa, and cyt2Ba. A 3542-bp fragment containing the cry4Aa gene was amplified from this strain. Phylogenetic analysis based on Cry4 toxin sequences showed that JW-1 Cry4 toxin belongs to the Cry4Aa toxin cluster. A Cry4Aa fusion protein was subsequently expressed in E. coli and purified using Ni-IDA affinity chromatography. A larval feeding assay showed that purified Cry4Aa was toxic to B. difformis larvae, with an LC₅₀ of 2.71 ng/mL. These results confirmed the identity and bioactivity of Cry4Aa from strain JW-1, offering a promising biological control agent against this major pest. Full article

A variety of pesticidal proteins derived from the bacterium Bacillus thuringiensis exhibit activity against the yellow fever mosquito Aedes aegypti and are used to control this insect vector. Several of these proteins, including Cry1Ca and Cry2Aa, additionally have activity against lepidopteran insects. Furthermore, the specificity of Cry2Aa has recently been shown to depend on domain I of the Cry protein, whereas it is generally recognized that domain II is the primary specificity-determining domain. This work has made use of disabled forms of three Cry proteins (Cry2Aa, Cry1Ca and Cry11Aa) and one naturally non-active protein (Cry2Ab) in an in vivo competition assay to investigate whether Cry2Aa and the dual-active Cry1Ca share a common receptor with the other pesticidal proteins. It was found that despite their differing specificities and potential modes of action, all of the Aedes-active proteins tested made use of a common receptor, although evidence is presented that Cry2Aa can use multiple receptors. When additional toxins (Cry41Aa, Cry1Aa, Cry1Ac) with no activity against this mosquito were tested, they too were found to share the same receptor, suggesting that Cry toxins may have evolved to utilize a common set of receptors in insects but that additional factors determine species specificity. Full article

Protein toxins are biologically active polypeptides produced by a variety of organisms, including bacteria, plants, fungi, and animals. These molecules exert potent and specific toxic effects on target cells and are primarily associated with pathogenicity and defense mechanisms of the organisms. In the past few decades, significant progress has been made in understanding their structure, mechanisms of action, and regulation. Among these, bacterial protein toxins have emerged as valuable tools particularly in the development of targeted therapies. A notable example is Botulinum toxin, originally known for its neurotoxic effects, which was approved as a therapeutic agent in 1989 for strabismus treatment, paving way for repurposing bacterial toxins for clinical use. This review provides an overview of the different classes of bacterial toxin-based therapeutics, with a particular focus on Pseudomonas exotoxin A (PE) from Pseudomonas aeruginosa and anthrax toxin from Bacillus anthracis. The modular architecture and potent cytotoxicity of these A-B type toxins have enabled their successful adaptation into targeted cancer therapies. The clinical approval of the PE-based immunotoxin, moxetumomab pasudotox, for the treatment of hairy cell leukemia, underscores the potential of this strategy. This review also discusses current challenges and outlines future directions for the advancement of bacterial toxin-based therapeutics. Full article

Phospholipase A1 (Ves a 1), a major toxin from Vespa affinis venom, poses significant risks to allergic individuals. Nevertheless, the epitope determinants of Ves a 1 have not been characterized. Thus, identifying its linear B-cell epitopes is crucial for understanding envenomation mechanisms. In this study, we predicted and identified B-cell epitopes EP5 and EP6 as potential candidates. EP5 formed an α-helix at the active site of Ves a 1, whereas EP6 adopted an extended loop conformation. Both synthetic peptides were synthesized and evaluated for their inhibitory effects using immune-inhibitory assays with polyclonal antibodies (pAbs) targeting both native (nVes a 1) and recombinant (rVes a 1) forms. The Ves a 1 polyclonal antibodies (pAb-nVes a 1 and pAb-Ves a 1) were produced, and their specificity binding to Ves a 1 was confirmed by Western blot. Next, ELISA inhibition assays showed that EP5 and EP6 significantly blocked pAb binding to both nVes a 1 and rVes a 1. Dot blot and Western blot assays supported these findings, particularly with stronger inhibition toward rVes a 1. Furthermore, enzymatic assays indicated that nVes a 1 and rVes a 1 retained phospholipase activity. Immunoinformatics docking showed that EP5 and EP6 specifically bind to a single-chain variable fragment antibody (scFv) targeting Naja naja PLA2. Molecular analysis revealed similar amino acid interactions to the template, suggesting effective paratope–epitope binding. These results support the potential of EP5 and EP6 for future diagnosis and therapy of V. affinis venom allergy. Full article

Infections caused by Acinetobacter baumannii are increasing worldwide. We evaluated the antibiotic resistance profile, biofilm production, and the frequency of 12 genes encoding carbapenemases and 13 virulence factors in 90 isolates from patients of three hospitals in various regions of Poland. Antibiotic resistance survey was performed using the disc-diffusion method, genes encoding resistance to carbapenems and virulence factors were detected with PCR, and biofilm formation was tested using microtiter plates. A total of 52.2% of isolates were resistant to all tested antibiotic groups (penicillins with β-lactamase inhibitors, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and trimethoprim plus sulfamethoxazole). Among the genes encoding carbapenem resistance, the bla_OXA-23 (68.9%), bla_OXA-40 (83.3%), and ISAba-bla_OXA-51 (18.9%) were detected. The ompA, ata, and recA genes responsible for biofilm formation, adhesion, and stress response, respectively, occurred in all isolates. Genes responsible for the production of other adhesins (bap—94.4%, espA—4.4%, chop—37.7%), biofilm formation (pbpG—90.0%), production of siderophore (basD—97.7%), toxins (lipA—92.2%, cpaA—1.1%), glycoconjugates (bfmR—84.4%), and inducing host cell death (fhaB—71.1%, abeD—93.3%) were also found. A total of 68.8% of isolates produced biofilm. The isolates from Masovia had more virulence genes than isolates from the other regions; moreover, all isolates from Masovia and West Pomerania were multidrug-resistant (MDR), including resistance to carbapenems. Full article

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin–antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae. Full article

Recombinant monoclonal antibody (mAb) botulinum neurotoxin (BoNT) antitoxins, consisting of three mAbs that bind non-overlapping epitopes, are highly potent. However, the three-mAb mixtures pose unique development and manufacturing challenges. Combining even more mAbs to create multivalent antitoxin drugs multiplies those challenges. We previously reported that a single tri-epitopic IgG1-based mAb (TeAb) containing the variable domains of the three parental BoNT/A mAbs and an Fc was as potent as the combination of three IgGs in the mouse neutralization assay (MNA). Here, we extended the tri-epitopic strategy to three other BoNT serotypes. Each TeAb (TeAb-B for BoNT/B, TeAb-E for BoNT/E, and TeAb-F for BoNT/F) binding was measured using fluorescence-activated cell sorting and flow fluorimetry, and the potency was tested in the MNA. The three TeAbs displayed binding affinities that were the same within error of the parental IgGs for each epitope, and all had higher avidity to each serotype of BoNT than that of the parental mAbs. The potency of the BoNT/B, BoNT/E, and BoNT/F TeAbs was similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/F in the MNA. We now have four examples of a single TeAb recapitulating the affinity and in vivo potency of a three-mAb antitoxin. The tri-epitopic strategy could be applied to streamline the production and bioanalytics of antibody drugs where three-mAb binding is required for activity. Full article