The Tolerance of Gut Commensal Faecalibacterium to Oxidative Stress Is Strain Dependent and Relies on Detoxifying Enzymes.

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O2- and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O2•- and improved the survival of Faecalibacterium longum L2-6 under high O2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O2 or H2O2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. IMPORTANCE Commensal bacteria in the genus Faecalibacterium have been proposed for use as next-generation probiotics, but efforts to cultivate and exploit the potential of these strains have been limited by their sensitivity to O2. More broadly, little is known about how commensal and health-associated bacterial species in the human microbiome respond to the oxidative stress that occurs as a result of inflammation in the colon. In this work, we provide insights regarding the genes that encode potential mechanisms of protection against O2 or ROS stress in faecalibacteria, which may facilitate future advances in work with these important bacteria.

Comparative meta-analysis of antimicrobial resistance from different food sources along with one health approach in the Egypt and UK.

BACKGROUND: Antimicrobial resistance (AMR) is a critical global issue that poses significant threats to human health, animal welfare, and the environment. With the increasing emergence of resistant microorganisms, the effectiveness of current antimicrobial medicines against common infections is diminishing. This study aims to conduct a competitive meta-analysis of surveillance data on resistant microorganisms and their antimicrobial resistance patterns in two countries, Egypt and the United Kingdom (UK). METHODS: Data for this study were obtained from published reports spanning the period from 2013 to 2022. In Egypt and the UK, a total of 9,751 and 10,602 food samples were analyzed, respectively. Among these samples, 3,205 (32.87%) in Egypt and 4,447 (41.94%) in the UK were found to contain AMR bacteria. RESULTS: In Egypt, the predominant resistance was observed against ß-lactam and aminoglycosides, while in the United Kingdom, most isolates exhibited resistance to tetracycline and ß-lactam. The findings from the analysis underscore the increasing prevalence of AMR in certain microorganisms, raising concerns about the development of multidrug resistance. CONCLUSION: This meta-analysis sheds light on the escalating AMR problem associated with certain microorganisms that pose a higher risk of multidrug resistance development. The significance of implementing One Health AMR surveillance is emphasized to bridge knowledge gaps and facilitate accurate AMR risk assessments, ensuring consumer safety. Urgent actions are needed on a global scale to combat AMR and preserve the effectiveness of antimicrobial treatments for the well-being of all living beings.

Therapeutic applications of nanobiotechnology.

Nanobiotechnology, as a novel and more specialized branch of science, has provided a number of nanostructures such as nanoparticles, by utilizing the methods, techniques, and protocols of other branches of science. Due to the unique features and physiobiological characteristics, these nanostructures or nanocarriers have provided vast methods and therapeutic techniques, against microbial infections and cancers and for tissue regeneration, tissue engineering, and immunotherapies, and for gene therapies, through drug delivery systems. However, reduced carrying capacity, abrupt and non-targeted delivery, and solubility of therapeutic agents, can affect the therapeutic applications of these biotechnological products. In this article, we explored and discussed the prominent nanobiotechnological methods and products such as nanocarriers, highlighted the features and challenges associated with these products, and attempted to conclude if available nanostructures offer any scope of improvement or enhancement. We aimed to identify and emphasize the nanobiotechnological methods and products, with greater prospect and capacity for therapeutic improvements and enhancements. We found that novel nanocarriers and nanostructures, such as nanocomposites, micelles, hydrogels, microneedles, and artificial cells, can address the associated challenges and inherited drawbacks, with help of conjugations, sustained and stimuli-responsive release, ligand binding, and targeted delivery. We recommend that nanobiotechnology, despite having few challenges and drawbacks, offers immense opportunities that can be harnessed in delivering quality therapeutics with precision and prediction. We also recommend that, by exploring the branched domains more rigorously, bottlenecks and obstacles can also be addressed and resolved in return.

Specific and sensitive detection of Influenza A virus using a biotin-coated nanoparticle enhanced immunomagnetic assay.

This study reports the development of a sensitive magnetic bead-based enzyme-linked immunoassay (MELISA) for the pan-reactive detection of the Influenza A virus. The assay combines immunomagnetic beads and biotin-nanoparticle-based detection to quantify a highly conserved viral nucleoprotein in virus lysates. At the capture step, monoclonal antibody-coated magnetic microbeads were used to bind and concentrate the nucleoprotein in samples. The colorimetric detection signal was amplified using biotinylated silica nanoparticles (NP). These nanoparticles were functionalized on the surface with short DNA spacers bearing biotin groups by an automated supported synthesis method performed on nano-on-micro assemblies with a DNA/RNA synthesizer. A biotin-nanoparticle and immunomagnetic bead-based assay was developed. We succeeded in detecting Influenza A viruses directly in the lysis buffer supplemented with 10% saliva to simulate the clinical context. The biotin-nanoparticle amplification step enabled detection limits as low as 3 × 103 PFU mL-1 and 4 × 104 PFU mL-1 to be achieved for the H1N1 and H3N2 strains respectively. In contrast, a classical ELISA test based on the same antibody sandwich showed detection limit of 1.2 × 107 PFU mL-1 for H1N1. The new enhanced MELISA proved to be specific, as no cross-reactivity was found with a porcine respiratory virus (PRRSV). Graphical abstract.

Applications of biosensors for bacteria and virus detection in food and water-A systematic review.

Biosensors for sensitive and specific detection of foodborne and waterborne pathogens are particularly valued for their portability, usability, relatively low cost, and real-time or near real-time response. Their application is widespread in several domains, including environmental monitoring. The main limitation of currently developed biosensors is a lack of sensitivity and specificity in complex matrices. Due to increased interest in biosensor development, we conducted a systematic review, complying with the PRISMA guidelines, covering the period from January 2010 to December 2019. The review is focused on biosensor applications in the identification of foodborne and waterborne microorganisms based on research articles identified in the Pubmed, ScienceDirect, and Scopus search engines. Efforts are still in progress to overcome detection limitations and to provide a rapid detection system which will safeguard water and food quality. The use of biosensors is an essential tool with applicability in the evaluation and monitoring of the environment and food, with great impact in public health.

Rapid point-of-need detection of bacteria and their toxins in food using gold nanoparticles.

Biosensors need to meet the rising food industry demand for sensitive, selective, safe, and fast food safety quality control. Disposable colorimetric sensors based on gold nanoparticles (AuNPs) and localized surface plasmon resonance are low-cost and easy-to-perform devices intended for rapid point-of-need measurements. Recent studies demonstrate various facile and versatile AuNPs-based analytical platforms for the detection of bacteria and their toxins in milk, meat, and other foods. In this review, we introduce the general characteristics and mechanisms of AuNPs calorimetric biosensors, and highlight optimizations needed to strengthen and improve the quality of devices for their application in food matrices.

Antimicrobial nanoparticles and biodegradable polymer composites for active food packaging applications.

The food industry faces numerous challenges to assure provision of tasty and convenient food that possesses extended shelf life and shows long-term high-quality preservation. Research and development of antimicrobial materials for food applications have provided active antibacterial packaging technologies that are able to meet these challenges. Furthermore, consumers expect and demand sustainable packaging materials that would reduce environmental problems associated with plastic waste. In this review, we discuss antimicrobial composite materials for active food packaging applications that combine highly efficient antibacterial nanoparticles (i.e., metal, metal oxide, mesoporous silica and graphene-based nanomaterials) with biodegradable and environmentally friendly green polymers (i.e., gelatin, alginate, cellulose, and chitosan) obtained from plants, bacteria, and animals. In addition, innovative syntheses and processing techniques used to obtain active and safe packaging are showcased. Implementation of such green active packaging can significantly reduce the risk of foodborne pathogen outbreaks, improve food safety and quality, and minimize product losses, while reducing waste and maintaining sustainability.

Bacillus subtilis Regulators MntR and Zur Participate in Redox Cycling, Antibiotic Sensitivity, and Cell Wall Plasticity.

The Bacillus subtilis MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we describe the global impact of mntR and zur deletions at the protein level. Using a comprehensive proteomic approach, we showed that 33 and 55 proteins are differentially abundant in ΔmntR and Δzur cells, respectively, including proteins involved in metal acquisition, translation, central metabolism, and cell wall homeostasis. In addition, both mutants showed modifications in intracellular metal ion pools, with significant Mg2+ accumulation in the ΔmntR mutant. Phenotypic and morphological analyses of ΔmntR and Δzur mutants revealed their high sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress. Mutant strains had a modified cell wall thickness and accumulated lower levels of intracellular reactive oxygen species (ROS) than the wild-type strain. Remarkably, our results highlight an intimate connection between MntR, Zur, antibiotic sensitivity, and cell wall structure.IMPORTANCE Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In Bacillus subtilis, Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of B. subtilis wild-type, ΔmntR, and Δzur strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of mntR and zur genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.

Advanced Methods for Detection of Bacillus cereus and Its Pathogenic Factors.

Bacillus cereus is an opportunistic foodborne pathogen causing food intoxication and infectious diseases. Different toxins and pathogenic factors are responsible for diarrheal syndrome, like nonhemolytic enterotoxin Nhe, hemolytic enterotoxin Hbl, enterotoxin FM and cytotoxin K, while emetic syndrome is caused by the depsipeptide cereulide toxin. The traditional method of B. cereus detection is based on the bacterial culturing onto selective agars and cells enumeration. In addition, molecular and chemical methods are proposed for toxin gene profiling, toxin quantification and strain screening for defined virulence factors. Finally, some advanced biosensors such as phage-based, cell-based, immunosensors and DNA biosensors have been elaborated to enable affordable, sensitive, user-friendly and rapid detection of specific B. cereus strains. This review intends to both illustrate the state of the B. cereus diagnostic field and to highlight additional research that is still at the development level.

Exploring the impact of Mg-doped ZnO nanoparticles on a model soil microorganism Bacillus subtilis.

The environmental contamination of soil by metal oxide nanomaterials is a growing global concern because of their potential toxicity. We investigated the effects of Mg doped ZnO (Mg-nZnO) nanoparticles on a model soil microorganism Bacillus subtilis. Mg-nZnO exhibited only a moderate toxic effect on B. subtilis vegetative cells but was able to prevent biofilm formation and destroy already formed biofilms. Similarly, Mg-nZnO (≤1 mg/mL) was moderately toxic towards Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and murine macrophages. Engineered Mg-nZnO produced H2O2 and O2•- radicals in solutions of various salt and organic molecule compositions. A quantitative proteomic analysis of B. subtilis membrane proteins showed that Mg-nZnO increased the expression of proteins involved in detoxification of ROS, translation and biofilm formation. Overall, our results suggest that Mg-nZnO released into the environment may hinder the spreading, colonization and biofilm formation by B. subtilis but also induce a mechanism of bacterial adaptation.

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria.

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification.

Turning off NADPH oxidase-2 by impeding p67phox activation in infected mouse macrophages reduced viral entry and inflammation.

BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS: IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity.

PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.

Advanced biosensors for detection of pathogens related to livestock and poultry.

Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal infection diseases and control management in farms and field conditions. Current techniques employed to diagnose pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to distinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR) and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments, being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we provide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at development level some are validated according to standards of the World Organization for Animal Health and are commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sensing systems for implementation in field condition are included in this review.

Identification of a Novel Complex between the Nucleoprotein and PA(1-27) of Influenza A Virus Polymerase.

The structure of the ribonucleoprotein complexes is crucial to viral transcription and replication of influenza virus, but association of the nucleoprotein (NP) with the polymerase remains to be characterized at the molecular level. Here, we identify a peptide of the polymerase acidic subunit PA(1-27) that associates with NP. Docking and molecular dynamics simulations suggest a similar NP binding site with PA(1-27) and PA(1-186). The PA(1-27)-NP complex is characterized by surface plasmon resonance and fluorescence using recombinant NP proteins and by pull-down assays in infected cells. The PA(1-27)-NP complex may have a role in the final steps of transcription and replication.

N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes.

PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1-90) and C-terminal PB1-F2 domain (53-90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1-52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain.

Pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties.

Th antibacterial activity of metal oxide nanoparticles has received marked global attention as they can be specifically synthesized to exhibit significant toxicity to bacteria. The importance of their application as antibacterial agents is evident keeping in mind the limited range and effectiveness of antibiotics, on one hand, and the plethora of metal oxides, on the other, along with the propensity of nanoparticles to induce resistance being much lower than that of antibiotics. Effective inhibition against a wide range of bacteria is well known for several nano oxides consisting of one metal (Fe3O4, TiO2, CuO, ZnO), whereas, research in the field of multi-metal oxides still demands extensive exploration. This is understandable given that the relationship between physicochemical properties and biological activity seems to be complex and difficult to generalize even for metal oxide nanoparticles consisting of only one metal component. Also, despite the broad scope that metal oxide nanoparticles have as antibacterial agents, there arise problems in practical applications taking into account the cytotoxic effects. In this respect, the consideration of polymetallic oxides for biological applications becomes even greater since these can provide synergetic effects and unify the best physicochemical properties of their components. For instance, strong antibacterial efficiency specific of one metal oxide can be complemented by non-cytotoxicity of another. This review presents the main methods and technological advances in fabrication of nanostructured metal oxides with a particular emphasis to multi-metal oxide nanoparticles, their antibacterial effects and cytotoxicity.

Electrochemical detection of the oligomerization of PB1-F2 influenza A virus protein in infected cells.

PB1-F2 is a nonstructural accessory protein of Influenza A virus described to enhance the mortality and the morbidity of the virus in a host-dependent manner. In this work, an electrochemical biosensor based on an immunodetection system was developed to follow the oligomerization of PB1-F2 during the viral cycle. The immunosensor was based on conductive polypyrrole modified with ferrocenyl groups as a redox marker for enhancing signal detection. Antibodies specific for monomeric or oligomeric PB1-F2 forms were immobilized on polypyrrole matrix via biotin/streptavidin layer. We demonstrated that this electrochemical biosensor sensitively detects PB1-F2 in both conformational forms. The linear range extends from 5 nM to 1.5 µM and from 5 nM to 0.5 µM for monomeric and oligomeric PB1-F2, respectively. The calculated limit of detection was 0.42 nM for monomeric PB1-F2 and 16 nM for oligomers. The biosensor platform allows the detection and quantification of PB1-F2 in lysates of infected cells during viral cycle. We show that at early stages of viral cycle, PB1-F2 is mainly monomeric but switched to amyloid-like structures at a later stage of infection. The quantification of two protein structural forms points out that PB1-F2 expression profiles and kinetics of oligomerization are cell-type-dependent.

Effects of water and cell culture media on the physicochemical properties of ZnMgO nanoparticles and their toxicity toward mammalian cells.

ZnMgO nanoparticles have shown potential for medical applications as an efficient antibacterial agent. In this work, we investigate the effect of water and two commonly used cell culture media on the physicochemical properties of ZnMgO nanoparticles in correlation with their cytotoxicity. In vacuum, ZnMgO nanopowder consists of MgO (nanocubes) and ZnO (nanotetrapods and nanorods) particles. Upon exposure to water or the Luria-Bertani solution, ZnO characteristic shapes were not observable while MgO nanocubes transformed into octahedral form. In addition, water caused morphological alternations in form of disordered and fragmented structures. This effect was directly reflected in UV/vis absorption properties of ZnMgO, implying that formation of new states within the band gap of ZnO and redistribution of specific sites on MgO surfaces occurs in the presence of water. In mammalian culture cell medium, ZnMgO nanoparticles were shapeless, agglomerated, and coated with surrounding proteins. Serum albumin was found to adsorb as a major but not the only protein. Adsorbed albumin mainly preserved its α-helix secondary structure. Finally, the cytotoxicity of ZnMgO was shown to strongly depend on the environment: in the presence of serum proteins ZnMgO nanopowder was found to be safe for mammalian cells while highly toxic in a serum-free medium or a medium containing only albumin. Our results demonstrate that nanostructured ZnMgO reaches living cells with modified morphology and surface structure when compared to as-synthesized particles kept in vacuum. In addition, its biocompatibility can be modulated by proteins from biological environment.

Recent Advances in Aptamer-Based Biosensors for Bacterial Detection.

The rapid and sensitive detection of pathogenic bacteria is becoming increasingly important for the timely prevention of contamination and the treatment of infections. Biosensors based on nucleic acid aptamers, integrated with optical, electrochemical, and mass-sensitive analytical techniques, have garnered intense interest because of their versatility, cost-efficiency, and ability to exhibit high affinity and specificity in binding bacterial biomarkers, toxins, and whole cells. This review highlights the development of aptamers, their structural characterization, and the chemical modifications enabling optimized recognition properties and enhanced stability in complex biological matrices. Furthermore, recent examples of aptasensors for the detection of bacterial cells, biomarkers, and toxins are discussed. Finally, we explore the barriers to and discuss perspectives on the application of aptamer-based bacterial detection.